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400 | (Spoilers ALL) Problems visualising yet-to-be-introduced-on-tv characters. | I was just curious if there are any examples of a poster spoiling a movies plot. | It can be with or without spoilers, or spoilers that they don't know are spoilers, but what scene is so single handedly awesome or enthralling that would hook someone as soon as they watch it without knowing anything else about it? | By side character I mean a character who have gotten at least a few episodes dedicated to them
Minor characters like crazy cat lady who rarely makes an appearance don’t count
View Poll | Its so stupid to see people saying "endorsi is here, you should watchout for the blanket guy, take care with rachel , this x character that was just shown is amazing"
ALL OF THOSE ARE CONSIDERED SPOILERS.
You dont need to say what will happen for it to be a spoiler.
If you bring the spotlight to something , people will create expetations about it. One of the coolest parts of a history is to find out that a character that you never thought existed is OP or cool asf.
Not even mentioning that if you bring a spotlight to something, most people will probably endup figuring out what eventually could happen.
Please, dont do that. Let they have fun figuring out each character in the story.
PS: sorry if any grammar errors, english is not my main language. | I only know of this because of the game, but I’m only on the beginning of season 2 of the show. I don’t know if they already establish this or if it will come up later. I’m asking because I don’t want to accidentally spoil this for my roommate who’s watching it with me.
EDIT: Thanks all! | So my bf only watched the series, and I had just finished ACoK and have gotten about 150 pages into ASoS. I kept wanting to talk about my favorite new character ACoK new character from ACoK who will be in season 2, so I pause from my reading and say, "Aw I want to discuss my favorite new character with you, but the character doesn't even pop up yet til next season..."
He looks up and says, unabashed, "Is it, ACoK new character?"
I glare at him, saying, "Um, no, but how do you even know about ACoK new character?"
He then says, "Well, I saw like a corner of your page in ASoS talking about ACoK spoiler."
I proceed to angrily reprimand him for even glancing at my book, but then he says "Well, it's not like the story's ruined for me, all I know about ASoS is that ACoK."
"..........yeah. Nothing was ruined for you. Heh."
I feel so bad, yet I can't even tell him...
Sorry for the overuse of spoiler tags, I don't even know what a spoiler is anymore..... | While no one should open a thread that says "Another shot from the set of Hawkeye," accidents happen. So please mark your [SPOILERS] from here on out. Spoilers include any story detail, even minor ones, and any set or costume shots. (More people than you realize like to go in blind.)
Speaking of which, that tag won't guarantee RES won't auto-expand a spoiler image, so I'm good with the use of the NSFW feature on spoiler posts.
Thank you, bros! | Does anyone else hate this? In the Voyager episode *Deathwish*, the opening credits spoil that John de Lancie and Jonathan Frakes make an appearance. So, the first time I watched it, Q(Quinn/Q2) showed up and I thought "O so now they're exploring other Q's without de Lancie, that's interesting" but then nope. The entrance of de Lancie is (not completely) ruined because of the credits already introducing him. Ditto for Frakes' appearance. There are other episodes that do this but you get the point.
Am I stupid for being annoyed by this? | 400 | Am I the only one who has trouble visualising the characters in the series that have not yet appeared in the show?
All the "new" characters, like Griff etc. just seem to be a blank figure in my head, like my brain is just waiting for the show to fill in the blanks.
Anyone else have this problem? or is my imagination just that shit? | Am I the only one not imagining the visual appearance of characters? Or anything else? | Anyone else disappointed by the format of the gameshow? | Does anybody else feel differently after an episode? | I loved this series but was constantly distracted by the lack ... | The plot holes of this show just keep getting deeper and deeper | Perhaps its just me, I thought the characters were ... | Weird nostalgic vibe when watching RWBY? | My ideas of what we'll see with a few of the characters in the series |
401 | // CreatedBy sets the optional parameter "created_by": | private function createDir($path, $chmod = 0777, $recursive = true)
{
if (!mkdir($path, $chmod, $recursive)) {
throw new RuntimeException(
sprintf(
'The directory path "%s" could not be created.',
$path
)
);
}
} | def project_create_handler(args):
""""""
if not db.setup(url=args.db, echo=args.db_echo):
return
if not _check_db_revision():
return
project_path = os.path.abspath(args.project_dir)
project_name = args.project_name
project = db.session.query(Project).\
filter_by(path_name=project_path).first()
if project is None:
project = Project.create(project_path, project_name)
else:
print("Path '{}' has already registered.".format(project.path_name)) | func createPR() (err error) {
if *prSubject == "" {
return errors.New("missing `-pr-title` flag; skipping PR creation")
}
if *prRepoOwner != "" && *prRepoOwner != *sourceOwner {
*commitBranch = fmt.Sprintf("%s:%s", *sourceOwner, *commitBranch)
} else {
prRepoOwner = sourceOwner
}
if *prRepo == "" {
prRepo = sourceRepo
}
newPR := &github.NewPullRequest{
Title: prSubject,
Head: commitBranch,
Base: prBranch,
Body: prDescription,
MaintainerCanModify: github.Bool(true),
}
pr, _, err := client.PullRequests.Create(ctx, *prRepoOwner, *prRepo, newPR)
if err != nil {
return err
}
fmt.Printf("PR created: %s\n", pr.GetHTMLURL())
return nil
} | def create_shipping_address(self, shipping_address):
""""""
url = urljoin(self._url, '/shipping_addresses')
return shipping_address.post(url) | def create(self, db_name, **kwargs):
db = PostgresDatabase(
db_name, host=self.host, port=self.port,
superuser=self.superuser, **kwargs)
db.ensure_user()
db.create()
return db | private function createRootFolders(OutputInterface $output)
{
$em = $this->container->get('doctrine.orm.default_entity_manager');
$repository = $this->container->get('ekyna_media.folder.repository');
$name = FolderInterface::ROOT;
$output->write(sprintf(
'- <comment>%s</comment> %s ',
ucfirst($name),
str_pad('.', 44 - mb_strlen($name), '.', STR_PAD_LEFT)
));
if (null !== $folder = $repository->findRoot()) {
$output->writeln('already exists.');
} else {
$folder = new Folder();
$folder->setName($name);
$em->persist($folder);
$em->flush();
$output->writeln('created.');
}
} | private function createNode(NodeInterface $node, $pathSegment, $uuid)
{
if ($node->hasNode($pathSegment)) {
return $node->getNode($pathSegment);
}
$node = $node->addNode($pathSegment);
$node->addMixin('mix:referenceable');
$node->setProperty('jcr:uuid', $uuid);
return $node;
} | public function create($params)
{
$client = new Client;
$client->fill($params);
$client->save();
// update datacite_symbol
$this->generateDataciteSymbol($client);
return $client;
} | 401 | func (c *DeleteManyBuildsCall) CreatedBy(createdBy string) *DeleteManyBuildsCall {
c.urlParams_.Set("created_by", createdBy)
return c
} | // DeleteGlobal deletes the first matching symbol found in current or parent scope. | // DeleteKeysetItem implements CAStore::DeleteKeysetItem | Delete function deletes some nodes in a linked list, but not others? glibc free() invalid pointer [C] | What is the difference between free and delete? | Callback for "product-class-delete" command | // removeDescedants deletes all entities of a given kind under the ancestor.
// If preserve is not nil and it returns true for a string id, the entity
// is not deleted. | // DeleteParams returns a map of parameters for an MaintenanceWindow that can be sent along. | // DeleteReminder deletes the provided Reminder. |
402 | who makes the bmw v8 for bentley arnage | Mans Series Options Identification of the chassis number The Bentley Arnage Le Mans chassis number follows the same identification as the Bentley Arnage Red label model: Bentley marked its 60 years of production at the Crewe factory with a special Diamond Series Arnage in 2006. 60 vehicles were planned, the majority for the United States, with diamond wood inlays, diamond quilted leather seats, a stainless steel front bumper, special 19 inch alloy wheels, and Union Jack badges on the front wings. In September 2008, it was announced that Arnage production would cease in 2009, once a final run of 150 | Rolls-Royce Holdings BR725 powering the Gulfstream G650, which received EASA Type Certification in June 2009. In 1996, Rolls-Royce and Airbus signed a Memorandum of Understanding, specifying the Trent 900 as the engine of choice for the then A3XX, now called the Airbus A380. On 6 April 2004, Boeing announced that it had selected both Rolls-Royce and General Electric to power its new 787. Rolls-Royce submitted the Trent 1000, a further development of that series. GE's offering is the GENX, a development of the GE90. On 13 June 2004, Rolls-Royce was awarded a £110m contract by the Ministry of Defence to supply engines | expected to get better fuel economy by U.S. EPA estimates, with (uncertified) projections of 12 miles per gallon (mpg) in the city and 20 mpg on the highway. Bentley Continental Flying Spur Bentleys have worn the Continental Flying Spur name on two quite different classes of four-door car. The Bentley Continental Flying Spur (2006 - 2013) is the second model after the 2-door Continental GT following Volkswagen Group of Germany's purchase of the coveted British brand in 1999. The Flying Spur is essentially a four-door version of the GT, with a stretched wheelbase and greater length for more spacious rear | Rolls-Royce–Bentley L-series V8 engine mass production V8-engined automobile. Rolls-Royce acquired Bentley in 1931 and continued to use Bentley engines alongside their own for a time, although none was a V8. Prior to World War 2, Rolls-Royce had developed a 7.3-litre V-12 for the Phantom III, which was succeeded by the inlet-over-exhaust B60 straight-6 and B80 straight-8 series of engines. The B80 powered the Phantom IV limousine, whilst the 4.3-litre B60 was used until 1955 to power the Rolls-Royce Silver Wraith and Silver Dawn and the Bentley Mark VI. The B60's bore was enlarged in 1955, increasing the displacement to 4.9 litres, that engine being | did not win another championship until 2008 the Ilmor-Mercedes engines won several races. In 2001 Paul Morgan was killed while landing one of his vintage airplanes, a Hawker Sea Fury at Sywell Aerodrome, Northamptonshire. In 2002 DaimlerChrysler increased its share to 55% and renamed the company Mercedes-Ilmor. In 2005 Daimler-Chrysler became the sole owner of Ilmor and renamed the company Mercedes-Benz High Performance Engines Ltd. Ilmor Engineering, Inc., a sister company to Ilmor U.K., was incorporated in 1990 (President - Paul Ray) with the primary goal of providing engineering support to customers using the Ilmor Ltd.-manufactured Chevrolet 265A Indy Car | BMW M6 The BMW M6 is a high-performance version of the 6 Series coupe/convertible, designed and developed by the motorsport division of BMW. The BMW M6 was based on the subsequent generations of the 6 series. In 1983 BMW took the M88/3 Straight-six engine, a modified version of the M88/1 from the BMW M1 and put it in the E24 chassis of the BMW 6 Series, thus creating the M635CSi (dubbed simply "M6" in North America and Japan). The first generation M6 was critically acclaimed throughout its lifespan for its elegant, aggressive "shark-nose" styling, its luxury equipment and its performance. | So my dad gave me what he's always described as a "92 BMW M3". I'm currently trying to sell it, and I'm not sure if it's really an M3.
pics
It has the M3 logo on the front grill, but it reads "325I" only on the back.
I found the vim number (WBACB331XNFE01038) and got this information from
> Type 325I (USA)
E series E36 (4)
Series 3
Type LIM
Steering LL
Doors 4
Engine M50
I've had people tell me that there's no such thing as a 92 M3 in the states. Just trying to alleviate some of this confusion and understand what I really have on my hands.
Thanks! | sedans, coupes and convertibles began production in 1966, and was based on a shortened version of the New Class Sedan platform. BMW New Class The BMW New Class () was a line of sedans and coupes produced by German automaker BMW between 1962 and 1977. These models ensured BMW's solvency after the company's financial crisis of the 1950s and established the identity of BMW automobiles as sports sedans. The first New Class vehicle was the 1500, a 4-door compact executive car with the new M10 (at the time called M115) OHC 4-cylinder engine. In 1965, the 2000C and 2000CS luxury | 402 | Bentley Arnage In a complete switch from tradition, these new cars would have bodies built at the Crewe factory, with its internal combustion engines built elsewhere. A number of potential engines were examined, including the GM Premium V engine, and a Mercedes-Benz V8 engine, before, in late 1994, Vickers selected a pair of BMW power plants. It was decided that the Rolls-Royce model, to be called the Silver Seraph, would use BMW's naturally aspirated V12 engine while the more-sporting Bentley model would use a special twin-turbo version of the 4.4-litre BMW V8, which was developed by Vickers subsidiary, Cosworth Engineering. On its | what is the difference between the bmw 3 and 4 series? | who built the bentleys for arnolt aston | who designed the engines for the volvo 960 | when did the bmw n74 come out | what kind of steering does the bentley gt use | what engine does the bmw 2 series have | when did rolls royce start using the l | GM E-Turbo engine |
403 | how many weeks did smack that stay in the charts | Disturbed discography Zealand RIANZ charts, and peaking at number two on the Canadian Albums Charts. It also was certified platinum in the US, Australia, and Canada. "Ten Thousand Fists" spawned singles such as "Guarded", "Just Stop", the Genesis cover of "Land of Confusion", and "Stricken". The latter charted at number 95 on the US "Billboard" Hot 100, and at number 88 on the UK Singles Chart. "Stricken" was later certified gold by the RIAA. Disturbed's fourth studio album, "Indestructible", was released in June 2008. Like its predecessor, it peaked at number one on the US and New Zealand charts; it also reached | Party Rock Anthem - reaching it in 68 weeks, just behind Adele's "Rolling in the Deep" which achieved it in 67 weeks - and the third-biggest selling digital single since Nielsen SoundScan began tracking digital sales in 2003. It has sold 8.1 million copies in the US as of October 2016 and over one million copies in the UK. It is the US's third all-time best-selling digital single. The song spent eleven weeks at number one in New Zealand and ten weeks in Australia. In New Zealand, it is the longest-running number one single since Smashproof's hit single "Brother" in 2009, with over | "Pain" is the second single from rock band Three Days Grace's 2006 album, One-X.
Meaning
According to former vocalist Adam Gontier, "Pain" is "a song about feeling like you're constantly numb to things around you, thanks to your own actions, and it's about being sick of that feeling."
Chart performance
"Pain" has become the band's biggest hit to date. It reached number one on the Billboard Modern Rock Tracks chart for four consecutive weeks, becoming their biggest hit on that chart to date but it stayed on the chart for 30 weeks where the prior single "Animal I Have Become" and next single, "Never Too Late" stayed longer on the chart at 41 weeks and 43 weeks respectively. On the Billboard Hot Mainstream Rock Tracks chart, it reached number one and stayed there for thirteen consecutive weeks. It also hit number 44 on the Billboard Hot 100, becoming their first single to chart in the top 50 of the Hot 100 and their highest charting single to date.
"Pain" also held the number one spot on many Canadian rock stations for weeks, and was the most-requested song ten weeks in a row. It also reached number one on the MuchMusic Countdown.
The song was featured on episodes of Criminal Minds, CSI: NY, and Ghost Whisperer, and is a playable song in the music video games Rock Revolution and Rock Band 3.
"Pain" was re-released to pop radio on June 3, 2008.
Music video
The music video, directed by Tony Petrossian, features the band playing the song in what looks to be an abandoned mansion, and it also features shots of troubled youths who are lip-syncing to the song. At the end of the song, everyone (youths and the band members alike) is shown to be tattooed with a red "X" on the back of their necks, signifying the name of the parent album, One-X.
Pain EP
The Pain EP is a digital exclusive. It features the main version of "Pain" along with stripped acoustic versions of both "Pain" and "Animal I Have Become".
"Pain" – 3:22
"Pain (stripped acoustic version)" – 3:18
"Animal I Have Become (stripped acoustic version)" – 3:44
Versions
There are currently three versions of the song "Pain", two of which are being played on radio stations – the original album version and an acoustic version. The acoustic version of "Pain" and "Animal I Have Become" are both available for download on most online music stores including iTunes. The third version runs 3:28 long and is titled "Pain (Pleasure Mix)".
Personnel
Adam Gontier – lead vocals
Brad Walst – bass guitar, backing vocals
Neil Sanderson – drums, organ, backing vocals
Barry Stock – lead guitar
Charts
Weekly charts
Year-end charts
Certifications
References
External links
2006 singles
2006 songs
Three Days Grace songs
Jive Records singles
Songs about suicide
Music videos directed by Tony Petrossian
Songs written by Adam Gontier | "Funky Friday" was reported to enter the UK Singles Chart at number two, a total of 2,000 combined sales behind "Promises" by Calvin Harris and Sam Smith. "Funky Friday" entered the UK Singles Chart at number one for the week dated 12 October 2018, with a combined number of 6.7 million audio and video streams. It became Dave's first number one and top ten single, and Fredo's first top forty entry. It is the first song by a British rapper to peak at number one on the UK Singles Chart as a lead artist since "Not Letting Go" by Tinie | December, 1963 (Oh, What a Night) 100 (matching the chart life of the original 1975 single). The peak position of the 1993 remix version was #14. Adding together the two 27-week chart runs for the 1975 original single and the 1993 remix version (for a combined total of 54 weeks, two more weeks than a full year) gave the song the longest tenure ever on the "Billboard" Hot 100 music chart up to that time. The tenure has never been surpassed. A music video was produced to accompany the original 1975 release. The video used the edited single version, which had a Phaser effect during Frankie's | Zealand, being certified platinum in its first week in both countries; and Canada, where the album went Platinum. Echoes, Silence, Patience & Grace Echoes, Silence, Patience & Grace is the sixth studio album by American rock band Foo Fighters, released on September 25, 2007 by RCA Records. The album is noted for a blend of regular rock and acoustic tracks with shifting dynamics, which emerged from the variety of styles employed on the demos the band produced. It also marks the second time the band worked with producer Gil Norton, whom frontman Dave Grohl brought to fully explore the potential | "Weekend" is a song by Dutch band Earth and Fire. It was released by Earth and Fire as a single in November 1979 and reached the number one spot in the singles charts in the Netherlands, Switzerland, Germany, Denmark and Portugal. It was written by keyboard player Gerard Koerts for the album Reality Fills Fantasy.
Track listing
Chart performance
Weekly charts
Year-end charts
Chips version
Weekend was first covered by the Swedish group Chips on their eponymously titled debut-album. Originally, the version was recorded in 1980, but was only available on the album's first printed issues, as all subsequent releases (now called "Sweets'n Chips") replaced the song with the track "Good Morning". It wasn't until the release of the 1997 Greatest Hits-album "20 bästa låtar" that the song became widely available again. The B-Side on the single was the Instrumental track "Tokyo".
Track listing
Scooter version
"Weekend" was also covered by German techno group Scooter as "Weekend!". It was released on 24 February 2003 as the first single from their ninth studio album, The Stadium Techno Experience (2003). The single reached number two in Germany and was a top-10 hit in Austria, Denmark, Finland, the Netherlands, Norway, and Sweden. In Norway, the song is certified Platinum for sales exceeding 10,000.
Music video
The video for the song shows how desperate for attention the group was. The video takes place on an illuminated part of a loam-covered floor encircled by dark. While Scooter are performing the song, there are Buddhist and Christian monks, nuns, Asian martial artists, topless women, traditional Indian female dancers and Ganesha dancing around them. H. P. Baxxter can also be seen wearing a costume resembling those worn by the Roman centurions. The puerile video was censored in the multimedia part of the CD single released in Germany.
Track listing
Charts
Weekly charts
Year-end charts
Certifications
Release history
Other versions
In 1980 the Belgian band De Strangers released a Dutch-language version of the song under the title "Pluchke".
The song was released in German version as "Kein Mädchen für das Wochenende" which was first sung by Conny Morin and later was covered by Daniela Dilow.
In 2002 Kid Q released the single "This Feeling", which contains a sample of "Weekend".
In 2008 Bloodhound Gang released a cover version of the Scooter cover for "Weekend!"
In 2012, Belgian electro producer Mickey released a cover version of the original track featuring Sylvie 'Billie' Kreusch.
In 2017, Dutch artist De Ambassade released a New wave/Synth-pop rendition of the song under the title "Jerney", after the lead singer of Earth and Fire Jerney Kaagman.
In 2019, German DSDS Star Sarah Lombardi released Weekend, a collaboration with DJ Herzbeat, as Schlager Song. The Song started on place 93 of the GfK Entertainment Charts and reached in the ITunes charts the Number 1, after only one week.
In 2020, German DJ LIZOT released a cover version of Weekend.
References
1979 singles
1979 songs
1997 singles
2003 singles
Dutch Top 40 number-one singles
Number-one singles in Belgium
Number-one singles in Denmark
Number-one singles in Germany
Number-one singles in Hungary
Number-one singles in Portugal
Number-one singles in Switzerland
Scooter (band) songs
Single Top 100 number-one singles
UK Independent Singles Chart number-one singles
Vertigo Records singles | The Big Bang (Busta Rhymes album) reason, the sound of a piano's lowest octave seems to evoke a profound sense of hopelessness. Dr. Dre understands that better than most." The album became Busta's first and only album to debut at number one on the charts with over 209,000 copies sold during the first-week of release. On August 30, 2006 the album was certified gold for shipments of over 500,000 units. The album has sold 823,000 copies as of November 22, 2011 The album became Busta Rhymes' highest charting album in the UK, debuting on the UK albums chart at number nineteen. His previous highest album peak | 403 | reaching its peak at number two, a position it held for three nonconsecutive weeks. The single lasted 26 weeks on the chart and earned a platinum certification from the Australian Recording Industry Association (ARIA). In New Zealand, "Smack That" debuted on the singles chart in New Zealand on November 20, 2006 at number 2. The song held the position for two more weeks before falling to number 14 in its fourth week and 21 in its fifth week. The single got a second wind, rising to number five in the following week. Two weeks later, "Smack That" reached the chart's | [Question] Can slap strumming damage my guitar? | Extra melee hit at Vindicta? | when was piledriver the wrestling album 2 released | Melee weapon: how to avoid damage when touching a weapon which isn't swinged? | "Smacked in the middle" | who is the record company for touch down 2 cause hell | Pretty sure it'll be a big hit | Do you think 'last hit' is absolutely necessary? |
404 | [Prevalence, features of circulation, and diversity of human parechoviruses in Nizhny Novgorod]. | encephalitis. Human parechoviruses are commonly spread and more than 95% of human cases are infected early in life, within two to five years of age. Parechovirus B has been proposed as a zoonotic virus, associated with diabetes and intrauterine fetal death in humans. However, the data regarding these features are currently limited and need to be confirmed. Parechovirus is a Biosafety Level 2 organism. The first parechoviruses (E22 and E23) were isolated in 1956, and recognized as a new genus in 1996. Parechovirus B was first isolated from bank voles ("Myodes glareolus", formerly "Clethrionomys glareolus") in the mid-1990s. Human parechovirus | Occurrence and identification of Impatiens necrotic spot tospovirus in the Czech Republic. | Development of a molecular assay for the general detection of tospoviruses and the distinction between tospoviral species | Review on Outbreak Dynamics, the Endemic Serotypes, and Diversified Topotypic Profiles of Foot and Mouth Disease Virus Isolates in Ethiopia from 2008 to 2018 | Ecological Factors Affecting Infection Risk and Population Genetic Diversity of a Novel Potyvirus in Its Native Wild Ecosystem | The Bulgarian vaccine Crimean-Congo haemorrhagic fever virus strain. | Nipah viruses (NiV) are emerging zoonotic viruses that cause severe and often lethal respiratory illness and encephalitis in humans. Henipaviruses can infect a wide range of species and human-to-human transmission has been observed for NiV. Nipah virus is an emerging zoonosis with the potential to cause significant morbidity and mortality in humans and major economic and public health impacts. According to World Organization for Animal Health (Office International des Epizooties: OIE), Nipah virus is a notifiable disease of importance to international trade. Keywords : Nipah virus, respiratory tract, CNS. |
plasma of acutely infected but asymptomatic donors, thereby resulting in the highly contaminated source plasma pools for fractionation, (2) it is highly resistant to virus inactivation or removal methods used in the manufacture of blood products, and (3) sometimes it can cause severe diseases in at-risk recipients. 2 To mitigate the risk of B19V transmission, most manufacturers have been performing nucleic acid testing (NAT) for B19V DNA as an inprocess test for source plasma pools used for manufacturing certain or all kinds of blood products to limit the virus load, according to the guidance or standard from European Pharmacopoeia, the Plasma Protein Therapeutics Association, and U.S. Food and Drug Administration. [3][4][5] Similar to B19V, PARV4 is also a frequent contaminant of source plasma pools for the production of blood products and the final products. 6 Baylis et al. 7 demonstrated PARV4 was even more resistant than B19V to virus inactivation strategies used during the manufacture of blood products, such as pasteurization and low-pH treatment. The transmission of PARV4 by virally inactivated clotting factor concentrates raised concerns among the patients with hemophilia or other recipients of such products. 6 14 recently reported a strong association of PARV4 with severe respiratory illness. Same as B19V, PARV4 has been classified into three genotypes. 15 All these three genotypes have been detected in human blood or blood products although they seem to have different epidemiology. Genotypes 1 and 2 are prevalent in North America, Europe, and some countries in Asia while genotype 3 seems to be endemic in Ghana. 16 However, data on the existence of different PARV4 genotypes circulating in Chinese plasma donors are limited.
In our previous article, the prevalence of PARV4 in Chinese plasma pools has been reported, but the genotypes of PARV4 were not identified. 17 The main objective of this study is to identify the genotypes of PARV4 circulating in Chinese plasma donors.
| B19V and PARV4 quantitative polymerase chain reaction assay
The DNA samples were initially tested for presence and quantities of B19V and PARV4 DNA using a duplex quantitative polymerase chain reaction (qPCR) assay, which has been proved to be able to simultaneously detect and quantify all the known genotypes of B19V and PARV4, with an equal limit of quantification of 5 copies/ml. 19 Serial log 10 dilutions of the B19V and PARV4 standard plasmids containing the qPCR target sequences and the samples were all analyzed in triplicate. On the basis of the standard curve generated by the standard plasmids and the quantification cycle value of each sample, the concentration of each virus DNA in copies/ml was calculated.
| PARV4 nested PCR and sequencing
A 161-bp region related to the NS1 gene of PARV4 (positions 1564-1724 in AY622943) was amplified by nested PCR with the primers described previously. 20 PCR amplification products derived from each sample were purified and cloned into the pMD18-T vector (TaKaRa Bio) and subsequently sequenced on an ABI 3730XL DNA Analyzer.
| Phylogenetic analysis of PARV4 sequences
The PARV4 genotypes were determined by phylogenetic tree ana-
| Quantity of B19V and PARV4 DNA in source plasma pools
The levels of B19V and PARV4 DNA in source plasma pools were shown in Figure 1. The quantity of B19V DNA varied from 2.56 × 10 2 to 2.30 × 10 9 copies/ml plasma. Levels of B19V DNA was as high as 2.30 × 10 9 copies/ml plasma, although 70% of the positive samples were at low levels (10 2 -10 4 copies/ml plasma). For PARV4, the level of virus DNA was lower than that of B19V: viral loads ranged from 2.42 × 10 2 to 2.28 × 10 5 copies/ml plasma, and most samples contained 10 2 -10 3 copies/ml plasma. In the one coexistence sample, levels of B19V and PARV4 were low, equal to 1.31 × 10 3 copies/ml plasma and 2.42 × 10 2 copies/ml plasma, respectively.
| Genotypes of PARV4 sequences in source plasma pools
The genotypes of PARV4 sequences in source plasma pool samples were identified. Of the 11 samples found to be positive for PARV4 sequences by qPCR (Table 1), only 2 were positive for PARV4 nested PCR and available for further analysis. The PARV4 DNA quantity of these two samples was 5.28 × 10 2 and 9.75 × 10 3 copies/ml plasma, respectively. While no targeted PCR products were amplified from five samples containing greater than 5.28 × 10 2 copies/ml plasma of PARV4 DNA. These unexpected results might attribute to the incomplete sequence of PARV4 genome in these samples, in view of the different target regions between PARV4 qPCR and nested PCR.
Phylogenetic analysis of 2 sequences obtained in this study, together with 24 sequences retrieved from GenBank, revealed that one sequence (pool-7) clustering together with genotype-1 reference sequences belonged to PARV4 genotype 1, and the other one sequence (pool-14) clustering together with genotype-2 reference sequences belonged to PARV4 genotype 2 ( Figure 2).
| DISCUSSION
Since discovered in 2005, PARV4 has received much attention. 22 Unlike B19V, which took nearly 30 years after its initial discovery in 1975 to identify its three genotypes, PARV4 genotypes 2 and 3 were identified within 3 years. 15 21 In this study, the PARV4 sequences in two PARV4-DNA positive source plasma pool samples were genotyped and the results demonstrated that at least two PARV4 genotypes, 1 and 2, were currently present in China. No genotype 3 was detected, which was not unexpected since this genotype was reported to be endemic in Ghana. 16 Besides, the possibility that genotype 3 might be present at an extremely low level in the sample, and, therefore, escaped identification can not be completely ruled out. Furthermore, it should be noted that this study was restricted to one region of China, and, therefore, could not reflect the whole national circulating status of different PARV4 genotypes. B19V genotype 3 is originally endemic to Ghana as well, but as research continues it shows a wider distribution and has been found in Brazil, France, North India, the United States, and China. 21,28 Whether PARV4 genotype 3 will show an increasing spread also outside of Ghana, just like B19V, needs further research with large sample size and a wide geographical area.
These results also showed that the positive rates and levels of B19V DNA in Chinese plasma pool samples were relatively higher than that of PARV4 DNA, indicating that the prevalence of B19V DNA in the Chinese population might be higher than that of PARV4, consistent with results detected in the general population from a previous report. 29 On the other hand, regarding the frequent coinfection with hepatitis C virus and HIV, some PARV4 positive plasma samples might be excluded by infectious agents screening tests before pooling. 6 The prevalence of B19V DNA in source plasma pool samples tested in this study was lower than that reported in 2015 (104/141, 73.76%), whose F I G U R E 2 Phylogenetic analysis of human parvovirus 4 (PARV4) nucleotide sequences. The phylogenetic tree was constructed based on the 161-nt NS1 region of PARV4 and the neighbor-joining algorithm using the Kimura two-parameter model. Two PARV4 sequences from this study (labeled with black circles) and a set of PARV4 sequences downloaded from GenBank (labeled with their GenBank accession number and isolate or strain name) used as references for the different genotypes were analyzed. Bootstrap replication frequencies are indicated above each node. Branch lengths are drawn to scale samples derived from the same manufacture were collected between 2008 and 2013. 30 Given that associated disease or specific symptoms have not yet been confirmed, there is no need to exclude PARV4 from source plasma pools, at least for the time being. 6 It should be noted that although the use of source plasma pools as materials in this study increases the potential for the detection of PARV4, the data regarding PARV4 containing plasma pools were still relatively limited. In further research, screen and sequence more source plasma pool samples by extracting larger volumes of plasma or concentrating virions by immune adsorption could address this limitation.
| CONCLUSION
In conclusion, the data present demonstrate the existence of PARV4 genotype 1 and 2 in Chinese plasma donors for the first time and also
show the relatively lower prevalence and level of PARV4 DNA in Chinese plasma donors in comparison with that of B19V DNA.
|
Source plasma pool samples A total of 78 source plasma pool samples (each comprising 2000-3000 donations) from one Chinese blood product manufacturer were analyzed in this study. All of such plasma samples were sourced from plasma donors in Central China and collected between April 2017 and June 2018. The collections of plasma samples were approved by the National Health Commission of the People's Republic of China, and all of the plasma donors provided informed consent. Every single plasma donation has gotten tested for infectious agents before and after the pooling and was confirmed to be qualified, according to the requirements of Pharmacopeia of the People's Republic of China. 18 2.2 | DNA isolation Viral DNA was isolated from a volume of 200 μl of each plasma sample with the High Pure Viral Nucleic Acid Kit (Roche Diagnostics) according to the manufacturer's instructions. The concentration and purity of extracted DNA were measured using the GeneQuant 1300 spectrophotometer (GE Healthcare Bio-sciences AB).
lysis based on the 161-nt NS1 region (positions 1564-1724 in AY622943) using MEGA version 6.0. Twenty-four PARV4 nonredundant sequences spanning this region were downloaded from GenBank (May 2020) and worked as the reference sequences. As reported previously, genetic distances of the sequences were calculated using the Kimura two-parameter method, and phylogenetic trees were constructed by the neighbor-joining method with 1000 bootstrap replicates. 21 JIA ET AL.
Prevalence of B19V and PARV4 DNA in source plasma pools Of 78 source plasma pool samples tested, 20 (25.64%) were positive for B19V DNA, 11 (14.10%) were positive for PARV4 DNA, only 1 (1.28%) was identified positive for both viral DNA, and 48 (61.54%) had no detectable levels of B19V and PARV4 nucleic acid.
,8 Given the clinical significance of PARV4 infection has not yet been confirmed, unlike B19V, there are no industry guidelines for restricting the level of PARV4 in source plasma pools. Despite the lack of clear evidence for PARV4-mediated diseases, a variety of potential clinical associations have been proposed, including encephalitis, early human immunodeficiency virus (HIV)-related symptoms, and fetal hydrops and hepatitis. 8-13 Moreover, Prakash et al.
,23-25 Research on the prevalence and distribution of different genotypes contributes to our understanding of human parvovirus evolution and diversification and enables good assay design for virus detection. Accumulating studies have reported the PARV4 genotypes prevalent in blood or plasma donors of many countries, however, no relevant data have ever been reported in This is the first report to identify the genotypes of PARV4 circulating in Chinese plasma donors. Source plasma pools are ideal materials for F I G U R E 1 Distribution of the B19V and PARV4 DNA load in virus DNA-positive source plasma pools. B19V, human parvovirus B19; PARV4, human parvovirus 4 T A B L E 1 Details of plasma pools containing PARV4China. 20,26,27
Sample No.
B19V
(copies/ml)
PARV4
(copies/ml)
Genotype of
PARV4
3
Neg
4.95 × 10 2
-
7
Neg
5.28 × 10 2
G1
10
Neg
2.70 × 10 2
-
11
Neg
2.28 × 10 5
-
13
Neg
3.12 × 10 2
-
14
Neg
9.75 × 10 3
G2
15
Neg
1.14 × 10 4
-
21
Neg
5.59 × 10 4
-
32
Neg
8.57 × 10 2
-
35
1.31 × 10 3
2.42 × 10 2
-
36
Neg
6.47 × 10 2
-
Note: "-" indicates that the genotype of PARV4 in the sample was not
identified.
Abbreviations: B19V, human parvovirus B19; PARV4, human parvovirus
4; Neg, negative.
blood-borne virus genotype studies. In our previous studies, the source
plasma pools were used as the samples for B19V genotyping, and first
reported the cocirculation of B19V 1a, B19V 1b, and B19V 3b, as well as
the putative B19V 1/3 recombinant and new strains in Chinese plasma
donations.
Given the duplex qPCR assay used in this study has high sensitivity, the diversity can be mainly attributed to the differences in the year and season of sample collection. Besides, differences in sample size and the number of plasma units within each pooled sample might be the influence factors on such diversity. Also, the prevalence of B19V DNA tested in this manufacturer was lower than that in other manufacturers in China (5.45%-100%) as well as other countries before NAT was introduced (56.10%-59.68%), reflecting the geographic and temporal differences in the prevalence of the virus, differences in detection methods, as well as the differences in thenumber of plasma units within each pooled sample and in the sample size. 31 For PARV4, the prevalence in samples collected between 2017 and 2018 in this study (11/78, 14.10%) was lower than collected between 2007 and 2010 from the same manufacture (39/101, 38.61%), indicating the seasonal epidemic variation, whereas was equivalent to other two manufacturers (3/20, 15% and 9/74, 12.16%, respectively) in China. 17 In our study, out of the 30 parvovirus DNA-positive source plasma pool samples, only one was identified positive for both B19V and PARV4 DNA. The low rate of coinfection indicated that the PARV4 was not associated with the infection of B19V. Thus, the implementation of B19V NAT assays was not able to reduce the possible risk of PARV4 transmission by blood products. The transfusion-mediated transmission of PARV4 remains a concern.
ACKNOWLEDGMENTSThis study was supported by the National Natural ScienceCONFLICT OF INTERESTSThe authors declare that there are no conflict of interests.DATA AVAILABILITY STATEMENTData are available on request from the authors. The data that support the findings of this study are available from the corresponding author upon reasonable request.ORCIDYuyuan Mahttps://orcid.org/0000-0002-9817-1830
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| 404 | A total of 5230 specimens from children with gastroenteritis collected in Nizhny Novgorod in 2006-2010 were screened for human parechoviruses (HPeV). HPeV were observed every year with mean frequency of 6.16%. The majority of HpeV (65.83%) was detected in children younger than 3 years. The typing of 71 detected HPeV with the use of partial sequencing of the VP3-VP1 region revealed the presence of HPeV1 (91.55%), HpeV6 (5.63%), HPeV3 (3.08%), HPeV4 (1.54%). HPeV1B was predominant among HPeV1, HPeV1A was identified rarely. Six stains of HPEV1 formed separate phylogenetic cluster, had sequence gomology with HPEV1A or HPeV1B not more than 88% and could be characterized as members of a separate genotype HPeV1. | [Detection of parechovirus in infectious gastroenteritis patients from Kanagawa Prefecture Region. Fecal specimens sent from the pediatric sentinel clinics from April 2008 to March 2011]. | Abstract Human Parechovirus (HPeV), a member of the Picornaviridae family, is an infectious agent mostly affecting children. There are 16 recognized genotypes which have globally spread. This study incorporated a total of 2957 nasopharyngeal (NP) swab and 759 fecal samples that were collected from different parts of Thailand. The NP of HPeV was detected in 0.4% of NP swab and 6.1% of fecal samples. The majority of HPeV infections occur in infants below the age of 2 years, while infections were detected in children above the age of 10 years as well. Various genotypes comprising 1A, 1B, 2, 3, 4, 5, 6, 10 and 14 have been characterized. This study revealed recombination events in 16 samples in which HPeV1B was shown as the highest frequency. In conclusion, HPeV can be detected in both the respiratory and GI tract. Moreover, HPeV which circulates in Thailand is highly diverse and subject to recombination. | The circulation of human parechoviruses (HPeVs) in the population was studied by environmental surveillance comprising of molecular analyses of sewage samples (n = 89) that were collected from 15 different locations in The Netherlands. Samples were taken from sewage originating from schools (n = 9) or from parts of municipalities (n = 6) during the Dutch school year 2010-2011. At 13/15 locations HPeV1, HPeV3, or HPeV6 RNA was detected at least once; however, sequence diversity did not reflect associations in time or place. A higher percentage of positives was observed in the samples originating from the municipalities. It was demonstrated that HPeV circulated in the studied population to a higher extent than would be expected from the current knowledge on infections predominating in young children. | Background ::: Emerging human picornaviruses, including human parechovirus (HPeV), Aichi virus (AiV) and salivirus (SalV) were found to be associated with gastroenteritis, but their roles in enteric infections are not fully understood. In addition, no report on the circulation of these viruses in Hong Kong is available. The objective of this study was to investigate the prevalence and genetic diversity of HPeV, AiV and SalV in fecal samples from hospitalized children with gastroenteritis in Hong Kong. | Molecular Detection of Human Calicivirus among Spanish Children with Acute Gastroenteritis | what level is the parechovirus considered in humans | INTRAUTERINE INFECTION WITH HUMAN PARVOVIRUS | The prevalence and genotype distribution of rotavirus A infection among children with acute gastroenteritis in Kunming, China |
405 | The Paris Conservatory | For anyone interested in the "Maitre's" huge project of the chapel in Vence,
this DVD is a MUST-HAVE.
Soeur Jacques-Marie is interviewed.. She gives a lively, personal account of her envolvement with the project
and with Matisse. She was truly a muse. I give this charming interview 5 stars. | With the goal of attracting more students to its French classes, the Department of French and Italian Studies at Emory University has capitalized on widespread interest in the world's number one tourist destination. The course, which has been offered in both French and English, is based on the study of representations of Paris from the Middle Ages to the present. It uses architecture as a point of departure, and explores the myth of Paris as expressed through a profusion of images in literature, painting, and film. This essay presents a sample unit on the Renaissance, followed by a full syllabus and bibliography. | Conservatoire national des arts et métiers Conservatoire national des arts et métiers The Conservatoire national des arts et métiers (CNAM; English: "National Conservatory of Arts and Crafts") is a doctoral degree-granting higher education establishment (or "grand établissement") and "Grande école" in engineering, operated by the French government, dedicated to providing education and conducting research for the promotion of science and industry. It has a large museum of inventions accessible to the public. It was founded on 10 October 1794, during the French Revolution. It was first proposed by Abbé Henri Grégoire as a "depository for machines, models, tools, drawings, descriptions and books in all the areas | conservatories train more than 1,200 students in structured programs, with 350 professors in nine departments. The Conservatoire national supérieur d'art dramatique (CNSAD) (National Superior Conservatory of the Dramatic Arts) is the conservatory for acting, drama, and theatre, known by its acronym CNSAD. It is located in the original historic building of the Conservatoire de Paris on the rue du Conservatoire at rue Sainte-Cécile in the 9th arrondissement of Paris. Free public performances by students at the CNSAD are given frequently in the Conservatoire's theatre. The Conservatoire national supérieur de musique et de danse de Paris (CNSMDP) (National Superior Conservatory of | Anthony Vidler (born July 4, 1941 in Salisbury Plain, United Kingdom) is Professor at the Irwin S. Chanin School of Architecture at The Cooper Union. He is an architectural historian and critic.
Education
Anthony Vidler received a B.A. and Dipl.Arch from Cambridge University and a Ph.D. from Technical University Delft.
Teaching
Vidler has taught at Brown University, The Cooper Union, UCLA, Cornell University, and Princeton University
He has been awarded fellowships with the Institute for Architecture and Urban Studies (1971–84), the New York Institute for the Humanities at New York University (1980–82), the John Simon Guggenheim Memorial Foundation (1985-86), the National Endowment for the Humanities (1989–90), the American Academy of Arts and Sciences (1995-present), and the Canadian Centre for Architecture in Montreal (2005).
Curatorial work
Vidler has curated several exhibitions since the late 1980s, including the part of the exhibition out of the box: price rossi stirling + matta-clark dedicated to James Stirling at the Canadian Centre for Architecture (2003-2004) and the exhibition Notes from the Archive: James Frazer Stirling which travelled to the Yale Center for British Art, the Tate, the Staatsgalerie Stuttgart, and the Canadian Centre for Architecture (2010-2012).
Publications
The Writing of the Walls. Architectural Theory in the Late Enlightenment (Princeton: Princeton Architectural Press, 1987). Paperback, 1990.
Ledoux (Paris: Editions Hazan, 1987). Foreign editions: Berlin, 1989, Tokyo, 1989, Madrid, 1994.
Claude-Nicolas Ledoux: Architecture and Social Reform at the End of the Ancien Régime (Cambridge, Mass.: MIT Press, 1990).
The Architectural Uncanny: Essays in the Modern Unhomely (Cambridge, Mass.: MIT Press, 1992).
L'Espace des Lumières: Architecture et philosophie de Ledoux à Fourier (Paris: Editions Picard, 1992). Translation and revised edition of The Writing of the Walls with new introduction and concluding chapter, 1992. Spanish edition: El espacio de la Ilustración. La teoria arquitectónica en Francia a finales del siglo XVIII, trans. Jorge Sainz (Madrid: Alianza Editorial, 1997).
Antoine Grumbach (Paris: Centre Georges Pompidou, 1996).
Warped Space: Art, Architecture, and Anxiety in Modern Culture (Cambridge, Mass.: MIT Press, 2000).
Claude-Nicolas Ledoux (Paris: Hazan, 2005).
Claude-Nicolas Ledoux: Architecture and Utopia in the Age of the French Revolution (Basel: Birkhäuser, 2006).
Histories of the Immediate Present. Inventing Architectural Modernism (Cambridge, Mass.: MIT Press, 2008).
Architecture Between Spectacle and Use, ed. Anthony Vidler, Clark Studies in the Visual Arts (New Haven and London: Yale University Press, 2008), “Introduction,” pp.vii-xiii; “Architecture's Expanded Field,” pp. 143–154.
James Frazer Stirling: Notes from the Archive (New Haven and London: The Yale Center for British Art and Yale University Press; Montreal: Canadian Centre for Architecture, 2010).
The Scenes of the Street and Other Essays (New York: The Monacelli Press, 2011).
References
External links
Anthony Vidler: How to Invent Utopia: The Fortunes and Misfortunes of Plato's Polis, Canadian Centre for Architecture, 17 May 2005
Alumni of the University of Cambridge
Living people
Delft University of Technology alumni
Brown University faculty
Cooper Union faculty
University of California, Los Angeles faculty
Princeton University faculty
Cornell University faculty
Fellows of the American Academy of Arts and Sciences
1941 births | Paris in the 17th century theater in Paris; it saw the founding of the Comedie-Française and the first productions of the works of Pierre Corneille, and Moliere. At the beginning of the century the pioneer Parisian theater company, the Confrérie de la Passion, installed itself in one of the buildings of the Hôtel de Bourgogne at 23 rue Saint-Etienne; it also rented out the space to visiting English and Italian theater companies. The first permanent theatre in Paris was created by Cardinal Richelieu in 1635, within his "Palais-Cardinal". It was located at the corner of rue Saint-Honoré and rue de Valois. The first performance, of | 'contest'). A piece of flute music was commissioned by Paul Taffanel to be performed at the Concours Pieces each year. Students would play this piece of repertoire before a panel of experts. This is why a dedication to Paul Taffanel appears on many of the following pieces of sheet music: Paris Conservatory Flute Concours The Paris Conservatory Flute Concours was the most highly regarded flute contest in Paris in the (musical) Romantic Period. In the 19th and 20th centuries, Paris was home to the world’s cultural elite. At this time the Paris Conservatoire was considered to be the finest institute | The night he arrives in Paris, the protagonist of Maria Edgeworth’s Ormond (1817) attends a tragedy at the Theâtre Francois in order to develop his urban literacy. There is a slight historical problem with Ormond’s intention to attend the Theâtre Francais: during the time at which the novel is set, the Comedie Francaise was housed not in the Theâtre Francais but in the Theâtre des Tuileries. What this means for Ormond is that Edgeworth and Harry Ormond should have attended two very different theaters. This chapter argues that Edgeworth’s representation of the French theater and Parisian space more broadly enacts the urban and literary project of Denis Diderot’s and Nicolas Cochin’s idealized community, re-situating Edgeworth’s novel within a new continental intellectual and urban environment. | 405 | The Paris Conservatory Orchestra goes next door to perform at the City of Music. Pierre Boulez (boo-LEZ) conducts a performance of the Adagio from the Symphony No. 10 by Gustav Mahler. This is the only movement Mahler completed of his Tenth Symphony. Recorded in January at one of the concerts celebrating the opening of the City of Music. (Radio France) | who invited prokovsky to join new york city ballet | At the Restoration , he was named composer of the royal chapel and conductor of the orchestra of the Opéra . | Is there a symphony orchestra at Chico State? | who conducted the last supper at the berlin state opera | which city is home to the victorian orchestra | when was the symphony in c written by stravinsky | arches is a musical composition by fred lerdahl in which form | where was the last of brahms' four pieces for piano, op. 119 |
406 | [Discussion] Server transfer | Hey all, been a while since i was here. Anyway. Is there any word on server transfers?
I ask because my colby character is pretty developed with gear that i got via armas a long time ago, not to mention various perm weapons.
I moved recently to eadt coast fro. West coast so ping issues would be horrendous. | Hi, so im plaing to server transfer in the next few days and i would like to know if theres anything i need to know to make it as smooth as possible.
Are there currently any known quests that can be messed up or bugged? Or is there anything that i show do before hand to prepare?
Ive only server transferred once back in burning crusade so i just dont want any thinrg to get messed up. | around the notion of equal "peer" nodes simultaneously functioning as both "clients" and "servers" to the other nodes on the network. This model of network arrangement differs from the client–server model where communication is usually to and from a central server. A typical example of a file transfer that uses the client-server model is the File Transfer Protocol (FTP) service in which the client and server programs are distinct: the clients initiate the transfer, and the servers satisfy these requests. Peer-to-peer networks generally implement some form of virtual overlay network on top of the physical network topology, where the nodes | my question is if i switch servers does my loot in my inventory transfer over to another server i joined. also might be considered server-hopping Heard of that But i just wanted to clear up any speculation so i don’t just lose all my gear 30+ hours in one server | Hey, I just logged on to PBE and I was just messing around in the shop, and then I clicked "Other" and I saw transfers to servers are available. Is this a glitch, do you keep your champions and skins, ip and rp if you transfer from PBE to any of the servers? | My main character is on Gilgamesh, and I have about 300,000,000 gil but I want to transfer to a different server on my Data Center like Adamantoise or Tonberry. Could I make a character on that server, put something silly that no one will buy for like 300,000,000 (or close to the gil i have) on the MB on that server...then go visit the server and buy it with the gil on my main? Thus transferring the gil to my alt? Would that work? | Ok, now I'm working with things that I'm not familiar with - migration. I can set up servers just fine if I'm starting anew, but I'm in the process of migrating three servers to two new machines. I'm not sure how to get all of the settings over.
For the file server, it should be as easy as copying files and re-setting share permissions. No big deal. Then update DNS records accordingly to point to the new server so users are none the wiser.
However, I have concerns about a Domain Controller and Exchange Server. Would I just set up replication between the two and then de-commission the originals? Or how would I go about transferring over all of the old mailboxes, settings, etc (between Exchange servers) and Active Directory (between DCs)? I'm completely unsure of how to migrate these, but I am aware that I will have to do this after hours so users aren't interrupted. Any help would be appreciated. | One of my favorite systems at PlanetSide Arena (although I've shut down the service) is that server transfer was free.
Of course, each server has a sufficient population at prime time.
It is not a problem for players playing at prime time to be unable to transfer servers.
However, the problem is that server transfer is not possible for players who play outside of prime time.
Creating a character for each server is, frankly, a waste.
Right now, I created 4 faction characters on 2 servers. It is a total of 8 characters!
For RPG, this may not be a serious problem. But please consider.
Like I said earlier, I liked PlanetSide Arena's free server transfer. | 406 | Apologies if I flared wrong.
How would people feel if c2u were to introduce a one time server transfer? - like when new servers were introduced.
Personally I'd like it. I wish I had transferred to EU when it opened. However I was away at the time and missed it. And God I hate the 5am rush hour on global lol.
Also it may freshen up the servers a little? New players/guilds moving.
Obviously I'm suggesting this as a free event. Other options could be to buy a server transfer ticket (one off).
Thoughts? | Let me once again ask some eu server questions; | Suggestion: How about waiting a few hours to open the server at an time that is agreeable for both EU and US players? | When is EU LCS exactly moving to Berlin? | West EU Server in a nutshell | Now I see two EU Central servers | Would you like it or love it if ArenaNet/NCSoft kept server transfers free? | European route E391 | Where are you transfering to? (EU) |
407 | Top surgery after a reduction? | what are the most common complications that happen with top surgery that everyone should know about? during surgery and also during recovery? | This is sort of a random post, but I've been a member of Nancy's Nook on fb for about a year and a half now and I just can't believe how good the people posting pics look after they've just had surgery. After both of my procedures I looked like I was just dug out of a grave. Granted I don't handle anesthesia well, I'm one of the 'lucky' ones that gets severe nasuea, but still. Did anyone here look perfectly fine getting out of surgery? | Hey guys, so I'm having top surgery in a few days... Crazy how time went by so fast. Super pumped for this...
If you guys have any recovery tips, please feel free to let me know!
Also, I just started my own you tube channel. Feel free to subscribe if you are a positive person!
There is much growing ahead but I'm so excited.... | 29 mtf Want to have bottom surgery by the end of the year. Just was wondering is that, possible. If not how long would it takes to approve bottom surgery.
Also any tips on how to go through the process with insurance and doctors | I have my reservations over bottom surgery, mostly from all the horror stories of the past still floating around. I know surgery is much better than a decade ago and techniques can only improve.
I've mostly only heard from the phallo camp and would love to hear more from the other surgery options out there.
Also how long was surgery?
How long was recovery?
How long before normal life resumed?
Did you get a choice in length/girth?
Did you choose pump or flexi rod? (Phallo)
I'm sure there are a million more questions that I could ask but I can't think of them right now.
Please go into as much detail as you like or not I appreciate it's a really personal thing to talk about.
Thanks guys | Hi ya'll,
I was wondering if anybody has experience with getting top surgery while working in the service industry. I am 24, and still covered by my parents health insurance for another 18 months. I feel like I should plan on getting top surgery while I still have insurance since most of the time the service industry doesnt have great insurance coverage.
Mostly though, I am concerned about how long it would take for me to recover. Bartending is my only source of income so I would need to plan and save for how much time I end up taking off for surgery and to recover. In addition, I worked so hard for the past year and half to get to where I am at and it would totally suck if I lost my position or ability to work. Being that it is such a physically intensive, and primarily upper body physically intensive job, has anybody else had experience with recovery while in this industry? Pls let me know how your healing experience went, how long it took, how you planned it, and how recovery/ resuming your job has been?
Thank you! Looking forward to hearing from you all. | Hi everyone, so a while back I came across a procedure called extended metoidioplasty which is similar to meta but creates a longer penis (about 2.4 to 4.7 inches in the study).
Here’s the article if anyone is interested ( but I have not seen anything additional about the procedure since that article was published.
Does anyone know if further research will be done? I think this would be a great alternative to the guys who are conflicted between choosing meta or phallo so it’s disappointing that there hasn’t been much research.
Since it’s not well studied I assume no doctors are doing this surgery. Does anyone know of surgeons who do this type of meta?
This article was also interesting to me because I want to know if it would be better to have bottom surgery soon or to wait until there are more advancements in surgical techniques. I bet a lot of other people are in the same position.
What are everyone’s thoughts about this? | After no less than 15 surgeries (all 3 kids combined) I can't tell a difference between when they had a blessing before the surgery and when they didn't. I'm starting to wonder if it really matters... :)
That is all. | 407 | I am cursed with 34FFs (at 5'0", no less!), but the upside to this is that insurance would cover a reduction. I'm still not 100% sure I want to fully transition, but I at least know that I want as little boobage as possible, and I'm thinking that a reduction might be a good compromise between my current situation and a full mastectomy. However, I'm worried that getting a reduction would complicate future top surgery, if I decide to eventually go that route.
Does anyone here know if a reduction would complicate future top surgery? I would especially love to hear from anyone who has had both surgeries! | Breast reduction rather than top surgery? | Is simultaneous bilateral mastoidectomy ever advisable | Mastectomy Plus Reconstruction Has Highest Complication Rate | What can you do if you have mastectomy with reconstructive surgery? | Modified radical mastectomy: definition and role in breast cancer surgery. | I'm considering a bilateral mastectomy. Are there any resources you would recommend? What can I expect physically in the long term? | Quick tip to anyone considering Rhinoplasty | Breast reduction questions |
408 | Liquid metal experiments with swirling flow submerged entry nozzle | Many theoretical and experimental studies have been conducted to investigate elements of swirl injector hydrodynamics, such as variations in liquid film thickness or air core diameter. From these studies, some theoretical relationships have been established through an approximate analytical solution of flow hydrodynamics in a swirl nozzle. However, experimental studies on elements such as the stability of internal flow have not produced conclusive results. In this study, the stability of the internal flow under tangential entry conditions was examined by visualizing the formation of the air core in the swirl chamber and measuring the liquid film thickness in the orifice. | Numerical Study of the acoustic characteristics of the entry diameter gas-liquid coaxial swirling injector | Pressure drop and liquid film thickness in air-water swirling flows in a one-fifth scale model of the steam separator are measured for a wide range of gas and liquid volume fluxes. Numerical simulations based on one-dimensional single-fluid and two-fluid models are also carried out to examine the feasibility of predicting the pressure drop and film thickness in swirling flows. The pressure drop in a single-phase swirling flow is about five times as large as that in a non-swirling flow due to the increase in the frictional pressure drop. The pressure gradient and liquid film thickness in a two-phase swirling annular flow at the inlet of the pick-off-ring of the separator are well evaluated by using a standard one-dimensional two-fluid model, provided that the interfacial and wall frictions in an ordinary two-phase annular flow are multiplied by appropriate constant values. | It works as a nozzle, but the gauge is inaccurate and the inside face with the numbers peeled off after sitting in my tool heat for a month. | Swirling Airflow Through a Nozzle: Choking Criteria | This paper concerns the experimental results of the outward and inward flows in radial-flow nozzles. With an air source of 500 mmAq gauge pressure, the overall pressure losses and pressure distributions are obtained for various flow rates. It follows that the results of the theoretical analysis obtained in the 1st and 2nd Reports are applicable to the practical problems with reasonable accuracy in spite of simple assumptions. However, when the flow is outward and the inner corner of a nozzle is sharp, a severe contraction will occur and experimental results will not be consistent with theoretical values. Since the clearances of the nozzles dealt with are extremely narrow, the velocity distribution in the clearance cannot be obtained by the experiment. | A submerged entry nozzle (20, 50, 90) for use in the continuous casting of a metal, the nozzle comprising: a) a body having a central bore (26, 66, 92) through most of the body the hole ends in a closed end (36, 76); b) a plurality of pairs of discharge outlets (30, 32, 60, 62, 64, 94, 96) which are arranged symmetrically about a longitudinal axis; the cross sectional area of the central bore decreasing between pairs of discharge outlets, and the ratio of height to width of any of the outputs being one or less characterized in that the cross-sectional area of the central bore (26, 66, 92) does not decrease around the entire circumference of the central hole. | Computational fluid dynamics,high-speed camera and LDV System are combined to simulate and test the inner flow field of a hollow cone pressure swirl nozzle so as to verify Volume-of-Fluid simulation method on this type of nozzle.Moreover,the processed results show fields of pressure,density and velocity.Tangential velocities regression equations on areas of sub-potential flow and similar solid region are also matched.So the study offers meaningful reference for expressing inner flow situation of swirl nozzle | 408 | The influence of a swirling flow inside the submerged entry nozzle on the structure and the stability of a liquid metal flow in a physical model of a slab casting mould are investigated. For visualisation of the flow, contactless inductive flow tomography (CIFT) is applied. As expected and desired, the swirling flow leads to a stronger upward fluid motion along the walls. At the same time, however, the oscillatory character of the flow becomes stronger. These flow features obtained with CIFT are shown to be in reasonable agreement with independent measurements using ultrasonic Doppler velocimetry. Preliminary results of numerical simulations also show a similar behaviour. | Examination of swirling flow using electrical resistance tomography | Hydrodynamic analysis of a spiral disc extruder | Research on the Inner Flow Field of a Hollow Cone Pressure Swirl Nozzle | Development of steady flow of liquid film over a domed cylinder | Study on the Effect of the Nozzle of Drag Bits on Bottom Hole Flow Field | Laminar flow over a periodic array of cylindrical surface roughness elements is simulated with an immersed boundary spectral method both to validate the method for subsequent studies and to examine how persistent streamwise vortices are introduced by a low Reynolds number roughness element. Direct comparisons are made with prior studies at a roughness-based Reynolds number Rek (=U(k) k/ν) of 205 and a diameter to spanwise spacing ratio d/λ of 1/3. Downstream velocity contours match present and past experiments very well. The shear layer developed over the top of the roughness element produces the downstream velocity deficit. Upstream of the roughness element, the vortex topology is found to be consistent with juncture flow experiments, creating three cores along the recirculation line. Streamtraces stemming from these upstream cores, however, have unexpectedly little effect on the downstream flowfield as lateral divergence of the boundary layer quickly dissipates their vorticity. Long physical relaxation ... | Frequency Domain Analysis of In-Line Forces on Circular Cylinders in Random Oscillatory Flow | Three-dimensional modeling of the flow and the interface surface in a continuous casting mold model |
409 | in which country was the first private copying system created | What divice that produce hard copy? | I've been dabbling with the idea of porting a model of mine, but am concerned about the prevalence of private model ripping. I understand the Cats Blender plugin had copy protection, but it was removed not long ago.
Does anyone know why this was and how effective today's private model rippers are? Are there any obfuscation methods currently available? I understand full protection is impossible, but I would like at least partial protection if it exists. | The present invention relates to a method of detecting whether a computer file has been copied, the computer file having an inode number. The inode number of the computer file is retrieved (330). From the computer file, a stored inode number is read (350), the stored inode number being the inode number of a file system from which the computer file should not be copied. The retrieved inode number and the read inode number are compared (360) and it is determined that the computer file has been copied if the retrieved inode number does not match the read inode number. Also provided are a method of enabling detection of the copying of a computer file, and devices (420) and software program products (430) corresponding to the methods. | Copyright legislation and licences relating to academic practice. Copyright information on electronic copies | Worshipful Company of Stationers and Newspaper Makers Protestant Reformation and toward the English Civil War. The Stationers' Charter, which codified its monopoly on book production, ensured that once a member had asserted ownership of a text or "copy", no other member was entitled to publish it, that is, no one else had the "right to copy" it. This is the origin of the term "copyright". However, this original "right to copy" in England was different from the modern conception of copyright. The stationers' "copy right" was a protection granted to the printers of a book; "copyright" introduced with the Statute of Anne, or the Copyright Act of | formalities toward the end of the 19th century. In response to their efforts, the 1908 Berlin text of the Berne Convention forbade treaty signatories from conditioning copyright on formalities, shifting copyright from a system of application (registration) to automatic copyright on fixation. Requirements for meeting copyright formalities were largely eliminated in many countries with the adoption of the Berne Convention, which granted a copyright for a creative work automatically as soon as the work was "fixed". Berne was first adopted in 1886 by eight countries, mostly in Europe. Acceptance grew over the course of the 20th century. (See List of | Who passed the first law for copyright? | There is this website which won't allow me to copy past stuff. It immediately shows warning popup if try to copy past or even press ctrl key. even If I open Developer Tools, It displays warning. After few warnings, My session might get terminated. I searched on the webstore for extensions to allow copy pasting and It showed me alot of them. Which one should I use ? I can't try every extension cause site login only get activated when my time slot comes which is only 1 hour. | 409 | the funds come directly from the state budget. As of beginning of 2012, the fees were (in Euros): There was no levy fee on mobile phones, computers, memory cards, game consoles, USB flash drives and CDs/DVDs. VAT of 9% is added to the levies. The world's first private copying system was created in Germany in 1965. It was a result of earlier successful litigation by GEMA against an audio equipment manufacturer in GEMA v. Grundig. Luxembourg is the only EU member state on the continent without a private copying levy, making it a popular "copying levy haven" for blank media | where was the private copying levy introduced in europe | ELI5: How come that Americans can reclaim the VAT after buying something in Europe and not vice versa? | what does russia's vat system not allow it to do | EU-VAT Exemption for Driving Lessons: Pending Case C-449/17, A & G Fahrschul-Akademie GmbH v. Finanzamt Wolfenbüttel | Inter Vivos Transfers and the 2009 German Transfer Tax Reform | The European Commission adopts a new block exemption on technology transfer agreements (Reg. EC No 240/96) | when must a flat designation fee be paid for a european patent | when did the tax on digital goods become permanent |
410 | Master's Ranked Episode 2: Jungle Role | I had picked my jungler in ranked, and I set up my masteries then clicked lock in. It then said I didn't have a champion selected, so it would random and pick one for me. I couldn't do anything to make it think I selected one, so I had to hit OK and then I was given Ashe. I dodged anyway to avoid screwing my team, but I thought they made it so you can't random in ranked at all. | Hey all,
Been trying to teach my wife the game and she recently hit level 12 so I thought I'd have her try out jungling. Obviously I intend to play with her and coach her but for reinforcement's sake, does anyone know of a good, up to date video introducing new players to the jungle role?
Thanks! | If your bot or top lane can't 1v2, then don't jungle. You're basically just forfeiting the game | Never played ranked before but M6 on my main champ(Poppy) in jungle and support. I wanted to start up a discussion around this video by Mobalytics (
Quick summary for those who don't want to watch it. The video rank orders all 5 roles, based on what they believe is the best role to utilize to climb within each rank/elo based on how much influence each role has within those elos and such.
Bronze/Iron - Mid, Silvir - Jungle, Gold - Mid, Plat - Mid, Diamond - Mid.
So, my question is, how much do people agree or disagree with this? Not just about the roles selected, but also with the general concept of using different roles at different ranks (they do list all 5 roles, I just typed in the top ones to make things easier to discuss over).
Personally, not too qualified to talk since I've never done ranked, but imo, I feel like this may work for someone who's a Mid lane main, since you're only really learning Jungle (as complex as it is) for a short period of time and then you just go back to doing what you've always been doing. On the other hand, if you're any of the other roles, it seems like you're chopping and changing all the time.
Does moulding yourself into a role that you don't naturally fit into make more sense than sticking to one role all the way up from Bronze and just getting stupid good at it? Kinda like one tricking a specific champion, with just one other as a back up to their counters, rather than having a massive champion pool.
As a parallel example, I do believe someone (maybe the same channel), put up a video a while ago suggesting to learn a few different jungle champions in order to learn skills that would cross over. During this time I was only doing jungle, but constantly changing between Master Yi, Zac, Warwick, and a few others. And surprise surprise, I never got past M5 with any of them, even after 150 games. On the other hand, when I settled down with Poppy, despite not being a stellar, peak of the meta jungler, doing 50 straight games with just her got me so intimately acquainted with her that Iam almost at M7 in the jungle.
So what do people think? Change roles and champions as you climb? Or stick to one role and get really damn good at it? (If we're talking about a role that's not Mid here) | I've seen the top streamers run jungle support. I tried copying it in ranked, but my hunter loses lane, and I up underleveled - lower level than the enemy support. I run the same routes Incon runs, but it never seems to work. Is it just something that doesn't work well on console, yet? | When people talk about it, it seems to always boil down to: Who clears faster, and who wins the 1v1 duel.
But in my limited jungle experience the winner of the "Jungle matchup" is decided by who has better players in their lanes.
How do I know if one champ has priority over the other in the jungle? (for instance if your team has an eve jungle vs the enemy lee sin) | I'm playing full time jungle since season 2. I want to share my opinion on what jungle should be / should not be.
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Right now, jungle role got nerfed. Some people will say it's still important because "dragons are important". It's right.
But the fact you get less xp and gold in jungle camps is a nerfed, and you can easily out level, it's harder to gank solo lanes and impact the game. I feel overall happy with this changes, the role become a bit more maccro oriented.
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Now, you don't have any bonus xp with jungle item, except on first jungle camp. This is fine, but you also don't have any malus xp from lane minions. The only malus you can get is gold from lane minions until you complete your jungle item.
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I would love to see some malus on xp from lane minions. I don't feel it's healthy that junglers can tax xp from lanners and get away with it.
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How you guys think we could improve jungle role without make it too annoying with perma ganking lanes ? | So I played a lot of fiddle jungle back in season 3,4,5 but now that I hear he’s getting a rework I feel as if I should get m7 to do the old sticks justice before he sails away. So, how do you jungle on this old scarecrow now, or are there any other alternative roles he can cheese his way to victory in. | 410 | Hey everyone, new episode devoted to the jungle role. I give my in depth analysis and commentary for the role. Hope you enjoy and all constructive feed back is appreciated.
LINK: | a very detailed jungle commentary | Extended chase scene through the jungle? | What was The Jungle about? | All in One Season 11 Brand Jungle Guide | Everything You Need to Know | Master Elo Gragas Jungle Gameplay w/ Commentary - Jungle Guides By Zazuu | Deep Jungle stories and experiences | Riot, can you take the time to explain what you're doing to the jungle, because it makes no sense to your player base. | Fun Jungle Gods to Learn the Role? |
411 | how far is highgate wood from charing cross | Check out this mockup image] ( and this [not-mine-but-identical real world image
Existing cross beams on the left. They run all the way down at a 45 degree or so angle, level to the bottom of the beams. My question is, can I replace them with ~60 degree angled crosses instead? I want to run some drywall for the ceiling and leave a couple of inches of beam exposed, but obviously cannot do that with the current cross beams. Will moving them up a few inches like this still provide adequate support? | Hornsey is public sector housing, surrounded by the late Victorian terraces developed by builders such as John Farrer. Between the western end of the High Street and the bottom of Muswell Hill, the character of the area changes; most being part of the Warner Estate built up with large late Victorian houses. To the south west of the High Street is Priory Park. The High Street has a number of shops, restaurants and pubs. The eastern section retains strips of grassed areas. The 13th-century tower is all that remains of St Mary's Church. The Tower has recently been used as The | Charing Cross tube station opened. The constituent stations also underwent a number of name changes during their history. The first part of the complex, the Bakerloo line platforms, was opened as "Trafalgar Square" by the Baker Street & Waterloo Railway (BS&WR) on 10 March 1906. The Northern line platforms were opened a year later, as "Charing Cross", by the Charing Cross, Euston & Hampstead Railway (CCE&HR, now the Charing Cross branch of the Northern line) on 22 June 1907. At its opening this station was the southern terminus of the CCE&HR which ran to two northern termini at and Highgate (now ) tube stations. | Charing Cross roof collapse On 5 December 1905, the iron-and-glass overall arched roof of London Charing Cross railway station collapsed during a long-term maintenance project, killing six people. The original roof was designed by Sir John Hawkshaw and comprised a single-span trussed arch with wrought iron tie rods. The roof was 164 ft wide by 510 ft long and was designed as a contained arch, with bowstring principals. At on 5 December 1905 one of the tie-rods of a main principal sheared, making a loud noise. Some passengers evacuated the station, although many remained. Shortly before , two complete roof | Margam Stones Museum which fills the cross stem. First mentioned in 1697, it was in Margam Abbey Churchyard, south of the Church, until it was moved into the museum. It is high, wide and thick, made from locally occurring Pennant sandstone. The cross head is in diameter. It is made from a single piece of Pennant sandstone, although a thin tenon on its base suggests it was made to fit into a pedestal socket. Stylistically the splayed arms and wide circular armpits are similar to 10th-century crosses from the north of England. Date: 10th century The largest of the Margam Stones, and with | A terrace crossing is a geographical zone between the sedimentation (downstream) part and the erosion (upstream) part of the river. This zone develops on the location where the transition of erosion to sedimentation takes place. Upstream of the crossing, terraces will exist. The highest terraces will be the oldest. Downstream of the crossing, older sediments will be buried beneath younger deposits.
See also
Riverterrace
Sedimentology
Fluvial landforms
Sedimentology
Geomorphology | fraction of an inch in rise over a one-foot run (e.g. 1/4 inch per foot). Cross slope Cross slope, cross fall or camber is a geometric feature of pavement surfaces: the transverse slope with respect to the horizon. It is a very important safety factor. Cross slope is provided to provide a drainage gradient so that water will run off the surface to a drainage system such as a street gutter or ditch. Inadequate cross slope will contribute to aquaplaning. On straight sections of normal two-lane roads, the pavement cross section is usually highest in the center and drains to | I'm trying to go north on the A5 from central london. All looks fine until the A5 approached the North Circular, \here\]([ . Looking on google street view it looks like a nightmare to stay on the A5 to get north of this area.
Any options on how to cross.
from what i can see i can turn left onto Oxgate lane, then North onto Coles Green Road which takes me to a pedestrian bridge which i cna cross and then swerve my way through an industrial estate get back on it to Hendon.
thoughts? | 411 | of the London Underground. It is adjacent to the A1 road and is situated approximately 6 miles (10 km) north of Charing Cross, well inside London's metropolitan area. Highgate Wood Highgate Wood is a 28 hectare (70 acre) area of ancient woodland in North London, lying between East Finchley, Highgate Village, and Muswell Hill. It was originally part of the ancient Forest of Middlesex which covered much of London, Hertfordshire and Essex and was mentioned in the Domesday Book. It lies in the London Borough of Haringey, but is owned and managed by the City of London Corporation. The London | when was beechwood house, highgate built | on which london railway line is woodgrange park situated | the bushwood area of leytonstone in east london is surrounded by the gre | when did the british go into trones wood | which woodlands in high wycombe were cordoned off for four months in | what is the name of the brook which rises in hadley wood in london | what is the name of the gated community that surrounds pine log mountain | how many trees were cleared from the site of raf woodbridge in 1943 |
412 | which animal is the main host of ixodes hexagonus | Distribution and Habitat of Ixodes pacificus (Acari: Ixodidae) and Prevalence of Borrelia burgdorferi in Utah | having large, square teeth like cattle, and lacking the typical facial glands. The addax are most prone to parasites in moist climatic conditions. Addax have always been infected with nematodes in the Trichostrongyloidea and Strongyloidea superfamilies. In an exotic ranch in Texas, an addax was found host to the nematodes "Haemonchus contortus" and "Longistrongylus curvispiculum" in its abomasum, of which the former was dominant. These animals are mainly nocturnal, particularly in summers. In the day, they dig into the sand in shady locations and rest in these depressions, which also protect them from sandstorms. Addax herds contain both males and | Molecular investigations of Rickettsia helvetica infection in dogs, foxes, humans and Ixodes ticks | Ixodes eadsi sp. n. is described from rodents in southern Texas. The new species of Ixodes here described was collected from several species of rodents in Cameron Co., Texas, by Dr. Richard B. Eads, U. S. Quarantine Station, Brownsville. We are greatly indebted to Dr. Eads for placing the materials at our disposal. All measurements given are in millimeters. | Ixodes holocyclus nutrition (except for a few Argasid genera in which the adult mouthparts are non-functional, i.e. Antricola, Otobius and Nothoaspis). Adults also require the blood for sperm or egg production. The feeding process of Ixodid ticks has first a slow phase for several days followed by a fast phase in the last 12–24 hours before detachment. There may be a tenfold increase in fed: unfed weights by the end of the slow phase, but there is an additional tenfold increase by the end of the final fast phase. Leaving the full engorgement as late as possible reduces the chances of detection | The author analyses the nosological complex "piroplasmosis" and emphasizes the differences between the two sub-orders and families of "piroplasms". He studies the life-cycle of these parasites in mammals and in their vectors, acari belonging to the super-family Ixodoidea. He lays out the mechanisms of piroplasmosis transmission to animals and, sometimes, to man and draws from these data the epidemiological features and means of control. | remain spread apart on the surface. The process by which Ixodid and Argasid ticks feed is termed telmophagy (= pool feeding). (This contrasts with the process of solenophagy, used by mosquitos, in which feeding is direct from a small venule.) The resultant pool expands as a result of the anticoagulants released from the salivary glands. In some Ixodid ticks a cementum is secreted into the wound within 5–30 minutes of cutting into the skin. This material hardens quickly into a latex-like covering around the mouthparts but excluding the palps that remain flattened out on the skin surface. "Ixodes holocyclus", however, | only i.e. "Ixodes holocyclus". The extent to which other species found in the area parallel "Ixodes holocyclus" in this and other aspects of their biology is not known. The graphs show the average seasonal prevalence of instars and types, observed over an eight-year period, which includes six years of detailed field observation, and collection, supported by information and specimens from many people. "Ixodes holocyclus" emerged from this survey as the dominant acarine ectoparasite of mammals and avians in the study area, its population dwarfing those of other tick species, and various species of mites. As distinct from instars (the life | 412 | Ixodes hexagonus nest-dwelling hedgehog specialist. It is also found on foxes, mustelids (including badgers), dogs and cats." "I. hexagonus" is found throughout western Europe as far east as Siberia. It is widespread over this area. It is closely associated with its principal host the hedgehog. "I. hexagonus" is endophillic; it is predominately a nest based parasite. It spends most of its life in the nest of the main hedgehog host. Thus it is buffered from the environmental conditions experienced by many other free ranging ticks such as "I. ricinus". The Hedgehog Tick is a potentially important reservoir for Borreliosis, the causative agent | Man-made barriers are well known for their effects on ecosystems. Habitat fragmentation, for instance, is a recognised consequence of modern-day infrastructure. The aim of the present study was to investigate the diversity and abundance of tick species, as well as the risks of acquiring tick-borne infections in habitats adjacent to a freeway. Therefore, ixodid ticks were collected from the vegetation at two-week intervals (in the main tick season, from March to June) in eight habitats of different types (forest, grove, grassland) along both sides of a freeway. Ixodes ricinus females were molecularly screened for three species of tick-borne bacteria. In the study period, 887 ixodid ticks were collected. These included 704 I. ricinus (79.4%), 51 Dermacentor reticulatus (5.7%), 78 D. marginatus (8.8%), 35 Haemaphysalis inermis (3.9%) and 19 H. concinna (2.1%). There was no significant difference in the abundance of tick species between similar habitats separated by the freeway, except for the absence of Dermacentor spp. on one side. In I. ricinus females, the overall prevalence of Anaplasma phagocytophilum was low, and (in part due to this low rate) did not show significant difference between the two sides of the freeway. Rickettsia helvetica had significantly different overall prevalence between two distant habitats along the same side of the freeway (12.3% vs. 31.4%), but not between habitats on the opposite sides. Borrelia burgdorferi s.l. showed significantly different overall prevalence between habitats both on the same and on the opposite sides of the freeway (8.6-35.9%), and the difference was higher if relevant habitats were also separated by the freeway. Importantly, the prevalence rate of the Lyme disease agent was highest in a forested resting area of the freeway, and was significantly inversely proportional to the prevalence of A. phagocytophilum (taking into account all evaluated habitats), apparently related to deer population density. Prevalence rates of | Detection of rickettsial DNA in ixodid ticks recovered from dogs and cats in Japan. | The prevalence of parasitic helminths in stray dogs in the Baghdad area, Iraq. | Ectoparasites of Rodents Captured in Hamedan, Western Iran | [The dynamics of important helminths in pig husbandry]. | Ixodes ricinus, the sheep tick, as a consequence of its habit of taking blood from mammalian hosts, can transmit disease from wild animals to humans. This is likely to be a particular problem in parks shared by humans and deer populations. These ticks were sampled, using cloth drags, from vegetation at 16 sites in Richmond Park, London, between 15 July and 22 August 2009. A total of 2436 ‘host-seeking’ ticks (2281 larvae, 151 nymphs and 4 adults; three males and one female) were collected, and attempts were made to identify the environmental factors affecting the distribution of these ectoparasites. Tick presence was closely related to soil moisture, light levels and humidity throughout the park. It is thought that improving our understanding of how these factors influence the presence of I. ricinus will facilitate methods of tick control and help to educate the public about where ‘hotspots’ for these parasites are likely to be within the park. | Background Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe.Methods Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene.ResultsOf the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis (n = 22).Conclusions Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis, which was found merely in stone martens and pine martens. None of the rodentassociated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/ or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs. | Further studies on the interaction of Toxoplasma gondii with neutrophils and eosinophils. |
413 | Development and Implementation of an Autonomy Supportive Training Program Among Youth Sport Coaches | The purpose of this paper is to provide a concise resource for coaches, coach educators, and coaching scientists by reviewing three common approaches to coaching: the mastery approach to coaching; autonomy-supportive coaching; and the transformational leadership approach to coaching. The theoretical foundations, purpose, evidence base, specifed behaviours, and translation into coaching and coach education of each approach are reviewed. Despite diverse theoretical foundations and variations in purpose, there is some overlap in the coaching behaviours prescribed by each approach. However, there is limited empirical evidence to support the use of the three approaches in coach education and this is detrimental to effective and evidence-based coach education. Efforts to integrate theoretical foundations are promising, and a comprehensive prescription of coaching behaviours based on an integration of the three approaches is possible. This approach can potentially lead to cumulative effects on positive athlete o... | The present investigation was designed to develop a profile of the coaching education background and self-perceived coaching education needs of elite U.S. amateur sport coaches. In all, 130 national team, Pan American, and/or Olympic coaches representing more than 30 U.S. Olympic structure sports were surveyed. Results revealed that the coaches were extremely interested in coaching education workshops and seminars, initiating mentor coach programs for potential elite coaches, and participating in a variety of coaching science courses. Few consistent differences were found between the various categories of coaches (individual vs. team sport, open vs. closed sport, experienced vs. inexperienced, male vs. female, and physical education degree vs. non physical education degree) in terms of their coaching education background and needs. Implications for university based coaching education efforts are discussed. | The literature in psychotherapy and sport psychology has supported the importance of self-awareness and countertransference management (Ellis, 2001; Leahy, 2001; Van Raalte & Andersen, 2000) and its applicability in all psychological settings (Hayes, 2004). This study was an audit of (n = 58) accredited UK sport psychology practitioners that explored the importance they attached to self-awareness and their behavior in practice that supported the management of these concerns. Results indicated that practitioners regarded self-insight and self-integration as important (Mdn = 4), but relied upon themselves and informal peer networks rather than regular supervision for professional support. Most practitioners never (Mdn=1) used counseling or therapy for personal support. Recommendations are made for piloting post-accreditation professional supervision in sport psychology and developing the provision of general counseling and sport psychology sessions for trainees. | Sport coaching is a multifaceted profession with many responsibilities. Coaches can have a profound effect on athletes that can be both positive and negative. Coaches have the ability to motivate athletes and increase their self-esteem. Conversely, negative effects of coaching may include athlete drop-outs, injuries, and loss of confidence. Coaches need to manage the coaching environment and create positive surroundings to ensure that athletes achieve their optimum potential. Managing a coaching environment refers to how coaches establish and maintain order. This paper explores the literature on behavior management in education and sport settings and aims to contribute to sport-coaching knowledge. General coaching tips for managing athlete behavior are suggested along with examples of potential coaching strategies. | AbstractIn terms of achieving wider health and social outcomes, sport coaching promises much for young people with disabilities. Despite this promise, the experiences and practices of those coaches who enter the disability sport arena are underexplored. This is particularly so for coaches who operate in community participation rather than competitive elite environments. Accordingly, this paper uses an autoethnographic approach to explore the experiences of a basketball coach (Colum), who enters a youth club for disabled participants for the first time. Utilising observational data, reflective field notes and interviews, five relativist vignettes are collaboratively constructed to represent Colum’s experiences across 12 basketball sessions. The vignettes reveal that the disability and community context disrupted Colum’s normative coaching behaviours. An emotional laborious journey is recounted that includes significant lessons, which may impact coaching practitioners, researchers and sport development offi... | The book provides a solid frame and coaching model for an easier and more predictable way of doing coaching.
It supports a clear and easy to understand approach of the human mind and behaviors, supporting the strengthening of the coaching practice, both for individuals and teams. | There is a paucity of sport parenting research that specifically examines the role parents play in the introductory stages of the youth sport experience, despite the fact that this is when youth involvement is at its highest. To fill this void in the literature, this study examined expert coaches’ v | A useful introduction of coaching approaches and application across context as well as personal reflection of their coaching learning process. | 413 | Studying perceived autonomy support, a basic tenet of self-determination theory (Deci & Ryan, 2000), provides some understanding as to how coaches can more positively influence youth athletes to enjoy and persist in youth sport. Borrowing insights from success in physical education and coaching-oriented interventions, the purpose of this paper was to highlight positive aspects and challenges of an innovative youth sport autonomy supportive training program for coaches. Positives included the initial training session and the use of an online training component. Challenges were the structure of the season, other coaches, and possibly the age of the athletes. Future training programs in youth sport coaching should increase in duration, provide specific examples of how to implement autonomy supportive coaching behaviors, as well as address solutions to the time constraints of the youth sport setting. | Autonomy-Supportive Pedagogical Approach to Sports Coaching: Research, Challenges and Opportunities | The applicability of self-determination theory to health coaching: a qualitative analysis of patient experiences | Changes in Children's Autonomous Motivation toward Physical Education during Transition from Elementary to Secondary School: A Self-Determination Perspective. | This study applied self-determination theory to investigate the effects of students' autonomous motivation and their perceptions of teacher autonomy support on need satisfaction adjustment, learning achievement, and cardiorespiratory fitness over a 4-month personal conditioning unit. Participants were 253 urban adolescents (121 girls and 132 boys, ages = 12–14 years). Based on a series of multiple regression analyses, perceived autonomy support by teachers significantly predicted students' need satisfaction adjustment and led to learning achievement, especially for students who were not autonomously motivated to learn in physical education. In turn, being more autonomous was directly associated with cardiorespiratory fitness enhancement. The findings suggest that shifts in teaching approaches toward providing more support for students' autonomy and active involvement hold promise for enhancing learning. | Personal coaching as a positive intervention | Mastery, Autonomy and Transformational Approaches to Coaching: Common Features and Applications | Extant research studies have found that autonomy support has a positive impact on the perceived competence and intrinsic motivation of students. However, few studies have investigated how autonomy supportive classrooms can be implemented. Montessori education is established upon the philosophy of helping each child attain self-mastery and independence. It emphasizes that students be given autonomy to engage freely with their learning environment. This case study of an upper-elementary Montessori classroom found that the Montessori philosophy of education guided how teachers used autonomy supportive strategies. Teachers supported student organizational autonomy by allowing them choice in terms of school work and work partners. They fostered cognitive autonomy by encouraging student independent thinking, encouraging self-initiation, and honoring students' voice. When implementing control, they acknowledged and respected student feelings, provided rationales for expected behavior, and suppressed criticism. Students surveyed rated themselves highly in terms of intrinsic motivation for schoolwork. Five guidelines are derived from this study to help teachers implement autonomy support in K-12 classrooms. | There is a body of research on the challenges that coaches face when trying to implement athletecentred coaching, but very little attention has been paid to the influence that the growing number of sport coaching degrees has on coaches' beliefs and practice in regard to athlete-centred coaching. While studies have been conducted on sport coaches' use of game-based approaches (GBA) to coaching, undergraduate sport coaching students' interpretation of this coaching innovation has been largely overlooked. This article takes a step toward redressing this oversight by reporting on a study that inquired into the influence of the experiential pedagogy used in a course on athlete-centred coaching on students' beliefs about coaching and their practice. The scholarship of teaching study adopted a constructivist grounded theory methodology to focus on five undergraduates in a sport coaching program with data generated through a series of three interviews with each participant. This study concludes that the experience-based course design was effective in influencing undergraduate students' beliefs about coaching and their practice outside university. |
414 | Microwave Effects Due to Anionic or Cationic Initiators in Emulsion Polymerization Reactions | This article contains original experimental data, figures and methods to the study of Microwave-assisted emulsion polymerization of styrene under the frame of “Enhanced Microwave Synthesis” (EMS), has been examined to investigate the advantages of Microwave (MW) power use in emulsion polymerization (Ergan et al., Eur. Polym. J. 69, 2015, 374–384). For comparative purpose, MW and conventional heating (CH) method experiments were conducted under similar conditions. By externally cooling the reaction vessel with 1,4-dioxane, constant and continuous MW power was successfully applied at isothermal condition during the polymerization. Here we give the MW power calibration data of MW-experimental system, the complete set of the experimental polymerization data and the analysis data obtained from different polymer characterization test devices (GPC, DSC and Viscometer). | Radiation-induced emulsion polymerization of styrene in soap-free system has beenstudied and the monodisperse polystyrene lattices were obtained. Both its purity and emulsionconversion are better than that of chemically initiated polymers. | The microwave synthesis represents a very convenient method of obtaining chemical compounds not very easy to synthesize by the means of organic chemistry. The high energy electromagnetic radiation can easily induce polymerization, dehydration, etc. In this study, the reaction of itaconic acid (IA) and N-hexyl amine was studied. The microwave synthesis leads to a mixture of imide and diamides while the oil bath reaction leads to mono and diamides. By simply adding an initiator a high purity polymer was obtained. This technique is particularly useful when working with low reactivity compounds, allowing a much shorter reaction time and high conversions. The possibility to control the power gives a good chance to maintain the temperature at low values. | New experimental data for nucleation in emulsion polymerization prove that the process is heterogeneous in nature so that water-borne oligomers precipitate at the surface of monomer drops. The particle morphology at the early stage of the process is strongly influenced by the initiator concentration. The hydrophilicity of the monomer is less important as methyl methacrylate, styrene, and 4 -tert-butyl styrene show similar behaviour. | Abstract With microwave irradiation, phthalic anhydride was obtained with a high yield of 67 mol% at ca. 563 K by the oxidation of o-xylene with air over V2O5/SiO2 system. It is found that dispersion of V2O5 on SiO2 is more homogeneous when the catalyst is prepared by microwave irradiating method. In the microwave catalytic process the optimum reactor bed temperature of the title reaction decreases to 563 K (653 K in the conventional process). The effects of the microwave electromagnetic field on the catalysts are discussed. | In the present studies a series of anion-exchange resins was synthesized in a microwave field. The 1,6-diaminohexane functionalized resins were obtained in presence of selected organic solvents, N,N-dimethyl formamide, dimethyl sulfoxide and 1-methyl-2-pyrrolidone. The resins were employed in batch and dynamic processes of Au(III), Pt(IV) and Pd(II) sorption from tricomponent systems in 0.1 M HCl. The experiment was designed in a way that allowed to determine the use of a specific solvent in the microwave field and how it impacts on properties of the anion exchangers. An influence of a reaction environment was discussed taking into account i.e., dielectric characteristics of the specific solvent, efficiency of the syntheses processes as well as the maximum sorption capacity of the resins. Ultimately the application of a specific reaction environment was set together with sorption of noble metals ability and evaluated using infrared spectroscopy. The proceeded analyzes allowed to determine which organic solvent from the selected ones is the most suitable for microwave-assisted synthesis of the anion-exchange resins. | In this article, we report on the effect of using ultrasound during emulsion polymerization. This work differs somewhat from that previously reported in that ultrasound is used in conjunction with conventional initiators. The aim is to observe the changes in the nature of polymerization and the synthesized polymer. In this work, reaction conditions and compositions typical of conventional emulsion polymerization are used. Azo-bisisobutyronitrile and potassium per sulfate are the initiators used. The initial indication is that the rate of polymerization and the final conversion are higher when ultrasound is introduced into the polymerization system. This effect is more pronounced at lower temperatures (50°C) and low initiator concentrations (0.01%). At higher temperatures (70°C) the polymerization rate is seemingly unaffected by the use of ultrasound. The final product in all the experiments is a latex. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 101–104, 2000 | The emulsion polymerization of some monomers can occur without the conventional free radical initiators under ultrasonic irradiation. However, the initiation mechanism is still under controversy. In this paper, the sources of free radicals arising from ultrasonically initiated emulsion polymerization were investigated. Experimental results show that ionic surfactants play a very important role in obtaining a high polymer yield. While monomer conversion is very low in the absence of surfactants or in the presence of nonionic surfactants, it increases significantly upon addition of a little amount of ionic surfactant. FTIR and a radical trapping experiment confirm that the free radicals involved in the irradiation process originate from the decomposition of the ionic surfactants. Under ultrasonic irradiation, ionic surfactants undergo bond scission between the alkyl and ionic group, where the bond is the weakest along the chain. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2617–2624, 2005 | 414 | Summary: Emulsion polymerization reactions were performed under microwave irradiation and conventional heating using anionic or cationic initiators and surfactants. Microwave irradiation promoted higher reaction rates for both initiators and surfactants, in comparison with the conventional heating. The effect of high power microwave irradiation was studied using a method of cycles of heating and cooling, where rapid polymerization reactions were obtained. In the reactions with anionic initiator and surfactant, a decrease in the particle diameters was observed with microwave heating, and even smaller particles were obtained using high power microwave irradiation. Moreover, the decrease in the particle size was acompanied by an increase in the polymer molecular weight. On the other hand, these effects were not observed for reactions with cationic initiator and surfactant. | Heating characteristics and polymerization of ε‐caprolactone under microwave irradiation | Microwave-mediated miniemulsion polymerization of styrene with consecutive periods of heating and cooling exhibits compared to continuously heated polymerizations a very unique behavior. For medium-hydrophobic azo-initiators under optimized conditions, polymer radicals survive the heating pulse and grow during the cooling period to ultrahigh molecular weights >107 g/mol. This “surviving radical effect” is accompanied by unexpectedly high conversion after the first polymerization cycle, comprising of a temperature pulse of less than 10 s duration and a subsequent cooling period. Besides the ultrarapid heating by the microwaves, the surviving radical effect is purely thermal in nature and can be explained by the elemental reactions of radical heterophase polymerizations. | RADIATION-INDUCED EMULSION POLYMERIZATION OF STYRENE IN SOAP-FREE SYSTEM | A rapid, highly efficient and green synthetic approach to polyol esters for lubrication oils is proposed. Using sulfuric acid and p-toluene sulfonic acid as a composite catalyst and C5–C9 straight-chain monocarboxylic acids, with pentaerythritol (PE) and dipentaerythritol (di-PE) as starting materials, a series of lubrication oil polyol esters were synthesized in the absence of solvent under microwave irradiation. Compared with the conventional synthetic method, our proposed microwave method exhibits advantages, including higher yields, shorter reaction times and lower reaction temperatures. The viscosity coefficients of the products at 40 °C and their refractive indices at 25 °C were investigated. In addition, the thermal behavior of the starting materials under microwave irradiation was also investigated. The results reveal that the microwave absorbance of n-pentanoic acid is stronger than that of n-hexanoic acid and that of n-heptanoic acid is stronger than that of n-octanoic acid. The microwave absorbance of di-PE is also stronger than that of PE. | Polymerization of Lactic Acid Using Microwave and Conventional Heating | ESR study of radiation-induced polymerization of styrene adsorbed on silica gel | Abstract Free radical polymerization of dialkyl fumarates (R:isopropyl, cyclohexyl, 2-ethylhexyl, 2-phenylethyl) under microwave irradiation was investigated. The polymerizations were carried out at different powers of irradiation and initiator concentrations (benzoyl peroxide, BP) and the effect of the monomer structure on the conversion, average molecular weights and the polydispersity index ( M w / M n ) was analyzed. A significant enhancement of the rates of polymerization was found, as compared with those obtained under thermal conditions. | Thermal degradation of γ-irradiated polymers—2: Polymethacrylonitrile |
415 | What does george want lenny to remember? | What tow things does george want lennie to remember? | When they got to where thet were going what does george tell lennie to do? | Why does george tell lennie to sleep in he barn? | Where is george in mice of men in chapter 44? | NPR's Tavis Smiley talks with the great George Benson about his long career and new album, <em>Irreplaceable</em>, which is dedicated to the younger crowd. | What favor does george ask candy in chapter 5 of mice and men? | Why did lennie and george have to flee from weed? | What does george do to stop being called koko? | 415 | What two thgs does george want lennie to remember? | What does george talk about to entice lennie to remember or do what he want? | When they got to where they goingwhat does george tell lennie to do? | Where is george when lennie is at the barn? | Why did george and lennie leave the last place? | Who does george order lennie not to talk when they get to the ranch? | Why are george and lennie different from the guys that work on ranches? | What does whit invite george to do tmorrow night? | Of mice and men what is george's role at work? |
416 | Differences in Patient Characteristics and Midterm Outcome Between Asian and European Patients Treated with Radiofrequency Ablation for Hepatocellular Carcinoma | Local Recurrence after Radiofrequency Ablation of Hepatocellular Carcinoma: Treatment Choice and Outcome | Meta-analysis of radiofrequency ablation versus hepatic resection for small hepatocellular carcinoma | Safety and efficacy of laparoscopic radiofrequency ablation of hepatocellular carcinoma in patients with liver cirrhosis. | The authors in the paper report their observation and nursing of 18 patients with hepatocellular carcinoma (HCC) treated by cooled-tip radiofrequency ablation (RF). According to the characteristics and principles of HCC. to ensure the success in treatment, such nursing measures should be strengthened as preoperative psychological nursing and other preparation, introperative monitoring of their conditions including HR, P. BP and the parameters of the machine, and postoperative observation and prophylaction of complications such as bleeding, abdominal pain, fever and renal dysfunction. | Efficacy and safety of percutaneous radiofrequency ablation versus surgical resection for small hepatocellular carcinoma: a meta-analysis of 23 studies | ObjectiveTo describe the safety and efficacy of radiofrequency ablation (RFA) to treat unresectable malignant hepatic tumors in 123 patients.BackgroundThe majority of patients with primary or metastatic malignancies confined to the liver are not candidates for resection because of tumor size, locati | Objective ::: The purpose of this study was to report on the clinical course and imaging findings of hemobilia after radiofrequency (RF) ablation in four patients with hepatocellular carcinoma (HCC). | Radiofrequency Ablation of Concomitant and Recurrent Pulmonary Metastases after Surgery for Colorectal Liver Metastases | 416 | Purpose ::: The aim of this study was to compare patient characteristics and midterm outcomes after RFA for unresectable Hepatocellular carcinoma (HCC) in Asian and European cohorts. | ObjectiveTo describe the safety and efficacy of radiofrequency ablation (RFA) to treat unresectable malignant hepatic tumors in 123 patients.BackgroundThe majority of patients with primary or metastatic malignancies confined to the liver are not candidates for resection because of tumor size, locati | Hepatocellular carcinoma (HCC) survival by etiology: A SEER-Medicare database analysis. | Purpose ::: This study aimed to compare clinically relevant outcomes following transarterial chemoembolization (TACE) and transarterial radioembolization (TARE) in patients with unresectable hepatocellular carcinoma (HCC) using only prospective randomized clinical trials as a source of information. ::: ::: ::: Materials and methods ::: A meta-analysis was performed to compare the efficacy of TARE and TACE in treating patients with unresectable HCC. Only prospective randomized trials were included in the quantitative analysis. Overall and progression-free survival, disease control rate, and transplantation rate were the variables under analysis. ::: ::: ::: Results ::: Overall survival at 1 year was similar between the two treatment groups (OR =1.31, 95% CI: 0.56-3.04, P=0.53). Progression-free survival at 1 year was also not statistically different between the two treatments (OR =0.23, 95% CI: 0.02-2.45, P=0.22). Although a higher proportion of patients underwent transplantation in the TARE group (30% vs 20.8%), this difference was not statistically significant (OR =0.68, 95% CI: 0.23-2.01; P=0.49). ::: ::: ::: Conclusion ::: TARE and TACE provide similar outcomes in unresectable HCC. The role of TARE should be explored in selected patient subpopulations in future clinical trials. | Purpose: Percutaneous ablation techniques, including microwave ablation (MWA) and radiofrequency ablation (RFA), are important minimally invasive treatment options for liver tumors. MWA is expected to provide a larger ablation zone than RFA in a shorter time. The aim of this study was to investigate the duration of ablation, the number of punctures, and technique efficacy of RFA versus MWA for hepatocellular carcinoma (HCC) and liver metastasis. sessions with 274 tumors (263 HCCs and 11 liver metastases) treated by RFA and 32 sessions with 34 tumors (26 HCCs and 8 liver metastases) treated by MWA were enrolled in this retrospective study. We investigated age, sex, Child-Pugh classification, number, and size of tumors. Technical success (TS), local tumor progression (LTP), size and shape of the ablation zone, number of punctures, and duration of ablation were compared. Postoperative follow-up was performed with imaging studies performed between January 2014 and March 2019.Results:The TS rate was 88.2% for MWA and 92.3% for RFA. The LTP rate after 3 months was 6.7% for MWA and 5.4% for RFA. The LTP rate after 6 months was 15.0% for MWA and 10.7% for RFA.There was no significant difference between MWA and RFA. The number of punctures per tumor was 1.42 ± 0.56 for MWA and 2.76 ± 1.27 for RFA. The duration of ablation per tumor was 10.40 ± 4.26 minutes for MWA and 23.20 ± 10.54 minutes for RFA. Compared with RFA, MWA caused spherical ablation in a shorter time with fewer punctures (P < 0.05). Conclusion: MWA had similar short-term outcomes with fewer punctures and duration of ablation than RFA. MWA has potential as a less invasive treatment for liver tumors. | The purpose of this study is to identify the optimal criteria of the radiotherapeutic parameters in patients with unresectable locally advanced hepatocellular carcinoma (HCC). 103 patients were enrolled in this study. All patients received RT delivered using the TomoTherapy Hi-Art system between March 2006 and February 2012. We evaluated the planning target volume (PTV), total dose (Gy 10 ), and NTNL-V BED20 (non-target normal liver volume receiving more than a biologically effective dose of 20 Gy 8 ) as significant radiotherapeutic parameters associated with hepatic function deterioration and local progression-free survival (PFS). A PTV of 279 cm 3 or 304 cm 3 , a total dose of 60 Gy 10 , and a NTNL-V BED20 of 40.8% were identified as the optimal cutoff values of radiotherapeutic parameters to prevent hepatic function deterioration and prolong local PFS. Based on these findings, patients were divided in a favorable and an unfavorable prognosis group. The differences in median local PFS, overall survival, and incidence of deteriorated hepatic function between the two groups were 11.2 months, 11.1 months, and 71.7%, respectively (p < 0.001 in each case). In conclusion, we suggest that the optimal criteria of the radiotherapeutic parameters for patients with unresectable locally advanced HCC are: PTV ≤ 279 cm 3 , total dose > 60 Gy 10 , and NTNL-V BED20 ≤ 40.8%. | Simple Summary: Textbook outcome (TO) is a novel composite measure that provides a comprehensive evaluation of a specific treatment which can be useful for procedures' standardization and reliable comparisons between different centers. This tool is gaining growing interest and widespread importance in many different fields. Considering liver surgery, however, an agreement was reached, and TO assessment was evaluated on a large scale only concerning liver resection. This study aimed to investigate the first TO for laparoscopic microwave ablation for hepatocellular carcinoma. Furthermore, the current study investigated the expendability of this tool for prognostic purposes.Abstract:In the context of spreading interest in textbook outcome (TO) evaluation in different fields, we aimed to investigate an uncharted procedure, that is, laparoscopic microwave ablation (MWA) for hepatocellular carcinoma (HCC). Absence of post-MWA complications, a hospital stay of three days, no mortality nor readmission within 30 days, and complete response of the target lesion at post-MWA CT scan defined TO achievement. Patients treated between January 2014 and March 2021 were retrospectively reviewed, and of the 521 patients eligible for the study, 337 (64.7%) fulfilled all the quality indicators to achieve the TO. The absence of complications was the main limiting factor for accomplishing TO. At multivariable analysis, Child-Pugh B cirrhosis, age of more than 70 years old, three nodules, and MELD score ≥ 15 were associated with decreased probabilities of TO achievement. A score based on these factors was derived from multivariable analysis, and patients were divided into three risk groups for TO achievement. At survival analysis, overall survival (OS) was significantly (p = 0.001) higher in patients who achieved TO than those who did not. Moreover, OS evaluation in the three risk groups showed a trend coherent with TO achievement probability. The present study, having assessed the first TO for laparoscopic MWA for HCC, encourages further broader consensus on its definition and, on its basis, on the development of clinically relevant tools for managing treatment allocation. | KeywordsHepatocellular carcinoma · Radiofrequency ablation · Overall survival · Two-dimensional supersonic shear-wave elastography · Liver stiffness measurement Abstract Purpose: To evaluate the prognostic value of liver stiffness (LS) measured using two-dimensional (2D) shear-wave elastography (SWE) in patients with hepatocellular carcinoma (HCC) treated by radiofrequency ablation (RFA). Methods: The Institutional Review Board approved this retrospective study and informed consent was obtained from all patients. A total of 134 patients with up to 3 HCCs ≤5 cm who had undergone pre-procedural 2D-SWE prior to RFA treatment between January 2012 and December 2013 were enrolled. LS values were measured using real-time 2D-SWE before RFA on the procedural day. After a mean follow-up of 33.8 ± 9.9 months, we analyzed the overall survival after RFA using the Kaplan-Meier method and Cox proportional hazard regression model. The optimal cutoff LS value to predict overall survival was determined using the minimal p value approach. Results: During the follow-up period, 22 patients died, and the estimated 1-and 3-year overall survival rates were 96.4 and 85.8%, respectively. LS measured by 2D-SWE was found to be a significant predictive factor for overall survival after RFA of HCCs, as was the presence of extrahepatic metastases. As for the optimal cutoff LS value for the prediction of overall survival, it was determined to be 13.3 kPa. In our study, 71 patients had LS values ≥13.3 kPa, and the estimated 3-year overall survival was 76.8% compared to 96.3% in 63 patients with LS values <13.3 kPa. This difference was statistically significant (hazard ratio = 4.30 [1.26-14.7]; p = 0.020). Conclusion: LS values measured by 2D-SWE was a significant predictive factor for overall survival after RFA for HCC.Excluded due to technical failure of liver stiffness value measurement by using Supersonic shear-wave elastography (n = 3) Immediate follow-up loss after RFA (n = 3) Undergoing liver transplantation after RFA during follow-up (n = 11) 134 patients with fewer than 3 HCC nodules, and Supersonic shear-wave elastography before RFA Liver Cancer 2018;7:65-75 Recurrence Outcome after RFA Of the 130 patients with HCCs in whom treatment success was achieved after RFA, LTP developed in 15 patients (15/130, 11.5%). For the treatment of LTP, 5 patients underwent repeated RFA, 2 patients underwent percutaneous ethanol injection, 6 patients underwent TACE, and the remaining 2 patients underwent hepatic resection. Of the 134 patients with HCCs treated by RFA, 79 patients (79/134, 59.0%) experienced IDR during follow-up and initial IDR was treated with the following modalities: percutaneous ethanol injection (n = 8); RFA (n = 17); TACE (n = 53); and best supportive care (n = 1). During follow-up, EM developed in 16 out of 134 patients with HCCs (16/134, 11.9%). The location of initial EM was: peritoneal seeding (n = 2), lymph node (n = 8), lung (n = 5), and adrenal gland (n = 1). Of these 16 patients, 9 were treated with systemic chemotherapy, 3 with radiation therapy, 3 with best supportive care, and 1 with surgery.The estimated 1-and 3-year recurrence-free survival rates after RFA in the 134 patients with 161 HCCs were 63.4 and 37.6%, respectively. The predictive factors for recurrence-free survival are summarized inTable 3. The development of LTP and previous treatment history for HCC were significant predictors of recurrence-free survival. | Citation: Hur, M.H.; Lee, J.-H.; Kim, J.Y.; Hong, J.H.; Park, M.K.; Cho, H.J.; Choi, N.R.; Kim, J.; Kim, M.A.; Nam, J.Y.; et al. Comparison of Overall Survival between Surgical Resection and Radiofrequency Ablation for Hepatitis B-Related Hepatocellular Carcinoma. Cancers 2021, 13, 6009. https://doi.org/10.3390/ cancers13236009 Academic Editor: Dan G. DudaSimple Summary: The effectiveness of surgical resection and radiofrequency ablation in early hepatocellular carcinoma is still controversial because previous studies show conflicting results. In addition, previous studies did not consider the antiviral treatment-related factors, even though there is now robust evidence that antiviral therapy is crucial for determining the prognosis of patients with chronic hepatitis B-related liver cancer. After adjusting for the antiviral treatment, we demonstrated that radiofrequency ablation may provide comparable overall survival to resection in the treatment of very early or early hepatocellular carcinoma, although recurrence-free survival is marginally shorter than in the resection group.Abstract: It remains controversial whether surgical resection, compared to radiofrequency ablation (RFA), improves overall survival (OS) in patients with early hepatocellular carcinoma (HCC). This study aimed to compare OS after RFA with that after resection for HCC. This retrospective study included patients who underwent RFA or surgical resection as initial treatment for hepatitis B virus (HBV)-related HCC at a very early or early stage. A total of 761 patients (RFA, n = 194; resection, n = 567) from Seoul National University Hospital (Seoul, South Korea) and 1277 patients (RFA, n = 352; resection, n = 925) from the Korean Primary Liver Cancer Registry were included in the hospital and nationwide cohorts, respectively. Primary and secondary endpoints were OS and recurrence-free survival (RFS), respectively. Additional analysis was performed when the history of the antiviral treatment and the type of prescribed nucleos(t)ide analogue were confirmed. The rate of complications was compared between the two treatment groups in the hospital cohort. Baseline characteristics were balanced, using inverse probability of treatment weighting (IPTW). In the hospital cohort, the RFA group had a smaller mean tumor size (1.7 vs. 3.9 cm) but a higher proportion of cirrhotic patients than the resection group (85.6% vs. 63.1%) (both p < 0.01). During 81.0 (interquartile range, 62.3-107.1) months of follow-up, there was no difference in OS (adjusted hazard ratio (aHR) = 0.870, 95% confidence interval (CI) = 0.400-1.897, p = 0.73) and RFA was associated with shorter RFS (aHR = 1.562, 95% CI = 1.099-2.219, p = 0.01) after employing IPTW. Antiviral treatment was independently associated with longer OS (aHR = 0.444, 95% CI = 0.251-0.786, p = 0.01) as well as RFS (aHR = 0.544, 95% CI = 0.391-0.757, p < 0.01) in the hospital cohort. In the nationwide cohort, there was no difference in OS (aHR = 0.981, 95% CI = 0.661-1.456, p = 0.92) between the Cancers 2021, 13, 6009. https://doi.org/10.3390/cancers13236009 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 6009 2 of 15two treatment groups when adjusted for antiviral treatment, which was a negative independent risk factor for mortality (aHR = 0.655, 95% CI = 0.451-0.952, p = 0.03) after IPTW. Among patients treated with tenofovir (n = 96) or entecavir (n = 184) in the hospital cohort, there was no difference in either OS (aHR = 0.522, 95% CI = 0.058-4.724, p = 0.56) or RFS (aHR = 1.116, 95% CI = 0.738-1.688, p = 0.60). The overall incidence of complications was higher in the resection group (26.3%) than in the RFA group (13.9%) (p < 0.01). RFA may provide comparable OS to resection in the treatment of very early or early HCC with a lower rate of complications, although RFS is marginally shorter than in the resection group after adjusting for antiviral treatment. Regardless of the type of NA, antiviral treatment in patients with HBV-related HCC is strongly associated with both OS and RFS. |
417 | why is the holding brake on la vu not working anymore | If I move the caliper to center on the wheel, it would stay (before the video) but when I pulled the lever, the right break would touch the rim, the left break would touch the rim. But when i release, only the left brake releases.
I've tried messing with the cabling tension and it doesn't help.
I also tried messing with the nut shown in the video and if I tighten it, it just locks the caliper so neither brake moves.
Is the spring tension the problem? I'm going a bit crazy over here.
Bicycle break problem | Brakes frequently will not release. Have to get a screw driver to release even if careful when locking. | 89 Chevy Pickup Brake indicator stays on? | This morning, on my way home from work, I had to brake pretty suddenly to avoid hitting a deer and now I have no pressure in the pedal.
I checked to make sure there was fluid, and the reservoir is full.
What went wrong and how can I fix it? | Does the ps3 brake? | What would cause brake pedal to stick on floor? | Long story short, I was bleeding my rear brake and the tube I was using popped off the bleeder. Like an idiot, I tried to grab it as it was falling with my hand that was holding the lever and let air into the system. The problem is that now I can't seem to get any rear brake pressure at all, no matter how much I pump the brakes (I pumped them for about 5 minutes straight). I tried to use a vacuum brake bleeder (hooked up to a compressor at 90 PSI), but when I do very little fluid comes out and I still have no brake pressure. Should I just keep trying to use the vacuum bleeder and hope that will eventually work? I've tried for about 2 hours and nothing has changed. | The brakes on my 05 lancer are fine until just after 60 mph, after this point the brakes feel hard to the touch and takes more effort to draw the car to a stop. The car is fairly new to me and the previous owner said that is sat for a month without being driven. has anyone experienced this before and what shoud I do | 417 | Six Flags Great America holding brake on V has not been used since September 2008 due to maintenance issues. On Déjà Vu, the riders were pulled backwards up a vertical tower and dropped through the station and into a cobra roll inversion, followed by a loop over the station and up another vertical tower. After being pulled up a bit more, the ride then repeated the course in reverse. The ride did not debut until October 7 that year due to mechanical and design issues, causing a public relations nightmare for Six Flags, including being threatened with lawsuits regarding false advertisement of the opening | how long does it take six flags great america to change the wheels | where is mardi gras in six flags great america | when did six flags re-open in new england | when did two face flip side open at six flags america | when was goliath built at six flags great america | when did the super 6 air on tv | how many flying dutchmans are there at six flags america | where was scream at six flags new england built |
418 | Seasonal variation, chemical composition and biological activity of the essential oil of Cordia verbenacea DC (Boraginaceae) and the sabinene | Abstract The chemical composition of the essential oils obtained from the fresh leaves of Cordia globosa (Jacq.) H.B.K., at different ontogenetic stages, were analyzed by GC and GC/MS. Twenty-three and 26 volatile constituents were identifed for the oils at the fowering and fructifcation stages, respectively. In both oils, the main compounds were bicyclogermacrene (22.7%-13.1%) and β-caryophyllene (11.9%-11.6%). | AbstractThe essential oils from wild Rhododendron tomentosum stems, leaves, and flowers in bloom and non-bloom periods from Northeast China were hydrodistilled and the chemical components were analyzed by gas chromatography and Gas chromatography-Mass spectrometry. A total of 52 constituents, accounting for 88.16-98.88 % of the oil yield, were identified from stem, leaf, and flower essential oils in bloom period; and 48 components which accounted for 94.40-98.21 % of the oil yield were found from stem and leaf essential oils in non-bloom period. The essential oils from different part and growth period revealed big differences both in composition and relative content. Predominant constituents in all oils were 4-thujene, 5-(1-methylethyl)- bicyclo[3.1.0]hex-3-en-2-one, α-thujenal and (-)-4-terpineol. The oils from stems, leaves, and flowers in bloom period were found to be the richest in (-)-4-terpineol (14.77 %), 4-thujene (27.49 %) and 4-thujene (49.51 %), respectively; oils from stems and leaves in non-b... | AbstractThe present study reports the variability of the chemical composition and the antioxidant properties of essential oils from an endemic species of North Africa “Pituranthos chloranthus”, col... | AbstractThe essential oils compositions from different parts of Pituranthos tortuosus (Coss.) Maire collected at the flowering and the fruiting stages were investigated. The essential oils were ext... | Infraspecific chemical variability in the essential oils of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) | Chemical composition and biological activities of extracts and essential oil of Boswellia dalzielii leaves | The essential oil composition of selected Hemerocallis cultivars and their biological activity | Chemical Composition of the Leaf Oil ofMentha rotundifolia(L.) from Algeria | 418 | Abstract Cordia verbenacea DC (Boraginaceae), popularly known as “Erva Baleeira,” has been widely studied with respect to its chemical and pharmacological properties, and its anti-inflammatory, analgesic and anti-ulcerogenic activities have been demonstrated. The objective of this study was to determine the essential oil content with regard to time of collection of leaves and to evaluate the antibacterial activity and antibiotic-modifying activity of the oil, sabinene and sabinene hydrate against multidrug-resistant strains. GC/MS analysis showed that in samples of the essential oil obtained at different times over one year, there was qualitative and quantitative variation in chemical composition, with statistical significance (p | Chemical Composition and Larvicidal Activity of the Essential Oil From Leaves of Cordia globosa (Jacq.) H.B.K. from Northeastern Brazil | Antimicrobial activity of essential oil of Cordia globosa | Antioxidant Activity of the Essential Oils of Different Parts ofJuniperus communis. subsp.hemisphaerica. andJuniperus oblonga. | Essential oil composition of Choisya ternata Kunth (Rutaceae) leaves | Houttuynia cordata Thunb is an important medicinal plant widely distributed in East Asia. The collected information is an attempt to cover recent developments in the pharmacology, phytochemistry and quality control of this species. During the past several decades, the medicinally important phyto-constituents have been identified including essential oil, flavonoids and other polyphenols, fatty acids and alkaloids. A survey of the literatures shows H. cordata possesses a variety of pharmacological activities including antiviral, antitumor, antimicrobial, anti-inflammatory, and antioxidative effects. Little attempt has been done to review the techniques used for its quality control. Future efforts should concentrate more on in vitro, in vivo studies and clinical trials in order to confirm traditional wisdom in the light of a rational phytotherapy. The information summarized here is intended to serve as a reference tool to practitioners in the fields of ethnopharmacology and natural products chemistry. | Comparison of different extracts leaf of Brassica juncea Linn on wound healing activity | Background:The genus Lippia comprises 150 species, most of which have interesting medicinal properties. Lippia sidoides (syn. L. origanoides) exhibits strong antimicrobial activity and is included in the phytotherapy program implemented by the Brazilian Ministry of Health. Since species of Lippia are morphologically very similar, conventional taxonomic methods are sometimes insufficient for the unambiguous identification of plant material that is required for the production of certified phytomedicines. Therefore, genetic and chemical analysis with chemotype identification will contribute to a better characterization of Lippia species.Methods: Amplified Length Polymorphism and Internal Transcribed Spacer molecular markers were applied to determine the plants' genetic variability, and the chemical variability of Lippia spp. was determined by essential oil composition.Results: Amplified Length Polymorphism markers were efficient in demonstrating the intra and inter-specific genetic variability of the genus and in separating the species L. alba, L. lupulina and L. origanoides into distinct groups. Phylogenetic analysis using Amplified Length Polymorphism and markers produced similar results and confirmed that L. alba and L. lupulina shared a common ancestor that differ from L. origanoides. Carvacrol, endo-fenchol and thymol were the most relevant chemical descriptors.Conclusion:Based on the phylogenetic analysis it is proposed that L. grata should be grouped within L. origanoides due to its significant genetic similarity. Although Amplified Length Polymorphism and Internal Transcribed Spacer markers enabled the differentiation of individuals, the genotype selection for the production of certified phytomedicines must also consider the chemotype classification that reflects their real medicinal properties. | The majority of essential oils obtained from vascular plants have been demonstrated to be effective in treating fungal and bacterial infections. Among others, Salvia hydrangea is an endemic half-shrubbelonging to the Lamiaceae family that has been widely used from ancient times in Iranian traditional medicine. The aim of this study was to compare the composition and antimicrobial properties of essential oils obtained from leaves or flowers of this plant, collected from the Daran region of Iran during June 2018. The oils were obtained using Clevenger apparatus, their composition was evaluated by means of gas chromatography/mass spectrometry (Gc/MS) and the antimicrobial properties were assayed by measuring inhibition halos, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The yield of leaf oil was ~ 0.25% and that of flower oil was ~ 0.28%. Oil composition was affected by the part of the plants used: the most abundant bioactives contained in leaf essential oil were (+)-spathulenol (16.07%), 1,8-cineole (13.96%), trans-caryophyllene (9.58%), β-pinene (8.91%) and β-eudesmol (5.33%) and those in flower essential oil were caryophyllene oxide (35.47%), 1,8-cineole (9.54%), trans-caryophyllene (6.36%), β-eudesmol (4.11%), caryophyllenol-II (3.46%) and camphor (3.33%). Both the oils showed a significant inhibitory and lethal effect on the Gram-negative bacteria Pseudomonas aeruginosa (MIC ~ 16 µg/mL), Shigella dysenteriae and Klebsiella pneumoniae (MIC ~ 62 µg/mL). Therefore, the essential oils obtained from both leaves and flowers of S. hydrangea may have potential application as bactericidal agents against some bacteria.Essential oils are mainly composed of aromatic and volatile compounds and can be obtained from different parts of plants, especially the leaves and flowers 1 . Indeed, in plants, they are either secreted directly from the protoplasm by the degradation of cell membrane and resin materials or by the hydrolysis of some glycosides 2 . In particular, glycosides produced by different species of plants and stored in different organs have a strict relationship with biosynthesis, metabolism and biological activity and are mostly affected by the environmental climatic conditions 3 . Essential oils are usually rich in terpenes, sesquiterpenes, esters, aldehydes, phenols, ethers and peroxides 3,4 ; they are mostly colorless or yellowish, less dense than water and soluble in organic solvents 5 . They are widely used in various industries, including the food and cosmetic industries among others 6 . Moreover, since ancient times, they have been widely used for the treatment of different disorders thanks to their well-known antioxidant, antimicrobial and antifungal properties 7-9 . Currently, essential oils have been proposed and tested in alternative medicine, especially as antimicrobial and antifungal products. Indeed, there is increased demand for safe and effective plant-derived bioactives as an alternative to antimicrobial synthetic drugs because their widespread and continuous use has led to the modification of microbes that have become resistant, thus reducing the therapeutic effect of these drugs 10 . Essential oils derived from plants have demonstrated promising antimicrobial therapeutic effects, which are generally accompanied by reduced side effects 11 . They have been screened and used in pharmacology, herbal pharmacology, medical microbiology and phytopathology 12 especially because of open Scientific RepoRtS | (2020) 10:15647 | https://doi.org/10.1038/s41598-020-73193-y www.nature.com/scientificreports/ their known insecticidal, antifungal, anti-parasitic, antibacterial, antiviral, antioxidant and cytotoxic properties 13 . These activities are related to the lipophilic nature of the hydrocarbon skeleton and of the functional groups of the bioactives. Indeed, the hydrophobic properties of the bioactives facilitate their interaction with bacteria and entrance into the cell, where they can exert their therapeutic effect. Rod-shaped cells and Gram-positive bacteria seem to be more sensitive than Gram-negative bacteria to these bioactives 5,14 . Lamiaceae Martinov is one of the largest plant families in the world (~ 252 genera and 6700 taxa) and has the main differentiation center in the Mediterranean and Irano-Turanian biogeographic regions[15][16][17][18]. The majority of Lamiaceae produce terpenes and a wide variety of other compounds, which are mainly stored in the epidermal glands of leaves, stems and reproductive organs 19 .One of the most important genera of Lamiaceae is Salvia L. with about 900 species worldwide and more than 70 species in Iran, 17 of which are endemic and exclusive to Iran 20,21 . Salvia, the name of which is derived from the word salwar which means healer, has been traditionally used as an anti-toxin and a restorative, aimed at strengthening the health and extending the longevity of both humans and soul 22 . The essential oils of Salvia species contain various bioactives such as terpenoids, steroids, flavonoids and polyphenols among others 23 and their concentration varies as a function of the part of the plant used 24 . Indeed, many Salvia species and their essential oils are commonly used in pharmaceutical and cosmetic products or used as additives for foods (seasonings and flavors)25,26.Previous studies reported that the main components contained in the essential oils from S. hydrangea DC. ex Benth. depend on the part of the plant used and the collection zone. Caryophyllene oxide and β-caryophyllene 10,27 ; and β-caryophyllene, 1,8-cineole, α-pinene and caryophyllene oxide 28 have been identified by different authors. Naphthalene, 1,8-cineole, camphor and α-terpineol are the main bioactives contained in plants at 2000 m above sea level, while 1,8-cineole, camphor, β-pinene, naphthalene and α-amorphene are those contained in plants at 1100 m above sea level 29 . Camphor, α-humulene 30 , α-pinene, 1,8-cineole, trans-caryophyllene and camphene 31 have been found in the essential oil from aerial parts of S. hydrangea and 1,8-cineole, caryophyllene oxide, α-pinene and β-pinene 32 are the major compounds detected in oil obtained only from the leaves of this plant.The oil obtained from S. hydrangea flowers shows an in vitro anti-malarial effect due to the presence of high levels of pentacyclic triterpenes (mainly oleanic acid) that inhibit the growth of the malaria pathogen 33 . The essential oil from the aerial parts of S. hydrangea is effective against different bacteria 10,28,30 .The present study aimed to investigate essential oil from both leaves and flowers of Iranian S. hydrangea. To this purpose, the chemical composition of oils has been determined and compared. Moreover, the variations in yield and antimicrobial activity as a function of the composition have been evaluated. |
419 | what are the symbols in a spider diagram | Where can you find spiders emblem in spiderman 3? | Symbol representing change? | Spider Lilies (film) decided to get the same tattoo, in the hope that it would help her brother's recovery. Nevertheless, Takeko finds herself drawn to Jade, and begins designing a new tattoo for her. Meanwhile, a young police officer is trying to ambush Jade and the rest of the girls working in the same website. However, he takes to speaking to her, listening to her childhood stories and connecting with her, thereby slowing down the investigation he is supposed to be working on. Eventually he falls in love with her, trying to tell her to get out before it's too late and before | Excuse me, complete noob at this crack the clue. But just thinking on the simplest levels, I was wondering if the arranged thetas might line up with woodcutting icons on the map. These icons are what I'm referring to. They just look an awful lot like a nature rune to me. | I have no idea what this symbol means, but its on some quests and not on others. When you go to select a hub quest, sometimes to the left of where it shows the monster drawing (on the right of the screen) there's a small symbol with a monster and a ? Over it. | My friend pointed out to me 2 Apothicon symbols that you can see from 2 of the dragon platforms. One to the left of the Dragon Command above the table and chairs. Another from the Tank Factory Platform to the right on the Giant Robots arm. The Symbols seem to disappear with an audio cue when you hit it with the fire from the Guard of Fanfir. Richtofen also said "You've made a grave mistake" after hitting them both. not sure if there is a third one.... Help?? | What are the symbols for love? | What does the symbol of a triangle inside a circle mean? | 419 | Spider diagram In mathematics, a unitary spider diagram adds existential points to an Euler or a Venn diagram. The points indicate the existence of an attribute described by the intersection of contours in the Euler diagram. These points may be joined together forming a shape like a spider. Joined points represent an "or" condition, also known as a logical disjunction. A spider diagram is a boolean expression involving unitary spider diagrams and the logical symbols formula_1. For example, it may consist of the conjunction of two spider diagrams, the disjunction of two spider diagrams, or the negation of a spider | In logic, there are various normal forms for formulae; for example, disjunctive and conjunctive normal form for formulae of propositional logic or prenex normal form for formulae of predicate logic. There are algorithms for `reducing' a given formula to a semantically equivalent formula in normal form. Normal forms are used in a variety of contexts including proofs of completeness, automated theorem proving, logic programming etc. In this paper, we develop a normal form for unitary Euler diagrams with shading. We give an algorithm for reducing a given Euler diagram to a semantically equivalent diagram in normal form and hence a decision procedure for determining whether two Euler diagrams are semantically equivalent. Potential applications of the normal form include clutter reduction and automated theorem proving in systems based on Euler diagrams. | Linear diagrams for syllogisms (with relationals) | Material Spider Diagram Link? | who is in the middle of the trinity diagram | On the basis of the combinational representation of spatial topological relations, the paper shows a further-perfected method for combinational reasoning with basic spatial topological relations. In addition, a spatial topological relations reasoning table is given, and a complete diagram about spatial topological relations between a line and an area is listed. | the table that shows the functional values of logical expressions is called the | what is the name of the second-order logic in which only the vertex and vertex | a logical hexagon represents how many statements |
420 | I've been very pleased. Fits well | Was thrilled when it fitted perfectly, so are very happy with my purchase. | Fits Perfectly; Has a good feel to it; Arrived Promptly | Love it. Fits very well, looks very nice. Something to be proud of. Thank you. | very pleased.
arrived earlier than expected.
fit better than I had thought it would.
looks like a factory accessory. | Was exactly as advertised. Perfect fit and match. | This was a gift for a friend. He says it fits really well and feels great. Can't ask for much more. | Totally as expected, good quality and fits great. | I was so impressed by the fit. Has a great look. Looking forward to getting more. | 420 | I've been very pleased. Fits well. Holds quite a few credit cards and cash. Has survived several drops and falls. | I have been using it for 3 days now and really like it. It fits in my front pocket and ... | Really love this. Fits very nicely and has never fallen ... | It is an uncomftorable fit and the credit card is super visible and hangs off edge | Great case. Had this brand on my 5- dropped ... | Great case. slips on easily and provides a nice ... | It fits a passport and a few credit cards and ... | ... is my 3rd wallet case and by far the best. It has plenty of room for cash and ... | Fits perfect. I have dropped it a few times and ... |
421 | but they looked great the whole time | PRETTY THOUGH - NOT AS PRETTY AS I THOUGHT - JUST OKAY USUALLY LOVE 1928 THOUGH THEY WERE OKAY EH | They looked good initially, but the painting eroded in a month when I solely used hand wash. I truly grow a concern about the quality and safety. | Appearance wise after mounting they were great!. Installation and how they are mounted, Sub Par!!!! They could have done a better job of engineering how the mounting brackets were attached. Knowing what I know now, I would have gone another route. | Everybody loved the style, mom, dad and baby. They're a little difficult to put on but they served their purpose well for the short time they were needed. | The leather wore out. But otherwise was perfect. | Front office...get back to the "traditional" look the Rams used to have please, these "stadium design" unis are hot garbage. It was very disheartening every game to have to wait and cringe at every combination you guys came up with. LA's three other original sports teams have iconic uniforms still, meanwhile the Rams look like their wearing wet toilet paper by the end of the third quarter, from the most elementary ass logo to the bullshit ass "bone" white and "Best Buy" blue...WE LOOK LIKE CLOWNS. #theuniformssuckmajorass | They feel line a dream.. Walking on clouds ... Wish they looked better however.. | They came out terribly overexposed and washed out. Probably good for an art project. Not for general purpose keepsake pictures. | 421 | They ripped after about a year of wear, but they looked great the whole time. At the price I paid for them, I most definitely would reorder. | My first pair of black Wolford Tights were perfect and haven't ripped yet | I bought these from Walmart last season ripped in between ... | they are pretty but they ripped pretty easy | I like them and would consider buying them again | Looked good, but they ripped after 1 month of light use. Cheaply made | Ripped before I even got to wear them... | I would purchase again though and like them for the price | ... they still look brand new- I wasn't sure how great they'd hold up since they're not very expensive |
422 | Great price for something so wonderful | Great purchase. Just what I expected. Best price anywhere! | Great price. This is the greatest stuff. I have been using it for many years. It is a must in our house. | Great price and value! At a fraction of what Sears or any other dealer would charge. | Great price, arrived right away and they are so easy to care for. They are growing nicely. Oh, and did I mention at an excellent price?!! | it was in great condition, and at such a low price it really helped me with my AP literature summer assignment. | Great item and value per price. Item arrived on stated time. | Really good price the lightning deal I can see us coming very handy when I need it for a laptop or some electronic | Good price for a very hard to find toy. My grandson loves it and it was in good condition except for some scratches which I did not expect for a new toy. However, my grandson loves playing with this toy. | 422 | You can't go wrong with this crockpot. Great price for something so wonderful! | If you like using crockpot | The best crockpot we have owned | Nice crock pot and not having to figure out where ... | Great crockpot! I was thinking that it wouldn't be ... | The crock looks great on our counter | Decent crock pot, but watch out for the lid | Delicious Crock Pot Recipes | and works great as a crock pot should |
423 | Anyway to change the MAC of a Unifi AP? | In an interesting situation here, and I hope someone can point me in the right direction (or even *a* direction).
I picked up ten Aruba AP-105 access points intending to flash them to OpenWRT. The conversion process involved dumping the SPI flash chip of one of the units, overwriting the first 256K of the dumped image with U-Boot, flashing that modified ROM back, and then using U-Boot to install OpenWRT via TFTP. Easy peazy (after destroying one fo the APs and my favorite RPi3 in the process).
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Rather than go through that for each of the remaining 8 APs, and possibly more in the future if this test project pans out, I installed the packages I'll need and staged the first AP. After staging the first AP, I dumped its SPI flash chip again and then flashed that image directly to the remaining APs. What a timesaver!\*
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That worked; they all boot and function as expected. Except for one thing I overlooked when I tested them individually post-flash: they all have the same MAC addresses, which is the MAC of the first unit. Since these are identical, I foolishly only took a backup of the first unit's flash chip. It never occurred to me that the MAC addresses would be stored in that flash and not re-detected dynamically; I approached the entire project from a server admin perspective rather than an embedded systems perspective, and I take full responsibility for that.
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My current workaround is to set `option macaddr 'MAC from sticker'` when I bring up the interfaces. That works, but it's cludgy and prone to mistake by omission.
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Not sure if this is a red-herring or not, but I just reviewed the boot logs from console, and it looks like the MAC is cloned there as well. I can "setenv eth0=CORRECTMAC" but that only seems to to save it for that boot.
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Any ideas that don't involve disassembling these and re-flashing them?
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^(\*Terms and conditions apply, apparently.) | What's up guys,
I was lent an Apogee Duet 2 interface recently but the person who lent it to me does not have the original driver / installer for mac, which I need to properly use it.
I tried downloading it from the apogee website and using his registered email but they failed to send it even after 24 hours of waiting and re trying.
Have also googled for the driver file and haven't been successful.
Do any of you guys know where I could possibly find this driver file for mac os?
Thanks in advance! help is much appreciated
Seb | I'm going to be somewhere with no wifi and have to burn a CDRW with a Mac. Also, I'm assuming Macs will burn CDRW's? Sorry for the dumb questions. | The description on the Amazon site failed to mention it does not work with a Mac. It doesn't even say on the box that the setup disc only works with a PC. Get with the program Hawking! | After 3 years of hardware problems, I took my 2014 27" iMac in and was happy to hear Apple would swap it out for a current model, complete with a 3 year Apple Care renewal. The new unit is a i5 instead of an i7, but I was told it has better overall performance and graphics, so I'm happy about that.
What I hadn't appreciated was the replacement of the Thunderbolt ports on the back with USB C. I purchased 2 adapters, and with a bit of fiddling, got my external Dell 4K display working. I am having trouble with my favorite feature of the duo -- getting the MBA 11 working as a target disk.
On the 2014 iMac, I could mount the MBA with its FileVault-encrypted drive under Sierra by entering the encryption passphrase once. After that, I'd plug in the MBA and it would be recognized. Things don't seem to be working the same way under Sierra on the 2017 iMac. Is there anything extra I should be doing to get the MBA to be recognized? | Does anyone know if the computing commons or somewhere else on campus has Mac’s w/ fcpx? | Yes, it's me again.
I still have my uncle's old Power Mac G5.
It seems to have OPTICAL INPUTS on the back.
Which I assume I can hook right up to my damn ADATs.
=== === ===
Does this mean that all I need is ProTools 7.3 LE,
and I'll be able to do things with ProTools?
Will the Mac OS automatically know how to pull in eight tracks of audio from the OPTICAL INPUT ? | Have spent two days trying to get Mojave beta 10 to install on my my iMac. The 2TB drive has been partitioned the old HFS way was, where one 1TB is a Core Storage Logical volume (mixture of the 128SSD and the platter), the other is the other 1TB HFS platter. I remember experiencing difficulties in achieving this old school way of using the hard drive (I like to separate work from apps and system). I think I found a solution on the AppleStackExchange two years ago when installing Sierra and have lived happily with this till Mojave came along.
Now it seems, APFS is going to be the new norm, everytime I install Mojave, everything is fine until the restart after installing and restoring , the iMac hangs on boot with just a black screen and Apple logo. Irrespective of a erase into HFS for the main volume, Mojave will convert to APFS on install. I have tried as many different ways in the last two days to get Mojave installed and working. After further investigation it looks like it won’t let me start up the Mojave system volume because it’s not blessed -Startup disk tells me so - but all other checks suggest everything is fine. In the end I have resigned myself to going back to High Sierra.
Does anyone have any experience of this? Do I have to actually join the two partitions again to make one APFS volume and add containers within that to enable Mojave functionality?
Any help, info, or advice much appreciated. | 423 | My google-fu is failing me. Does anyone know how to change the MAC address of a Unifi WAP? | How do you find a mac adress? | Android 10 includes a feature to change the MAC address for each Wi-Fi | If I want to change resellers, does my old need to release my MAC address? | Need to find a MAC address | how to change microsoft office user name mac? | who makes the wap web browser for windows | WAF Loadbalancer VPN/Proxy checker | Why does someone change their MAC address? |
424 | My dogs loved these. Not much more to say then that | I'm sorry of indifferent about these but my dog sure seem to love them. Wish they would've Lasted longer, but apparently my dogs are beasts :-). | My dog loves these! Recommended by my vet! | dogs loved them and kept them busy for a few hours only Too expensive for a few hours of quiet time. | Dog went crazy over these! Even though they say for cats dogs LOVE them too! | My dog loves them and they can really take a beating | Our dogs love these.
One thing of note, when you get these for your animals and have more than a few, pay attention to how they place them when done. We find the dogs like to arrange them. | It was a gift for our daughters dog. I guess she liked them. | Bought them for my cat. My dogs love them. The cat, not so much. I reordered for the dogs. | 424 | My dogs loved these. Not much more to say then that. They like them which means I like them. I personally haven't tasted them though so I can't say how good they really are. Then again I'm sure they eat some of my food and think it might taste nasty too. So I guess it's a good thing they like these. | i can't say they taste good but my dog loves them | My dogs love these treats and I love the fact they are ... | These are flavored like food so the dogs will think of it as ... | My dogs love the way these treats taste | My dog and I enjoy them as a treat | my dog absolutely LOVES them-- probably because they smell like roadkill. | The dogs really enjoy them. | My dog LOVES these. Just wish she didn't consume ... |
425 | Do you think the dude will get $500,000 funding | I’m listening to a podcast about the potential for life of some kind on Venus (due to the recent detection of phosphene gas). The scientists are calling for an investment in comprehensive missions to Venus at a cost of $billions. What’s the return on that type of monetary investment? | Tucker Carlson says something that makes sense for once.
Commentators come close to making the connection that maybe the government shouldn't be handing out money to any rich people, not just universities. Will they finish the leap?!? | I would say yes, because Trump is a conservative, and conservatives believe in hard money like gold money and bitcoin/digital gold. I don’t think Trump likes fiat money. | Westpac to raise $500m | In light of the recent headline about the Koch brothers spending $900 million on the upcoming elections, how could that money better help Americans? | The problem is actually Kjaer. Kjaer wants 4 million after tax. He currently gets 4 million before tax (about 2.5m) in Spain. If we solve this issue, we will likely loan Kjaer.
Link to tweet: | Barry Diller political causes. As of January 2018, Diller's estimated net worth was $3.3 billion. Diller is an outspoken critic of President Donald Trump. In 2011, the Diller-von Furstenberg Family Foundation announced a donation of $20 million to support the completion of the High Line park project in Manhattan. In 2012, Diller donated $30 million to the Hollywood Fund, which provides health and social care to retired individuals from the show-business world. In 2015, Diller and his wife committed to donate $113 million toward a floating public park and performance space on a pier in the Hudson River, New York. It is | Do mike Tyson still have his money? | 425 | Just read from a blog ( ) that a guy is planning to make iron man armor suit and he needs $500000 funding. Do you think he will get anything close? | what is the name of the defense contractor in iron man | Looking to make an iron man (finally) | who makes iron man hero squad figures | how much money did iron man 2 make | [Suggestion] Let Ironpeople upgrade their Ironperson armor! | Do you think Capcom will ever make use of Alex Wesker? Would you like to see it? | The future of Iron Man in the MCU? Is he still set for the Guardians film? | What's the current state of Iron Man? |
426 | Pack is awsome!! Only reason why I have ... | Great pack. Quality and performance exceeds expectations. You cannot go wrong with this. | Awesome pack! Video review: https://www.youtube.com/watch?v=LFrOv03XvuI | This pack has great items in an inexpensive price which is always a plus. The students at my child's school loved them. Which made me very happy and my son was the star of the day. Great product!! | This pack is awesome! Looks great. Holds all my gear. I get compliments on it every time I go camping. | Love the variety pack. I order these to my office and keep them at my desk at work for a snack. :) | One of the most versatile packs I have ever used. This pack provides great comfort and the key feature for me was the ability to access the inside of the pack with out taking it off you waist. Just take your arms out of the shoulder straps and spin it around to access the inside. | I love having everything in one pack like this. The combination is just what I would want, as a middle aged woman. | best pack in the business. If you have been on the fence as to whether or not you should get one, just hit add to cart. Totally worth it. Make sure you buy the bottle front pouch for added storage. | 426 | Pack is awsome!! Only reason why I have given a 4 star rating is because the hydration tube is flat earth, would be way nicer if it were black to match the pack. | ... give 3 stars because this is the 1st pack like this I've ever bought | ... a 4 star really as build quality is very good. A sturdy shoe with bright colours | I give it a 4 star because its not pure ... | This scarf was beautiful but four stars only because it had a very ... | 4.5 stars - great size water bottle | This is the second such pack I've owned and have not been disappointed yet! | Only four stars bc although I love it, many people I've showed it to are ... | Garmin Forerunner 10 Review - Four Stars |
427 | Scalping Lawns That Never Go Dormant | Does something like this exist? I have two acres in east San Diego county and the entire two acres gets covered with weeds every spring that grow like 2-3 ft tall if I don’t mow them. In the summer, the ground turns into a bunch of dirt basically because everything dies. I was wondering if I can put down seeds of some sort of grass that will outcompete the weeds but maybe only grow like a foot tall. Then in the summer it will die but come back in the spring and out compete the weeds again? Ideally this would be my zero maintenance “lawn”. I don’t care about having a nice green lawn all year. I just don’t want to mow weeds several times a year to prevent them from getting 2-3 ft tall.
Someone mentioned Zoysia grass. But there are many types from the quick research I’ve done. | Recently bought a house in Northwest Arkansas (zone 6b). When I bought last August, grass was in good shape. Went dormant over the winter and it's starting to come back back these flowers have popped up all over my yard. What are they and what's the best way to get rid of them? Moved here from Southern California so I'm not familiar with them and how to control them.
flowers
flowers2 | Remarkable From the street looks like my dead lawwn grew overnight. Lawn lift will be used on all my lawns in this draught | Hi All,
I'm in Austin TX and my front yard needs a restoration. About 1/3 of it is under a tree and is actually quite healthy St Augustine. The other 2/3 is full sun and is 110% weeds. Stickers, thistle, those weeds that shoot 3' tall seed heads, everything but grass.
I've decided I'm going to fertilize and put pre-emergent on my healthy-ish St Augustine grass for the fall/winter, but I want to completely nuke the rest and fix the soil underneath this winter before re-sodding in the spring.
Does this sound like a solid plan?
1) Right now, scalp the weed-side of the lawn as low as I can with the mower and scrape it up and discard. Spray Glysophate all over and water every other day for the next week or two.
2) Spray again with Glysophate as weeds reappear, keep pulling/killing anything that appears. This should be done by end of September
4) Over the course of the fall/winter, I need to repair parts of the yard. There is a big rotted out hole in the middle where a tree was removed at one point. I will fix all of these potholes and low points, amending the soil best I can
5) Install a sprinkler system (should be easier now that it's just dirt, even if it is hard winter ground)
6) Once spring is coming (April/May), plant St Augustine sod and go from there
In the meantime, I'll be maintaining the 1/3 of the yard separately and hope they mesh back together happily once all is done.
Does this sound like a plausible plan? When is the best time to nuke the yard and kill everything? I read somewhere that if I spray poison once the yard is dormant it won't actually do anything, so I'm assuming I should spray now and deal with having a dirt landscape all winter.
I can't really afford sod until next year, would it make more sense to scalp now and do all the repair and sprinkler installation now, then spray poison to kill/re-sod next year as the lawn returns to life?
Thanks people! | For some context, it's his yard, and this was done because we just moved to PA from Florida. I'm 23 and have the back of an 80 y/o bull rider. That is, to say, I'm disabled, that's why I still live with my parents (always feel like I need to explain that). Anyway, the tree was a black walnut with about a 2 ft diameter surrounded by buried rocks that were also up to 2 ft in diameter. You couldnt stick a shovel in the ground more than 4" without hitting a rock.
By the time he was finished, what was soft, lush, green grass is now a brown clay wasteland.
So, is there a way to uncover the grass beneath the clay (which I assume there isnt) or is the only remaining option just to find out what type of grass it is and replant? | Leaves the lawn real ugly. About four weeks later lawn starts to look better than before. | Had my Pilea for something over two years now, and it surprised me by flowering for the first time a few months ago. The buds are now starting to fall off when I move the plant. What are you supposed to do with the stalks once all the flowers have died? Should I prune them off or just leave them to do their own thing?
Thank you, Pilea pals :-)
| Hello All,
I’ve got quite the problem. Through the end of last year, my lawn was looking great. Now, a large patch in the front looks pretty much dead. Lawn
The first picture is what it currently looks like, and the second is from last year around September. I know it’s a bit early in the season, but my neighbors all have pretty green and nice looking lawns.
As the title states, the lawn is pretty much all St Augustine, and I live in San Antonio.
Is there anything I can do? Or have I killed it?
Thanks! | 427 | I also have St. Augustine and bahia grass, but my main concern is for Bermuda and zoysia grass because they grow in a way that usually necessitates scalping during the spring green-up. However, there is no dormancy or spring green-up here in South Florida if the lawn is irrigated during the winter drought.
Scalping is necessary to lower the HOC, and it’s suggested to scalp prior to aerating and top dressing. Would it be a good idea to scalp anyways while everything is green, or should I force my lawn into dormancy (next year) by letting it dry out?
If I can scalp without spring green-up, when should it be done? The drought and cold weather around here is only in the month of January, so the start of February is when I apply the first round of pre-emergent. I plan on applying another round in May to control spurge. My last round would be in early November, after the heat and rain have eased up. | when do the azaleas bloom in wayah bald | Been picking scalp for 13 years, finally addressing my habit and I feel incredibly free. | Should I still take treatments if my hairloss isn't advancing? | Last Green Lawn In The Fall & First Green Lawn In The Spring | [Help!] Dry curly hair that loves falling off. My wedding is in january | Womens hairdressers in Norwich | how long before i can wash my hair after yuko straightening? | do i need to use clarifying shampoo before keratin? |
428 | Method and device for monitoring text editing in real time | What appears automatically and contains commands to change the appreance of text? | We present the design and evaluation of a gesture-based interaction technique for text editing on mobile devices equipped with touchscreens. The technique includes both taps and gestures to be drawn on the top of the soft keyboard to perform the basic editing actions: moving the cursor, selecting text, and using the clipboard. The technique is compatible with gesture writing and is suitable for bi-manual text entry. We carried out a user study including several practical tasks to evaluate it. Results showed a gain in performance for small font with respect to the technique currently available on most mobile devices with Android OS. | PROBLEM TO BE SOLVED: To provide a method for interactively correcting a text and for providing user guidance in a word processor and the other application program. SOLUTION: In the method, the monitoring of user input is contained and a previously decided event is identified. The previously decided event in a rule base 58 is scheduled for evaluation in response to the identification of the previously decided event. The evaluation of the event generates a subordinate event and a scheduled rule. The evaluation of the rule contains decision whether a condition on the rule is satisfied or not. In the case of approval, the other event or rule is scheduled in accordance with the rule, or an operation for automatically corrected text is started, or information useful for the user is displayed. | Feature that changes the selected text when the pointer? |
Introduction
Since the diffusion of smart phones and tablets, we often read and edit text with these touch screen devices for email, blog, SNS and so on. Most of the touch screen device provides virtual keyboard for inputting text. However, due to the lack of the physical tactile feedback, the inputting text with the virtual keyboard afford insufficient usability rather than physical keyboard. In order to reduce the amount of typing and burdens of inputting, we usually make full use of copy-and-paste technique especially for making quotation and reusing text during the editing.
Conventional copy-and-paste technique for touch screen devices utilizes region handles to specify text snippet. For example, Apple iOS and Google AndroidOS provide region handles when the user taps and holds him/her finger on a word (see Fig. 1). In both tablet OSs, the initial selected region is decided where the place of the tap-and-hold. If the place is on a "word," the initial selection becomes the word. After the initial selection, region handles appear on the screen. The user can move the region handles for further selection. After the moving, the user press a "copy" or a "cut" button to keep the region in the clipboard.
In iOS, the user can precisely select the region by seeing the magnifying glass. The magnifying glass is an effective solution of fat-finger problem 1 . However, in the both tablet OSs, the minimum unit of the region handle movement is still a "character" while the further selection task. The "character-based selection" may decrease the efficiency of the text selection because it requires precise and careful control of the handles by fingers. If the tablet OSs provide a different minimum unit of the text selection by considering the context of the text, the usability of the text selection task can be improved.
We propose a context-sensitive text-selection method for the tablet OSs. In this paper, we mainly focus on a "word" as a context of the text. The "word" is a fundamental unit of a sentence, and it can be acceptable in various cases and situations of the text-selection task.
Context-sensitive text-selection and its application to Tablet OSs
In this section, we describe the concept of the context-sensitive text-selection, and word snapping method as an instance.
Context-sensitive text-selection
The concept of the context-sensitive cut-copy-paste technique was presented by Wallace et al. in terms of programming 2 . In their research, the editor recognizes the context of the source code, and enables the programmers to select a possible source code block (region) by simple repetitive click operations. Their research aimed to reduce the burden as well as the errors caused by the cut-copy-paste operations on the source code. Kerr and Stuerzlinger enhanced the approach to automatically fix the error caused by the difference of context when pasted 3 .
The direction of enhancing cut-copy-paste edition by considering context is similar to our approach. We will apply the direction to a multi-touch interface.
Word snapping
As we described above, the usability of the text selection task can be improved if the tablet OSs consider context of the target text. We propose a word snapping method for text selection on the tablet OSs. The word snapping method changes the minimum unit of text selection as "a word" rather than "a character" while moving the region handles (see Fig. 2). Since most of the text selection is performed by the meaningful text like words or sentences, the proposed method reduces an irrelevant selection of text for cut-copy operations. The proposed method can also relief the burden of precise region handle controls. In this paper, we investigate basic characteristics of the proposed word snapping method on several texts written in natural languages.
Related works
Baby-face problems 4 and fat-finger problems 1 had been recognized for designing handheld interfaces including PDA and cellular phones. For making pointing operations accurate, several approaches have been investigated 5,6 . However, for test selections on tablet-OSs, not so many researches were performed yet.
Fuccela et al. 7 proposed a gestural text editing technique for touch-screen devices. The (multi-touch) gestures drawn on the soft keyboard area are interpreted as commands for moving the caret and text selections. The gestures also control the clipboard. Since the input area was different, the proposed technique can coexist with conventional widget-based input methods. We consider that these gestures affect as similar to the alternative short-cut keys (ex. Ctrl+C, Ctrl+V) which are omitted on the smaller screen keyboards.
Scheibel et al. 8 presented a virtual stick controller for precise caret positioning tasks. The virtual stick that emulates the function of joystick on a touch screen was implemented, and the performance was revealed. By comparing the finger tapping, the method had advantages when the movement distance was shorter, and the font size was smaller. Cockburn et al. 9 analyzed the fundamental performance of three input devices (the mouse, the stylus and the finger) across three different types of target acquisition activity (tapping, dragging, and radial dragging) on touchbased interactions. They revealed that the finger was fastest for tapping activities, but slowest for dragging. They also investigated that the errors in tapping activities was worst for the finger. We think that these results support that our proposed word-snapping method is appropriate for text-selection tasks because it will accept inaccurate and lower positioning resolutions by the fingers.
Experiment
We have conducted an experiment to verify the improvement of the usability of the text-selection interface according to the word-snapping technique. To evaluate the usability, we compared our method with a conventional text selection method. This chapter describes the procedure and results of the experiment and discussion.
Procedure
We asked 10 participants (all belonging to a student) to read documents on a 7-inch tablet (Nexus 7) in two different methods. Nine participants had been experienced with the (multi-)touch operations like tap, pinch, and swipe on a tablet. The participants utilize tablets or smart-phones in their daily life. For text-selection and cut-and-paste functions on the tablet/smart-phone device, five participants utilized daily, and other four participants utilized once a week.
In order to conduct the experiment, we have developed a prototype application specialized for the experiment with Processing for AndroidOS environment. During the experiment, the participant were required to select a text snippet specified by blue-colored text with white-colored background (see Fig. 3). Hereafter, we call the text snippet as "target text." When the participant tapped on the document, a character (or a word) at the point was selected, and two selection handles were appeared at the beginning and ending of the selected region. The range of the initial selected region was determined by the "snapping mode." The selected region was represented as a red-colored text with yellow-colored background (see Fig. 4). After the initial tapping, the participant dragged the handles so as to select all target text specified. If the "word snapping mode" was selected, both handles were snapped at the word boundary. When the participant precisely select the target text, he/she taps on the "Answer" (or "Start") button shown below the touch screen to proceed to the next task. If the selection was wrong, the overshooted region(s) were highlighted by pink-colored background (see Fig. 4). If the participant pressed the "Answer" button with overshooted, the system notices the error by playing sound.
We collected (1) time to complete a task, (2) touch count, and (3) overshoot time for each method. Since the purpose of this application is to estimate the fundamental properties of the proposed method, the all texts used in the experiment were pre-determined, and word separations of Japanese text were inserted by manual. In future, these separated text data can be generated by a morphological analyzer such as KAKASI 1 and MeCab 2 .
We also conducted a questionnaire survey to the participants. The following items were asked in 5 Likert scale.
1. Do you think the character mode was easy to select texts? (1-5) 2. Do you think the word-snapping mode was easy to select texts? (1)(2)(3)(4)(5) 3. How much you want to use the word-snapping mode for future similar tasks? (1-5) Fig. 6. Average task complete time. ** denotes 1% significance, and * denotes 5% significance. Fig. 6 shows average complete time of the tasks by 10 participants. The "task type" represents the number of lines and words of the target text. For example, "L2w7" means 7 words were included, and layed out in 2 lines. Appendix shows all screenshot of the tasks. The error bar denotes standard errors. Asterisk mark after the task number denotes significance level from conducting pairwise t-tests.
Results and discussion
Regarding the average complete times, the word-snapping was significantly faster than the character-based in the three tasks (No. 2, No. 3, and No. 10). But in the two tasks (No. 5 and No. 13), the word-snapping was significantly slower than the character-based. We consider that the phenomena were caused by the following reasons. Firstly, the word-snapping method worked well if the target text is a word. Because the participants could select the region by onetapping near the word. Also the word-snapping method was suitable for single line target. Because the participants could finish the task by moving handles horizontally. However, in the case of multiple lines target text, the participants should move the handle vertically. By our system design, the word snapping point was defined at the lower-middle of the word. When a word consist more characters, the gap between the word snapping point and the end point of the word was increased. Since we found that the gap distance influenced the performance of the word-snapping, we will fix the issue for future experiment. Secondly, the word-snapping method decreased the level of finger-tip feedback while dragging. This was caused by the reduction of the number of the possible snapping points for text-selection. In our implementation, the region handles were always sticked at one of the snapping points, and no extra carets/cursors were displayed. We can relief the issue by feedback of finger-tip position as well as the possible snapping points determined by the word separations. Fig. 7 and Fig. 8 show average touch count and overshoot count during experiments, respectively. Regarding the touch count, no significant differences were observed except the task No. 13. The significance also caused by extra touches during moving the region end handle. Regarding the overshoot count, a few words tasks were advantageous for word-snapping method. The task No. 13 (2 lines, 3 words) was the most unsuitable task for word-snapping method. Table 1 shows the result of the questionnaire survey. We performed a Mann-Whitney test for Q1 and Q2 answers under 5% significance levels. Consequently, were significantly differed (U = 8, p < .01). The result implies that the word-snapping method has a potential to relieve the burden of text-selection tasks if the current issues are solved.
Conclusion
In this paper, we introduced a word-snapping method, which considers text context for relieving burdens of textselection on the touch-sensitive screens with tablet OSs. The word-snapping method provides the snapping of the region handles at the boundary of word. The method can relief the precise control of the region handles.
We conducted an experiment to compare the word-snapping method with a conventional character-based method. The experiment revealed the word-snapping method can reduce the text-selection time if the target text consists of one or two words, and no line breaks exist. We could clarify the issues of the word-snapping method. If the issues are solved, the merit of the word-snapping method will be increased. In future work, we will also investigate the further extension by adding controlling handles by gestures.
Fig. 1 .
1Region handles for text selection (left: Google AndroidOS, right: Apple iOS).
Fig. 2 .
2Proposed method.
Fig. 3 .
3Initial screen of the task.Fig. 4. Region handles and overshooted text.
Fig. 5 .
5Scene of the experiment.
Fig. 5
5shows the scene of the experiment. The participant sat on the chair, held the tablet by the non-dominant hand, and put the hand on the
Fig. A. 9 .
9Task No.01 Fig. A.10. Task No.02 Fig. A.11. Task No.03 Fig. A.12. Task No.04 Fig. A.13. Task No.05 Fig. A.14. Task No.06 Fig. A.15. Task No.07 Fig. A.16. Task No.08 Fig. A.17. Task No.09 Fig. A.18. Task No.10 Fig. A.19. Task No.11 Fig. A.20. Task No.12 Fig. A.21. Task No.13 Fig. A.22. Task No.14
table. The participant used his/her dominant hand to tap-and-drag operations.ϲ
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ge of Time (unit : sec)
Character
Word Snapping
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Averag
Task Type
Task Number (Significance Level)
Fig. 7. Average touch count. * denotes 5% significance.Fig. 8. Average overshoot count. ** denotes 1% significance, and * denotes 5% significance.Touch Count
Character
Word Snapping
Task Type
Task Number (Significance Level)
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Character
Word Snapping
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Task Type
Task Number (Significance Level)
Table 1 .
1Result of the questionnaire survey (N = 10) Q3 How much you want to use the word-snapping mode for future similar tasks? (1-5) 0 1ID
Questionnaire Item
1 2 3 4 5 Ave
Var
Q1
Do you think the character mode was easy to select texts? (1-5)
1 1 7 1 0
2.8
0.75
Q2
Do you think the word-snapping mode was easy to select texts? (1-5)
0 0 1 7 2
4.1
0.54
0 6 3
4.1
0.83
http://kakasi.namazu.org/ 2 http://mecab.sourceforge.net/
Appendix A. Tasks
Fat finger worries: how older and younger users physically interact with PDAs. K A Siek, Y Rogers, K H Connelly, Human-Computer Interaction-INTERACT. SpringerSiek, K.A., Rogers, Y., Connelly, K.H.. Fat finger worries: how older and younger users physically interact with PDAs. In: Human-Computer Interaction-INTERACT 2005. Springer; 2005, p. 267-280.
Smarter Cut-and-Paste for Programming Text Editors. G Wallace, R Biddle, E Tempero, ISBN 0-7695-0969-XProceedings of the 2Nd Australasian Conference on User Interface; AUIC '01. the 2Nd Australasian Conference on User Interface; AUIC '01Washington, DC, USAIEEE Computer SocietyWallace, G., Biddle, R., Tempero, E.. Smarter Cut-and-Paste for Programming Text Editors. In: Proceedings of the 2Nd Australasian Conference on User Interface; AUIC '01. Washington, DC, USA: IEEE Computer Society. ISBN 0-7695-0969-X; 2001, p. 56-63. URL: http://dl.acm.org/citation.cfm?id=545646.545654.
Context-sensitive Cut, Copy, and Paste. R Kerr, W Stuerzlinger, 10.1145/1370256.1370283doi:10.1145/1370256.1370283Proceedings of the 2008 C3S2E Conference; C3S2E '08. the 2008 C3S2E Conference; C3S2E '08New York, NY, USAACMKerr, R., Stuerzlinger, W.. Context-sensitive Cut, Copy, and Paste. In: Proceedings of the 2008 C3S2E Conference; C3S2E '08. New York, NY, USA: ACM. ISBN 978-1-60558-101-9; 2008, p. 159-166. URL: http://doi.acm.org/10.1145/1370256.1370283. doi:10.1145/1370256.1370283.
Baby Faces: User-interface Design for Small Displays. A Marcus, J V Ferrante, T Kinnunen, K Kuutti, E Sparre, CHI 98. Marcus, A., Ferrante, J.V., Kinnunen, T., Kuutti, K., Sparre, E.. Baby Faces: User-interface Design for Small Displays. In: CHI 98
10.1145/286498.286547doi:10.1145/286498.286547Cconference Summary on Human Factors in Computing Systems; CHI '98. New York, NY, USAACMCconference Summary on Human Factors in Computing Systems; CHI '98. New York, NY, USA: ACM. ISBN 1-58113-028-7; 1998, p. 96-97. URL: http://doi.acm.org/10.1145/286498.286547. doi:10.1145/286498.286547.
Shift: A Technique for Operating Pen-based Interfaces Using Touch. D Vogel, P Baudisch, 10.1145/1240624.1240727doi:10.1145/1240624.1240727Proceedings of the SIGCHI Conference on Human Factors in Computing Systems; CHI '07. the SIGCHI Conference on Human Factors in Computing Systems; CHI '07New York, NY, USAACMISBN 978-1-59593-593-9Vogel, D., Baudisch, P.. Shift: A Technique for Operating Pen-based Interfaces Using Touch. In: Proceedings of the SIGCHI Conference on Human Factors in Computing Systems; CHI '07. New York, NY, USA: ACM. ISBN 978-1-59593-593-9; 2007, p. 657-666. URL: http://doi.acm.org/10.1145/1240624.1240727. doi:10.1145/1240624.1240727.
Precise Selection Techniques for Multi-touch Screens. H Benko, A D Wilson, P Baudisch, 10.1145/1124772.1124963doi:10.1145/1124772.1124963Proceedings of the SIGCHI Conference on Human Factors in Computing Systems; CHI '06. the SIGCHI Conference on Human Factors in Computing Systems; CHI '06New York, NY, USAACMBenko, H., Wilson, A.D., Baudisch, P.. Precise Selection Techniques for Multi-touch Screens. In: Proceedings of the SIGCHI Confer- ence on Human Factors in Computing Systems; CHI '06. New York, NY, USA: ACM. ISBN 1-59593-372-7; 2006, p. 1263-1272. URL: http://doi.acm.org/10.1145/1124772.1124963. doi:10.1145/1124772.1124963.
Gestures and Widgets: Performance in Text Editing on Multi-Touch Capable Mobile Devices. V Fuccella, P Isokoski, B Martin, Proceedings of the SIGCHI Conference on Human Factors in Computing Systems; CHI '13. the SIGCHI Conference on Human Factors in Computing Systems; CHI '13Fuccella, V., Isokoski, P., Martin, B.. Gestures and Widgets: Performance in Text Editing on Multi-Touch Capable Mobile Devices. In: Proceedings of the SIGCHI Conference on Human Factors in Computing Systems; CHI '13. 2013, p. 2785-2794.
Virtual Stick in Caret Positioning on Touch Screens. J B Scheibel, C Pierson, B Martin, N Godard, V Fuccella, P Isokoski, Proceedings of the 25ième Conférence Francophone on L'Interaction Homme-Machine. the 25ième Conférence Francophone on L'Interaction Homme-MachineScheibel, J.B., Pierson, C., Martin, B., Godard, N., Fuccella, V., Isokoski, P.. Virtual Stick in Caret Positioning on Touch Screens. In: Proceedings of the 25ième Conférence Francophone on L'Interaction Homme-Machine;
ISBN 978-1-4503-2407-6. 10.1145/2534903.2534918doi:10.1145/2534903.2534918IHM '13. New York, NY, USAACM107IHM '13. New York, NY, USA: ACM. ISBN 978-1- 4503-2407-6; 2013, p. 107:107-107:114. URL: http://doi.acm.org/10.1145/2534903.2534918. doi:10.1145/2534903.2534918.
Understanding Performance in Touch Selections: Tap, Drag and Radial Pointing Drag with Finger, Stylus and Mouse. A Cockburn, D Ahlström, C Gutwin, 10.1016/j.ijhcs.2011.11.002doi:10.1016/j.ijhcs.2011.11.002Int J Hum-Comput Stud. 703Cockburn, A., Ahlström, D., Gutwin, C.. Understanding Performance in Touch Selections: Tap, Drag and Radial Pointing Drag with Finger, Stylus and Mouse. Int J Hum-Comput Stud 2012;70(3):218-233. URL: http://dx.doi.org/10.1016/j.ijhcs.2011.11.002. doi:10.1016/j.ijhcs.2011.11.002.
| I don't even know if AHK is enough for this, I may need to learn another language to even make this smooth (no opening windows, no needing to use "sleep", mouse clicks and stuff). To me this is very complicated so I'll explain it with it broken down into a numbered list because that's how my mind works.
---
1. A box comes up when I press a hotkey. That box has a text field that is automatically ready for writing in. I want to be able to feed this into the box: "1600" (for the time 16:00, 4PM). After I press enter it will feed the date, time stamp under the column named "Monday", "Tuesday", etc. (automatically knowing what what day it is today).
2. It feeds the data into a spreadsheet program (Excel or Google Sheets preferably) with time stamp and date under the column named "Monday", "Tuesday", etc. (automatically knowing what day it is today). Visualization:
|avg. o the time|avg. o the time|avg. o the time|avg. o the time|avg. o the time|
---|---|----|----|----|----|----
Monday | Tuesday | Wednesday|Thursday|Friday|
| [Time] [Date] | [Time] [Date] | [Time] [Date] | [Time] [Date] | [Time] [Date] | || || |
| [Time] [Date] | [Time] [Date] | [Time] [Date] | [Time] [Date] | [Time] [Date] | || || |
| [Time] [Date] | [Time] [Date] | [Time] [Date] | [Time] [Date] | [Time] [Date] | || || |
(Date is in another cell though, but it's hard to format that apparently)
How do I feed the time into one cell, and then the date into the cell next to it?
That is what I need help with.
---------
For those that want/need context:
I'm trying to track when a certain thing happen every day and then get the average of when it happens every day to know when I'll be expecting the thing to happen.
Thank you for all the help you can offer. | that constantly changes. Therefore, another system is needed. Real-time databases may be modified to improve accuracy and efficiency and to avoid conflict, by providing deadlines and wait periods to insure temporal consistency. Real-time database systems offer a way of monitoring a physical system and representing it in data streams to a database. A data stream, like memory, fades over time. In order to guarantee that the freshest and most accurate information is recorded there are a number of ways of checking transactions to make sure they are executed in the proper order. An online auction house provides an example of | I've like that the Sony SMS app has "mark as read" in the notification, but I haven't found any other good apps with this. Tried Textra and Google's Messenger. It's strange that other apps don't have this useful feature. | 428 | The invention discloses a method and device for monitoring text editing in real time. The method includes the steps that a text file is opened or edited by a monitored end in a built monitoring set; the text file is then transmitted to a server by the monitored end; the text file is received by the server, and whether files with the same name exist in the server or not is judged; if files with the same name exist in the server, the text file is directly forwarded to a monitoring end by the server; if files with the same name do not exist in the server, a file with the same name is built in the server, and then the text file is forwarded to the monitoring end by the server; the text file forwarded by the server is received and opened by the monitoring end. In this way, the method and device for monitoring text editing in real time can achieve real-time monitoring of the text file editing process. | Standardization of real-time text in instant messaging | Exploring Consistency of Read-Only Transactions in Real-Time Systems | We present a multimodal real-time monitoring system called MMM that describes server activity by multimodal representation and supplements traditional ways of conveying sonification and peripheral information to Webmasters. We also describe a prototype and plug-in that MMM's three-level distributed architecture implements. | what is the difference between real time smart posting and batch? | Research of Real-time Monitoring System Integration Based on Event Notification Service | Research and Development of Realtime and Multitask Configuration Software for Monitoring in Industry | We study the problem of online runtime verification of real-time event streams. Our monitors can observe concurrent systems with a shared clock, but where each component reports observations as signals that arrive to the monitor at different speeds and with different and varying latencies. We start from specifications in a fragment of the TeSSLa specification language, where streams (including inputs and final verdicts) are not restricted to be Booleans but can be data from richer domains, including integers and reals with arithmetic operations and aggregations. Specifications can be used both for checking logical properties and for computing statistics and general numeric temporal metrics (and properties on these richer metrics). We present an online evaluation algorithm for the specification language and a concurrent implementation of the evaluation algorithm. The algorithm can tolerate and exploit the asynchronous arrival of events without synchronizing the inputs. Then, we introduce a theory of asynchronous transducers and show a formal proof of the correctness such that every possible run of the monitor implements the semantics. Finally, we report an empirical evaluation of a highly concurrent Erlang implementation of the monitoring algorithm. | Program for real time logging of disk usage? |
429 | Good book in simple spanish? | I purchased this book as a gift for a Spanish speaking friend so he can learn English. It is so clearly written and easy to use! It will help me with my Spanish too! All thumbs up! | Very strange book but super interesting. It's helpful to have a little background in the Spanish language but not necessary | This book is an excellent spanish - english reference. It is very easy to use for a student or for the office. I highly recomend this book for anyone doing english - spanish or spanish - english translations. | Not the best Spanish book I've seen, but does a good job. We are using this in an informal Spanish class. | This is the beginner book to teach Spanish to your child. Was recommended to us by a cousin who is a teacher. | I've been practicing Spanish for a couple years now and wanted a book that just had some normal phrases and such to help keep my mind fresh between weekly classes. This book is perfect for that! | Good reference book for Spanish language beginners. It takes the hard stuff, and makes it sound easy. Just as, in English, we know just by the way it sounds, whether it's proper or not. If we're past 5 years old, we know "We was all watchin them trucks," is not proper grammar. This books helps me to recognize when Spanish sounds proper. I'd recommend it. | Excellent books for people learning Spanish. I have a collection and am adding to it all of the time. I recommend it to anyone whether young or old. | 429 | Hi. I’m trying to get back into Spanish, I took it through high school and college but much of it has faded. However, whenever I do try to get back into it (this is not my first time), I’d rate my reading comprehension skills as intermediate. For reference, I can usually slowly read my way through a BBC espanol article with a few assists from google translate. Can anyone recommend a good book that might suit? I was thinking YA fiction but could be anything. Basically a good story with simple spanish. Thank you!
P.S. I’m willing to reread, so if there is something obvious that you assume I’ve already read, please still flag it. Except Harry Potter because I literally just reread the series. | One of the best books for learning Spanish that I have seen | Book recomendation in spanish | A great book if you want to see how much Spanish ... | [Free Book] 20 Short Stories in Spanish (With Vocabulary, Q&A and English Summaries) | Best books for tutoring/teaching spanish? | Can speak Spanish with decent grammar, but a weak vocabulary. Any suggestions for beginning/intermediate books? | Solid Book for Intermediate Spanish | Best Guide Book to anyone interested to learn Spanish language |
430 | Douglas versus Manning: The Ideological Battle over Medicare in Postwar Canada | This Handbook chapter aims to describe and explain the course of reform in North American healthcare from the Great Depression of the 1930s to the years 2013–2014. First, there is a brief discussion of the place of such a compari- son in the broader field of comparative policy studies. Our approach, which follows a ‘close comparison’ mode of analysis, takes up in turn the decades of dispute over the proper role of government in the financing of healthcare services to its citizens. That history constitutes the bulk of the comparative nar- rative, but is supplemented by two additional elements: a case study of the role of courts in North American healthcare reform, and an extended discussion of the role of political institutions in explaining policy change and continuity more generally. | Jonathan Cohn is a senior editor at The New Republic. Suppose I told you one of the political parties was determined to increase wasteful government spending by hundreds of billions of dollars, to pay the salaries of countless extra bureaucrats and to degrade the quality of medicine in the U.S. If you've been paying attention to politics for the last few months, you'd probably assume I was talking about the Democrats. Not so. I'd actually be talking about the Republicans who want to repeal health care reform. Confused? Well, don't blame yourself. The Republicans and their allies have spent a lot of time -- and a lot of money -- attacking the Affordable Care Act and promising to undo it. And they have done so with such a fury that almost nobody seemed to notice they are making a pair of arguments that are fundamentally incompatible. The Republicans start their calls for repeal with a familiar, thematic critique of government. The new health law, in this telling, represents an unconscionable government intrusion into the private sector and, ultimately, an encroachment on individual liberty. The federal government will be dictating everything from how employers conduct their businesses to how doctors treat their patients. And, oh yes, the government will be spending a ton of money it does not now have, increasing the deficit and/or laying new burdens on the taxpayers. The argument is hyperbolic and, in places, downright inaccurate. But, at least, it is consistent with longtime conservative principles about the role and size of government. But that's not all the Republicans have been arguing. They've also been attacking the health overhaul for what it will do to Medicare. And instead of accusing Democrats of trying to dump more money into a government program, as Republicans would typically do, they've attacked Democrats for doing the very opposite -- noting that the Affordable Care Act will reduce spending on Medicare somewhere around $400 billion over the next 10 years. Apparently government-run health care is awful, except, um, when it isn't. To be fair, the Republican argument makes perfect sense if you think like a campaign operative. Senior citizens are, at the moment, the most conservative age group in the electorate. They were least likely to support President Obama in 2008 and, during the health care fight, were most likely to oppose enactment. Republicans seized on that fact and have gleefully proclaimed themselves champions of Medicare, despite a long history of opposing it and, as Newt Gingrich once put it, letting this universal social insurance program "wither on the vine." Seniors are playing along, since they figure reform means taking money once targeted for Medicare and diverting it to help people under-65 pay for their medical care. But here's where things could get complicated for the advocates of repeal. Consider what undoing the cuts in Medicare would entail. It would start, first of all, with restoring higher payments to the insurers that provide private coverage for people in Medicare, through what's known as the Medicare Advantage plans. There's a reason the health law reduces those payments: Repeated independent studies, including those by the well-respected Medicare Payment Advisory Commission, determined that the government was paying the insurers too much. Restore those payments, and you're wasting taxpayer dollars. And a lot of those wasted dollars will go to hiring new people to work at insurance companies. They won't be government bureaucrats, obviously. They'll be insurance company bureaucrats. But is that really better? Is the Tea Party in favor of waste as long as its lines the pockets of insurance executives rather than Uncle Sam? Meanwhile, restoring the other cuts to Medicare would mean rescinding payment reductions designed to make the program more efficient. Remember, a major goal of the health reform measure is to push against higher spending while simultaneously promoting higher quality care. In the case of Medicare, that means slowing down payment increases to providers and penalizing those that provide substandard treatment; while, at the same time, boosting payments to primary care doctors and providing bonuses for those who actually treat patients better. Reasonable people can argue how well these efforts will work. But allowing Medicare to continue going along as it has been for the last ten to twenty years -- which is what repealing the new health law would do -- would almost surely force a choice between much higher taxes or much worse access to care. If you don't believe me, just look at the plan proposed by Republican Representative Paul Ryan, who is forthright enough to admit that the GOP alternative to the Democrats' approach to Medicare is to reduce radically its guaranteed benefits. Of course, the Republican Party's leadership hasn't embraced Ryan's plan in any specificity. And, at least for the short term, it seems unlikely they'll advocate such a path, le | History of Saskatchewan against the CCF's medical care program and to Canadians' general historic reluctance to vote for progressive change. In the 1964 Saskatchewan provincial election, the Liberal party, led by Ross Thatcher (1917–71), swept to victory, ending 20 years of CCF government. The Liberals had launched a strong party membership drive and engaged in vigorous campaigning on a platform demanding more private enterprise and industrial development; it promised substantial tax cuts. The CCF's internal factionalism, together with lingering reaction to the medical care crisis of 1962 and the separate school issue, contributed to the CCF defeat. The impact of the Douglas government | During whose presidency was medicare enacted? | Who prime medicare or group? | Woodrow Stanley Lloyd (July 16, 1913 – April 7, 1972) was a Canadian politician and educator. Born in Saskatchewan in 1913, he became a teacher in the early 1930s. He worked as a teacher and school principal until 1944 and was involved with the Saskatchewan Teachers' Federation, eventually becoming its president.
He was first elected as a Member of the Legislative Assembly of Saskatchewan in 1944. He served as Education Minister and then Treasurer in Tommy Douglas's Co-operative Commonwealth Federation government between 1944 and 1961. He succeeded Douglas as Saskatchewan Premier in late 1961. Lloyd is best remembered as the man who piloted Canada's first Medicare program from legislation to implementation in 1962, and overcoming the Saskatchewan doctors' strike that summer strike to enable it to continue.
Lloyd was defeated in the 1964 Saskatchewan general election and served the next six years as the Leader of the Official Opposition. He stepped down as the New Democratic Party's leader in 1970 (the CCF changed its name in 1967), and from the Legislature in 1971. He was appointed to a United Nations post in South Korea, where he died of a heart attack in 1972.
Early life
Lloyd was born in Webb, Saskatchewan on July 16, 1913. He initially studied engineering, but due to the Great Depression, switched his studies to teaching and graduated with a BA from the University of Saskatchewan in 1936. He started teaching school that year, and eventually became a school principal in the early 1940s at Stewart Valley, Vanguard and Biggar. He was also active in the Saskatchewan Teachers' Federation and held many positions in the organization including the presidency from 1940 to 1944. He also served on the University of Saskatchewan's Senate, and was the president of the Saskatchewan Educational Conference in the early 1940s.
Douglas government
In 1944, Lloyd was elected to the Saskatchewan Legislature as the Co-operative Commonwealth Federation member for the constituency of Biggar, a seat that he held until his retirement in 1971. Lloyd became the youngest cabinet minister in Saskatchewan history, up to that point, when he was appointed to cabinet as Minister of Education by new Premier, Tommy Douglas. Lloyd served as Minister of Education for the next 16 years and oversaw the complete overhaul of the Saskatchewan education system. The most controversial measure he introduced was the amalgamation of over 5000 of Saskatchewan's local school boards (units) into 56 larger school units in 1944–1945. The measure was instituted to create more equitable educational opportunities for students across the province by providing students greater opportunity to receive instruction by specialized teachers and access to increased education resources, including provincial grants.
However, the move was opposed by some in rural Saskatchewan who resented the loss of local control over schools, as the move to large school units resulted in the closure of nearly all rural one-room schools over the next two decades.
After the 1960 election, Douglas appointed Lloyd to be the provincial treasurer. In 1961, Douglas resigned as premier to assume the leadership of the newly-formed federal New Democratic Party (NDP). Lloyd was elected as leader of what was now called the Saskatchewan CCF-NDP and easily defeated Olaf Turnbull.
Premier of Saskatchewan
As Premier, Lloyd was responsible for implementing the universal health care plan that Douglas had introduced. Lloyd's government had to cope with the July 1962 Saskatchewan doctors' strike, when the province's physicians withdrew service in an attempt to defeat the Medicare initiative. Lloyd and his government refused to back down on the concept of a universal public health care system and persuaded the doctors to settle after 23 days.
Medicare was implemented, but the political turmoil did lasting damage to the Lloyd government and contributed to its defeat at the hands of Ross Thatcher's Saskatchewan Liberal Party in the 1964 provincial election. Medicare was later extended to all provinces and territories in Canada as a result of the Saskatchewan experiment.
Lloyd was the first premier of Saskatchewan to have been born in the province after its accession to Confederation in 1905.
Later life
After his government's defeat, Lloyd became Leader of the Opposition, a post he held until 1970 when Allan Blakeney was elected leader of the Saskatchewan NDP. On his retirement, Douglas gave him the ultimate compliment by saying that Lloyd was "the conscience of the government and the conscience of the party." After retirement from the Saskatchewan Legislature in 1971, Lloyd was appointed as representative for the United Nations Development Program in South Korea. However just months after assuming that post, he died suddenly in Seoul, South Korea.
Electoral history
Saskatchewan general elections, 1944 to 1960
Lloyd led the CCF in two general elections: 1964 and 1967. The CCF was defeated both times.
1964 General election
The 1964 election was very close in the popular vote, with a difference of only 660 votes between the Liberals and the CCF. The distribution of votes in the ridings gave the Liberals a majority, ending the CCF's seventeen year term in office. Ross Thatcher defeated Lloyd and became Premier. Lloyd became Leader of the Opposition.
1 Leader of the Opposition before election was called; Premier after election.
2 Premier when election was called; Leader of the Opposition after election.
3 Rounds to zero.
1967 General election
In the 1967 election, Lloyd again led the CCF, now re-named the NDP, against Ross Thatcher and the Liberals. The Liberals were returned to office, the last time the Liberals formed the government. Lloyd resigned as party leader before the next election, being succeeded by Alan Blakeney.
1 Premier before election was called; Premier after election.
2 Leader of the Opposition when election was called; Leader of the Opposition after election.
Saskatchewan constituency elections
Lloyd stood for election to the Legislative Assembly in seven general elections, all in the constituency of Biggar. He was elected in all seven elections, from 1944 to 1967.
1944 General election: Biggar
E Elected.
1948 General election: Biggar
E Elected.
X Incumbent.
1952 General election: Biggar
E Elected.
X Incumbent.
1956 General election: Biggar
1960 General election: Biggar
E Elected.
X Incumbent.
1 Rounding error.
1964 General election: Biggar
E Elected.
X Incumbent.
1967 General election: Biggar
E Elected.
X Incumbent.
Citations
References
Premiers of Saskatchewan
Lloyd, Woodrow S.
Lloyd, Woodrow S.
Leaders of the Saskatchewan CCF/NDP
Saskatchewan Co-operative Commonwealth Federation MLAs
20th-century Canadian politicians
Saskatchewan New Democratic Party MLAs | Because health policy is the result of a tremendous variety of shifting forces, it is necessary to be eclectic in the approach used to study it. Analyzing the Canadian case in this broad perspective reveals that the influence of ideological and institutional forces, and principal actors is more important than most commentators believe, and that the dynamism in health policy formulation has resulted in a great deal of internal conflict as well as politicization and provincialization. | What year was Medicare enacted? | 430 | The accepted narrative of the history of medicare in Canada does not do justice to the struggle between premiers Tommy Douglas of Saskatchewan and Ernest Manning of Alberta over two very different models of universal health coverage. Douglas and Manning were committed advocates of their respective models for ideological reasons, but these political differences had their origins in their conflicting interpretations of Christian teachings and biblical interpretation. These differences are examined in detail in order to arrive at a richer understanding of the values and the key policy design features of their respective models of medicare. Ultimately, Douglas’s model of medicare would be adopted in the rest of Canada even though Manningcare was the preferred choice of doctors, insurance companies, the business establishment, the majority of provincial governments, and fundamentalist Christians such as Manning who believed that Douglas’s model resulted in an abdication of individual responsibility and moral c... | what type of health insurance did the alberta medical association create | Medicare and Medicaid - The Difference | List 2 possible advantages of having Medicare in Canada? | What is the neoliberal view on single-payer healthcare vs a public option? | What is the difference between Obamacare and medicare? | Medicare versus private insurance: rhetoric and reality. | Libertarian views on rights tend to rule out coercive redistribution for purposes of public health care guarantees, whereas liberal conceptions support coercive funding of potentially unlimited access to medical services in the name of medical needs. Taking the "priority of liberty" seriously as supreme political value, a plausible prudential argument can avoid these extremes by providing systematic reasons for both delivering and limiting publicly financed guarantees. Given impending demographic change and rapid technical progress in medicine, only a two-tier system with explicitly limited public guarantees and optional privately financed health services seems acceptable. | what is the difference in original medicare and medicare advantage? |
431 | when did la paz became a global city | La Paz, Honduras La Paz () is the capital city of the La Paz Department of Honduras. The town, founded in 1792 and has a population of 46,264 (2015 est.). The town is 750m (2461 feet) above sea level on the Comayagua River near the Cordillera de Montecillos in an area that has mountainous terrain with thick jungle cover. The town dates back to 1750 when two Spanish colonies existed in the area. The town's title was given on 14 September 1848 when the name "La Paz" was officially recognized by a decree from Comayagua and in 1861 it was | La Paz Department (Bolivia) of La Paz is divided into 20 provinces ("provincias") which are further subdivided into 85 municipalities ("municipios") and - on the fourth level - into cantons. The provinces with their capitals are: The chief executive office of Bolivia's departments (since May 2010) is the Governor; before then, the office was called the Prefect, and until 2006 the prefect was appointed by the President of Bolivia and now currently is elected by the voters. The current governor, César Cocarico of the Movement for Socialism – Political Instrument for the Sovereignty of the Peoples was elected on 4 April 2010 and took | La Paz is a small town in the province of Córdoba, Argentina. Its population is 1189 inhabitants (2010 Census) and it is located from Villa Dolores.
The town grew around a Catholic church, San Juan de las Talas, built there in 1720, during colonial times.
Economy
The main activity is tourism and the production of medical vegetables. One of its tourist attractions is Cerro Loma Bola (Loma Bola Hill), the Piedra Blanca stream, and wide native woods, as well as its local gastronomy.
Each August 29 the town celebrates the Virgen de la Merced.
Populated places in Córdoba Province, Argentina
Tourism in Argentina | Siege of La Paz The Siege of La Paz was a Mexican siege of their own city of La Paz in Baja California Sur. Mexican militia forces attempted to destroy the United States Army garrison, occupying the peninsular town. The siege occurred over a twelve-day period in November and December 1847, at the end of the Mexican–American War. Captain Manuel Pineda Munoz, of the Mexican Army had been drafting Mexican peasants to serve in his campaign on the western coast of Mexico. After his militia army was defeated twice at the Battle of La Paz and the Battle of San | Alonso de Mendoza Alonso de Mendoza (Garrovillas de Alconétar, Spain, c. 1471–1476 – Tipuani, Bolivia, 1549) was a Spanish captain, conquistador, and the founder of the city of Nuestra Señora de La Paz, current capital city of Bolivia. He was appointed by Pedro de la Gasca, the "Peacemaker," to found the city to commemorate the peace in the Peruvian colonies after the defeat of the Pizarro brothers. Alonso de Mendoza was born between 1471 and 1476 in Garrovillas de Alconétar (Cáceres, Spain). He left the peninsula attracted by the news about the wealth of the New World, and by the | the rewards they could secure through fidelity to the Crown. Murillo and the other leaders were beheaded and their heads exhibited to the people as deterrent. La Paz revolution The city of La Paz (modern Bolivia, then part of the Viceroyalty of the Río de la Plata) experienced a revolution in 1809 that deposed Spanish authorities and declared independence. It is considered one of the early steps of the Spanish American wars of independence, and an antecedent of the independence of Bolivia. However, such revolution was defeated shortly afterwards, and the city returned to Spanish rule. In 1781, for a | the rewards they could secure through fidelity to the Crown. Murillo and the other leaders were beheaded and their heads exhibited to the people as deterrent. La Paz revolution The city of La Paz (modern Bolivia, then part of the Viceroyalty of the Río de la Plata) experienced a revolution in 1809 that deposed Spanish authorities and declared independence. It is considered one of the early steps of the Spanish American wars of independence, and an antecedent of the independence of Bolivia. However, such revolution was defeated shortly afterwards, and the city returned to Spanish rule. In 1781, for a | History of Bolivia the Bolivian state sought to strengthen its role in rural areas, implementing an extensive public health campaign that specifically included indigenous Bolivians. Twelve more tumultuous years of national reform left the country bitterly divided and in 1964, a military junta led by vice-president René Barrientos overthrew President Paz Estenssoro at the outset of his third term; an event that many assert brought an end to the National Revolution and marked the beginning of nearly 20 years of military rule in Bolivia. Many scholars have looked to the CIA in explaining the November 1964 coup, but an increasing number of declassified | 431 | Beirut, Doha, Durban, Havana, Kuala Lumpur and Vigan. La Paz is listed on the Global Cities Index 2015, and is considered a global city type "Gamma" by Globalization and World Cities Research Network (GaWC). This area had been the site of an Inca city, located on a major trading route. Although the Spanish conquistadors entered the area in 1535, they did not found La Paz until 1548. Originally it was to be at the site of the Native American settlement, Laja, with the full name of the city being "Nuestra Señora de La Paz" (meaning "Our Lady of Peace"). The | when did la paz arizona became a town | what position does paz y paz hold in guatemala | where is theatro da paz located in south america | when was the last copa paz del chaco played | who led the spanish conquistadors who crossed the rio paz | What is the capital of La Paz y Sucre located at? | when did che guevara arrive in la paz | what is the county seat of la paz county arizona |
432 | what was the luff award given to brett for his philatelic research | was elected a Fellow of the Royal Society (FRS) in 1936. As a result of the development of flash photolysis, Norrish was awarded the Nobel Prize in Chemistry in 1967 along with Manfred Eigen and George Porter for their study of extremely fast chemical reactions. One of his accomplishments is the development of the Norrish reaction. At Cambridge, Norrish supervised Rosalind Franklin, future DNA researcher and colleague of James Watson and Francis Crick, and experienced some conflict with her. Ronald George Wreyford Norrish Ronald George Wreyford Norrish FRS (9 November 1897 – 7 June 1978) was a British chemist who | Edward Calvin Kendall its structure. He also isolated several steroids from the adrenal gland cortex, one of which was initially called Compound E. Working with Mayo Clinic physician Philip Showalter Hench, Compound E was used to treat rheumatoid arthritis. The compound was eventually named cortisone. In 1950, Kendall and Hench, along with Swiss chemist Tadeus Reichstein were awarded the 1950 Nobel Prize in Physiology or Medicine for "their discoveries relating to the hormones of the adrenal cortex, their structure and biological effects." His Nobel lecture focused on the basic research that led to his award, and was titled "The Development of Cortisone As | board of trustees and also chairs The Institute's advisory council. He was also the recipient of the Golf Writers Association of America's 2008 Charlie Bartlett Award. In 2009 Norman was inducted into the Queensland Sport Hall of Fame. In 2015, the PGA of Australia established the Greg Norman Medal, which is awarded to the best Australian male or female golfer in a given year. He also received the Australian Global Icon Award and the National Golf Course Owner's Association Award of Merit both in 2015. Norman had a bold and aggressive style of play. He is widely regarded as one | Cormac O'Ceallaigh he was mentored by Lord Rutherford and concentrated on nuclear physics. He got a lectureship at University College Cork in 1937 and returned to Ireland in 1938. He remained at Cork until 1947 and then took a position at the University of Bristol working in a group assembled by the Nobel Prize winning particle physicist C F Powell. Bristol was at the time the worldwide centre of cosmic ray research, and O'Ceallaigh, nurtured by Rutherford and Powell, two of the greatest experimental physicists in history, soon became one of its leading figures. Their research into cosmic rays, involving pions, kaons, | Dinner with Friends with Brett Gelman and Friends Dinner with Friends with Brett Gelman and Friends is a 2014 American television special created and written by Brett Gelman and Jason Woliner for Adult Swim. The special features Brett Gelman as a demented version of himself, along with several guests, who also play fictionalized characters of themselves. Gelman and Woliner had frequently collaborated on other projects before producing the special. On its broadcast on April 24, 2014, the special was positively received by critics. A sequel, entitled "Dinner with Family with Brett Gelman and Brett Gelman's Family", aired February 13, 2015, | Carl Wieman Alison Marjorie Fry in the United States and graduated from Corvallis High School. His paternal grandfather Henry Nelson Wieman was a religious philosopher of German descent and his mother had white Anglo-Saxon Protestant family background. Wieman earned his B.S. in 1973 from MIT and his Ph.D. from Stanford University in 1977; he was also awarded a Doctor of Science, "honoris causa" from the University of Chicago in 1997. He was awarded the Lorentz Medal in 1998. In 2001, he won the Nobel Prize in Physics, along with Eric Allin Cornell and Wolfgang Ketterle, for fundamental studies of the Bose-Einstein condensate. | the first academic researchers of biorhythms was Estonian-born Nikolai Pärna, who published a book in German called "Rhythm, Life and Creation" in 1923. The practice of consulting biorhythms was popularized in the 1970s by a series of books by Bernard Gittelson, including "Biorhythm — A Personal Science", "Biorhythm Charts of the Famous and Infamous", and "Biorhythm Sports Forecasting". Gittelson's company, Biorhythm Computers, Inc., made a business selling personal biorhythm charts and calculators, but his ability to predict sporting events was not substantiated. Charting biorhythms for personal use was popular in the United States during the 1970s; many places (especially video | three children together, Jeff, Lisa, and Tom and seven grandchildren at the time of his death. Cheek was inducted into the Blue Jays Level of Excellence in 2004 with the number "4306" next to his name, signifying his streak. Canada's Sports Hall of Fame established the "Tom Cheek Media Leadership Award" shortly before his death, for "playing a key role in promoting Canadian sports", with Cheek being named the recipient of the first award. During the 2006 season, the Blue Jays wore a white circular sewn on patch with the letters ' TC ' and a radio microphone in black | 432 | of the United States Stamp Society (previously called the Bureau Issues Association) during 1966 and 1967, then continued on as chairman and emeritus chairman until his death. And from 1961 to 1963 he was a member of the Citizens’ Stamp Advisory Committee. Brett was honored with the Lichtenstein Medal in 1983, the Luff Award for distinguished philatelic research in 1978, and numerous other awards. He was named to the Writers Hall of Fame in 1979 and the American Philatelic Society Hall of Fame in 2006. George Wendell Brett George Wendell Brett (May 30, 1912 – January 14, 2005), of Iowa, | when did edward calvin kendall win the nobel prize | when was jan bach awarded a presidential research professorship grant | how did george brett go in the 1985 american league championship series | when was brett boyko born in los angeles | when was the hans christian andersen writing award established | in which country was german experimental nuclear physicist robert dopel sent to work on | when did oscar janiger found the albert hofmann foundation | during which war did frank peregory receive the medal of honor |
433 | who wrote the dance piece in talk to her | also performed during the opening shows of the first leg of Beyoncé's Mrs. Carter Show World Tour. Dance for You "Dance for You" is a song by American singer Beyoncé for the deluxe edition of her fourth studio album, "4" (2011). It was written by Beyoncé, Terius "The-Dream" Nash and Christopher "Tricky" Stewart, while production was handled by the former two. "Dance for You" is a midtempo R&B and synthpop song, in which Beyoncé adopts sensual vocals. The instrumental elements used on it include echoing drum patterns and clapping synthesizers. In "Dance for You", Beyoncé, as the female protagonist, speaks | How do you ask a girl for a slow dance? | chart. 7" Single 7" Single, 10" Picture Disc 12" Single Can the Rhythm "Can the Rhythm" is a song by British female pop duo Girl Talk. The song was written solely by group member Karen Wright, who wrote it at the age of 12. The song was initially released as Girl Talk's debut single in 1983, produced by Mick Clarke and released on the label Park Records. At that point, the group was composed of Karen Wright and Leigh Pearce, who were aged 12 and 13 at the time. Wright sings the first verse and Pearce the second, with both | Girl Talk (musical group) Girl Talk was a British girl group, formed by Karen Wright and Leigh Pearce. The girls, aged 12 and 13 respectively, released their debut single, "Can The Rhythm", in 1983 on Park Records. By the next year Pearce had been replaced by Karen's sister, Julie Wright. Signed to Innervision Records, the pair released the single "Marvellous Guy" in 1984, produced by Peter Collins, but it failed to chart. Their only hit in the UK was their second Innervision single, a re-recorded "Can The Rhythm", which reached #92 in October 1984. The track was an early production | I can't dance. But she mentioned it couple times in the past. What should I do?
I was maybe twice in club and have zero experience. | on 28 May. Musically, "We Dance On" is an uptempo British hip hop, dance and R&B song with a dance-orientated beat. "We Dance On" has a distinctly faster tempo than previous N-Dubz singles. The song opens with the Baroque-styled string section sample which transitions into Dappy saying the N-Dubz trademark line "Na-na-nai". Tulisa is the most prominent contributor to the vocals in the song, with her verse "Fox on a mission" being said by critics as one of the highlights in the track. "We Dance On" was described by Gavin Martin of the "Daily Mirror" as having "unifying floorfiller" and | Dance (Alexandra Stan song) of the song's melody and lyrics in Paris, and later brought her idea for further development by her team at Fonogram Records, led by producers Alex Cotoi and Mika Moupondo. The song is a dance track, also incorporating house music influences into its sound. The track opens with acoustic guitar chords, which are then followed by a pop strophe. The refrain consists of a saxophone drop, which is backed by cut saws and plucks. "Dance" ends with the last lyrics of the song being backed by acoustic guitar. Lyrically, the track speaks about the art of dancing, but also features | the end of a semester, students present dances they have choreographed. Lydia Johnson Dance Lydia Johnson Dance is a contemporary dance company that performs the choreography of Lydia Johnson, primarily in New York City and New Jersey. It is notable for combining ballet and modern dance, sometimes isolating and reworking "components of classical ballet technique." The company was founded in 1999 by Johnson, a choreographer. She has choreographed dance works to various composers including Beethoven, the alternative rock band Cake, Philip Glass, Argentine composer Osvaldo Golijov, Polish composer Henryk Górecki, and others. Since 2008 the company has received annual support | 433 | Talk to Her Language and the Golden Globe for Best Foreign Language Film while Almodóvar won the Academy Award for Best Original Screenplay. It is now generally regarded as one of the finest films of the 2000s. The story unfolds in flashbacks, giving details of two separate relationships that become intertwined with each other. During a performance of "Café Müller", a dance-theatre piece by Pina Bausch, Benigno Martín and Marco Zuluaga cross paths, but the two men are no more than strangers. Still, Benigno notices that Marco cries. Marco is a journalist and travel writer who happens to see a TV interview with | How likely do you think Lisa’s solo will be in her native language? | New italian interview for Mr. Uchikoshi about him, Zero Escape and World End Club | what is the title of silvia azzoni's ballets | which 2005 novel by kate mosse has been translated into more than 37 languages, | How do you say in Italian 'Talk soon'? | what language do most arab brazilians speak | [Suggestion] Implement a system where you vote for what language X person speaks. (Instead of language preferences) | All-star vote: Should European voters consider the players ability to communicate in English when choosing who to vote for? |
434 | Marvel legends best of 2019 | the quality of detail in the HeroClix figures. The first game set, Marvel's "Infinity Challenge", was released in 2002 and included figures and maps. The original "HeroClix" figures were all from comic books printed by Marvel Comics, but later expanded to include sets from DC Comics and from various independent comic book publishers such as Image Comics and Dark Horse Comics. Later expansions also added new card-based mechanics such as "Feats" and "Battlefield Conditions", expanding the game beyond the addition of new characters. The Original HeroClix won three awards at the 2002 Origins Awards including "Best Science Fiction or Fantasy | Who is the greatest character of marvel? | Deadpool vs Hawkeye #2 came out today. Also out today is AXIS #5, Deadpool however does not make an apperance in this issue.
Also of note, Superior Iron Man came out today for those interested in more of the inversion stuff. Its pretty good. | There has been a recent trend of posts in the sub about how MCU movies are "SAFE" , "CHILDISH" and "NOT LIKE REAL MOVIES" thanks to a certain someone trying to be relevant in the news.
Before I defend MCU movies, I would like to state that movies and their enjoyment are subjective. I personally can attest to that as I didn't enjoy AoU and Loved the Original Avengers in the same way as I was bored of Hugo but thought Departed was a classic.
1. Marvel movies are too safe.
No they are not. They look safe because they have earned that goodwill through the years. Comic book movies(CBM's) were a risky venture ( and still are to an extent) before the MCU. Having a well rounded , emotionally appealing superhero movie was rare to come by because these movies often wanted to be spectacle and action first rather than story first.
(
Famous Kevin Smith rant on how superhero movies were made)
2. Marvel can't do mature storytelling.
I really don't know where people get such ideas. Marvel studios and MCU movies have dealt with loss, betrayal, authoritarianism (still odd Civil War released in China), PTSD, broken families, moral and ethical conundrums all in the fantasy setting of the superhero genre. These movies are loved because of this mature storytelling. Mature storytelling doesn't mean blood , gore or F bombs, it means storytelling that can make you think and ponder or open your eyes to a different worldview.
3. Marvel movies are too funny.
This one I believe is more open to one's opinion. I find it has the right amount of humor but I understand if someone else feels otherwise.
4. Marvel movies are all good vs evil and no grey areas.
Again I don't know what people who say this smoke exactly. Thanos, Loki, N'Jadaka and Zemo were all grey villains. They all had their own reasonable motivations. These are motivations are normal person would consider abhorrent to act on, but would not call them unjustified. Similarly our so called heroes weren't perfect. Happy literally calls out how imperfect Tony was. Thor was a mess. Cap hid and lied to Tony about crucial information for the greater good when it suited him.
5. Marvel movies are all about spectacle!
I am paying 20$+tax, plus paying for my nieces and nephews and the overpriced popcorn and drinks to watch this in a large screen in a premium format. Fuck yeah I want my spectacle! These were things I imagined how i wanted them to be when i read comics or saw them in cartoons in the 90's. Superhero movies without a good spectacle and action set pieces is like removing the cheese off a cheeseburger. Perfectly fine but lacks something in the end.
Marvel Movies have been consistently higher quality than the normal fare and it's the reason for their success. You may not like it and that is your opinion. Not everything appeals to everyone. But saying Marvel movies are safe or whatever other bullshit reason you wish to justify is not the way to go.
MCU movies have ushered in a golden age for the genre, something which wasn't considered possible earlier.
Now this doesn't mean I agree with Disney's poor business practices which is the true source of all this strife but that is a topic for another post. I still maintain Marvel Movies are top tier high quality movies worth the admission price. | It'll be wonderful to have Marvel's First Family and it's most iconic villain and herald back into their rightful home. Where they will be treated with care and respect. Since every try at a movie was considered a failure, it would be smart for Fox to give Marvel back the rights. Then right after the Infinity War, Galactus and his herald Silver Surfer can be the main villains of Phase 4. What do you think? | The best of the Avengers movies, and maybe the next best Marvel movie, after Iron Man 1. My opinion.... | This movie is up there with The Avengers, as the Best Super Hero movie of all time! It's just that good! | The initial line was Doctor Strange, Ms. Marvel and Black Widow followed by Silver Surfer and Guardians of the Galaxy. #10 - #14 also feature Ms. Marvel & Nick Fury's Civil War II stories. It goes: Every Thursday per 4 weeks Release: April 5, 2018 Issue 1: 100-PAGE-SPECIAL! Issue 2: Issue 3: Issue 4: Coming Next: Issue 5 - #6 will be part of the 'Secret Empire' event and feature Champions Vol. 2 #10 - #11. The Mighty World of Marvel The Mighty World Of Marvel (commonly shortened to MWOM) was Marvel UK's first-ever title, debuting on 30 September 1972, | 434 | What do you think will be the repackaged wave for the MCU figures from 2019, I think the best lineup would be:
Captain America (quantum suit), Ronin, Rescue, War Machine, Shuri ,Spider Man (red and black suit), Mysterio | Probably the weakest recent Marvel Legends BAF Wave. No real standouts | Post Avengers 4 dream lineup. | Shouto Todoroki (My Hero Academia) vs Captain America (Marvel 616) | Sentry, Gladiator, Hyperion, and Blue Marvel vs Superman, Wonder Woman, Martian Manhunter, and Shazam | What would be your dream team X-men? | I was hoping for something as good as the wave prophecy 2 | Trying to remember if Agent Brand is in any MCU movies/shows. | [Spoilers All] What Characters would you like to see return in DA4? |
435 | Who gets to be a writer? Exploring identity and learning issues in becoming a fiction author | What questions do you have about the craft of writing novels or stories for young adults? What do you need to know about getting published—either self or traditionally? Ask here! | Education you need to become a writer? | What is being a novelist like? | As graduate students, writers, and editors, we identify that writing for coursework and writing for publication are two fundamentally different tasks. Likewise, publishing from one | I need an outlet for writing humor. I've dabbled in stand-up comedy & acting, and if I'm going to pursue those further, I need to shake off the writing cobwebs and get into a regular routine.
So... how did you manage to make a hobby out of creative writing? What habits did you develop? What works?
MOST IMPORTANTLY: How do you start writing when you don't know what to write about?
Any & all advice is appreciated! :) | I just convert my daily activities into prose. I figure future generations will be so enthralled by my writing that they'll want to know about my life. | It can be fiction or non-fiction. I just need something to teach me how to accept my shortcomings. Thanks all. | It can be either about the technical side of writing, or marketing and promotion, or anything else as long as it somehow relates to writing.
I've learned that writing is something that is inside of me that I really enjoy doing. And even if i try to stop (which I have) I will always come back. | 435 | Drawing upon a research study on lifelong learning, citizenship, and fiction writing, this paper explores issues around identity and learning in becoming a fiction author. Five main thematic areas are discussed: (1) envisioning a writing career, (2) compelled to write, (3) learning the craft, (4) getting published, and (5) online identity. The challenges, hurdles, and motivational factors in pursuing a career in a field as tenuous as fiction writing are explored. The paper argues that fiction writers, like many people who work in the creative sector, have a strong desire to engage in work that they consider to be meaningful. Those who succeed demonstrate great perseverance. As the impact of new technologies and social media shape and change the publishing sector, there are new challenges as well as opportunities that writers will need to learn about and address as they develop their career trajectories. | Has anyone here transitioned to creative writing from non-fiction? Can it be done? | Aspiring Content Writer - Academic Niche | Do you need education to be a writer? | The Writer Within You: A Step-by-Step Guide to Writing and Publishing in Your Retirement Years | Reddit, I want to be an established author someday. Any advice? | What is the proses in becoming a novelist? | I want to write non fiction and I don't know where to start | IWTL how to get a career writing comic books |
436 | My mom keeps calling me a dropout and it is affecting me. | My mom keeps making me cry saying that I was stupid to be confident that I would go to JHU and that I shouldn't be hopeful for any Ivy's cause they probably hate me and that they've thrown out my application. I was trying so hard to cope with the rejections and keep a positive attitude but my mom isn't helping. Guys I am really trying here but I'm breaking from my mom's comments | I'm 16, and she always yelled at me. She's been yelling at me for no reason or over something small since I was 14. And you would think I got used to it by now, but I haven't. I don't like being yelled at and it's sometimes embarrassing. And just yesterday, we were at Walmart. And we were getting some shelf's for the bathroom and there were 5 in total. And she drove up to the building so I could put them away. And I was putting them in and my mom starting yelling and repeating over and over again going: "PUT IT IN THE CAR! NO, DON'T PUT IT IN LIKE THAT! IT DOESN'T GO IN LIKE THAT! WHY ARE YOU IGNORING ME?!" When she was yelling at me I did nothing. I just put it in my own way.
And some people who were leaving the store were looking over at us. It was embarrassing. And the same thing happens when I put away the dishes and do chores. I never do anything. I don't know the root of the problem or why in the first place it was just out of the clear blue. It makes me not want to be around my mom sometimes. | Anyone else experience this? I get an incoming call and when I answer, the call drops. When I eventually connect with the caller, they report modem/fax sounds from the previous call. I think it only happens on wifi. | I feel bad saying this, but my mom is not a good singer and she thinks she’s amazing, constantly shitting on other singers who are so much better than her and she wants to perform in bars and stuff but she’d just and up embarrassing herself, which I do not want to happen.
Should I tell her? Any advice? | While me and my girlfriend were arguing she thought it would be ok to call my mom and grandma bitches but I don’t think she understands where I’m coming from | Im only sixteen how do you tell your parents im pregnant? | ^throwaway ^for ^obvious ^reasons
My mom screams at me alot and i dont know if this is normal but she screams alot to me and when she feels like screaming she just gets mad at me for the smallest things and when her arguments dont go as she wants to she starts screaming and i cant move out cause im not old enough so what can i do now to try to avoid this? | So last night I attempted the EE in Zetsibou no Shima but ran into a annoyance. When I would try and get the cog that is on the docks near the bunker I simply just couldn't get it to drop me. I electrified the panel and tried meleeing and pressing and holding the ACTIVATE button. I tried changeing my controls as well as hooking up my 360 controller I use for rocket league. No matter what button I pushed or held it would not let me drop. I attempted this ~30 times before I decided I was sick of it. Anybody else on PC have this issue / know a fix? | 436 | I am 18m, my past senior year has been utter shit from depression to abuse to being forced to take part in a parental separation to moving houses and living in a friends house. I go to a charter school where below an 80 is failing and there is no way i can catch up. I am transferring to an 'alternate school' instead of repeating the year and my mom keeps calling me a dropout. I keep saying it isn't dropping out because im transferring but she just keeps saying it and it is making me feel bad about myself. I dont know what to do | Dropping out of Uni due to severe depression, not sure what to do next? | I don't know how to tell my parents i want to transition | My parents can’t afford my private education anymore, and I have to go to public school. What should I use as an excuse for why I left? | I’m [18F] moving to a residential college, my divorced parents [50M, 51F] have me torn... please help | Am I dooming myself by dropping out of college? Everyone but my parents seems to think so. | Dropping out of college for two years for financial reasons. What should I do for two years? | Hi, please I neeed real answers, I feel like college suddenly became overwhelming...This is my first semester and I dropped two of my classes which I missed the deadline so I had write a petition to grant my request....and I'm planning to drop my history classs and keep my art history as my only subject left. I know this doesn't look good but I feel like I wasn't honestly ready to start school. Beinig a first generation college student to every step foot in a high institution, in fact I am the first to graduate high school in my family, they put alot of pressure in me to succeed in school. I planned to retake my dropped or failed classes next year and during the summer do you think this is ok? Thank you, I need peoples advice.. and help. | How do I tell my parents I'm dropping out of college for the Marines? |
437 | the arhuaco bag is the cultural symbol of colombia, what magazine is | Bogotá celebrated and maintain the city active year-round . Amongst the most populars is "The Alternative Theater Festival". Bogotá has its own film festival, the Bogotá Film Festival, and many theaters, showing both contemporary films and art cinema. The main cultural center of the city is the La Candelaria, historic center of the city, with a concentration of universities and museums. In 2007 Bogotá was designated the Ibero-American cultural Capital of Iberoamerica. Before the Spanish conquest, the beliefs of the inhabitants of Bogotá formed part of the Muisca religion. From the colonial period onwards, the city has been predominantly Roman Catholic. | What are colombia's traditional clothes? | I've lived in Colombia for over ten years and find this book entertaining and true to my own observations over the last decade. If you are visiting Colombia and want to get a bit more out of it, this book is a must-buy. | Music of Colombia include Grupo Socavón, Grupo Gualajó, and Grups Bahia Trio. A well renowned figure among the old marimbero masters in Colombia is Baudilio Cuama Rentería from Buenaventura Colombia. In the United States two Colombian Bands performing this genre with authentic traditional instruments are La Cumbiamba NY, on the east coast (New York), and Aluna Band in the west coast (San Francisco). In 2010, Currulao has been added to the UNESCO list of Masterpieces of the Oral and Intangible Heritage of Humanity. Bambuco is a type of music with Basque influence, sometimes known as Música del interior. It is not clear the | Who was President in Colombia in 2011? | Alright, this is fairly specific. Is anybody aware of a speciality chocolate store that might sell Colombian brands of fine chocolate? I could order online but I'm just wondering if you guys know of a local shop anywhere in the city that might have them.
there's a picture in the article here of some of the brands I'm thinking of
any help would be greatly appreciated. | Chuspas A "chuspas" (which is Quechua for bag) is a pouch that is used to carry coca and cocoa leaves, used primarily in the Andean region of South America. Both textiles and coca are very important to the people in Andean South America. These "chuspas" are a vital piece of culture and are especially important to combat the bitter cold in the mountainous zones of the Andes. These bags are also a way to showcase the cloth which in itself is a primary artistic medium. Highland textiles are traditionally woven from the hair of native camelids, usually the domesticated alpacas | Carnival in Colombia offend the ruling elites. The result was the uninterrupted celebration of carnival festivals in Barranquilla ("Barranquilla's Carnival"), and other villages along the lower Magdalena River in northern Colombia, and in Pasto, Nariño ("Blacks and Whites' Carnival") in the south of the country. In modern times, there have been attempts to introduce the carnival in the capital, Bogotá, in the early 20th century, but it has always failed to gain the approval of authorities. The Bogotá Carnival has had to wait until the 21st century to be resurrected, this time, by the authorities of the city. Colombia is recognized by its | 437 | the geographical arhuaco, penetrated large Colombian cities (especially Valledupar, Santa Marta, La Guajira and Barranquilla), and is used primarily by young people today as a way to claim their indigenous culture. In 2006, the backpack was nominated as the Arhuaco cultural symbol of Colombia in the contest organized by the magazine "Semana". Arhuaca mochila The mochila arhuaca (English: Arhuaca knapsack), or tutu iku in Ika, is a popular Colombian artisan bag made by the Arhuaco people of the Sierra Nevada. In recent years, the bags have turned into a cultural symbol for Colombian identity. Although the whole arhuaco community is | how much money did mohan bag in maya | what is the name of the constellation in which bagu is formed | Mammoth Tusk Beads and Vintage Elephant Skin Bags: Wildlife, Conservation, and Rethinking Ethical Fashion | Where and how are Gucci bags made? | I've always loved kipling and this is the right purse to carry ... | what was in the ridolfi plot bag | Awesome poop bags for our boxer | Bagot goat |
438 | Can't get enchantment levels over 2 | So uh, I have a Geisha and a Lightin that has both reached level 33 (My Lightin is blue and Geisha is Green 1), and has reached max EXP, but can't level up for some reason. I know that Lightin can still level up from the fact that one of her blue items needs her to be level 35, but no matter what I do, I can't level her up. My account is level 34 btw, so even if the level capacity of the girls depends on my level, I should still be able to at least make them level 34, but I can't. Why? | How do you get level 2 riding howrse? | I'm currently in merciless lab. I am wondering how to tell if the enchant will raise the level requirement of the item? I notice sometimes it does, and sometimes it doesn't. | So i've tried enchanting a few things on my world at level 30, but none of them are tier 4 stuff (Eff IV, etc) they're all tier 3. Is this intended now or is my enchanting just broken?
| I've been jungling for a couple of months now and I can't figure out why sometimes I'm level 2 after I take blue and sometimes I'm not? So how do I know when my exp gets shared? Sometimes I don't get all the cs and still get level 2. (I go Wolfs > Smiteless Blue > Red > Gank)
And when counter jungling, when do I want to leave a creep so it doesn't respawn and when do I want to take all the exp/gold? | I have a problem/bug , My character wont level up . The bar stopped moving. Not a millimeter and I have been slatering vampires all the time. I do have a few mods, but no one that go on touch my leveling skills. | Ive just reached level 10 on my Con Tromboncino but I’ve not gotten any mastery assignments? Ive tried restarting the game and nothing seems to be showing up?? | Awnsers to level 2 on howrse? | 438 | i want to enchant my gear to the best possible but i cant get it past 2 why is this happening and how do i fix it
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edit: i mean by using anvils, i cannot enchant items over the second level no matter what | why cant I disenchant half my gear? | Really confused about enchanting/anvil | Can someone tell me what I'm doing wrong with my enchanting setup? | Help with enchanting! Please. | Level 30 Enchanting broken? | What are the Limits to Enchanting? | Can't enchant Lycanites equipment | help me learn enchantress because i committed a big dumb and bought her by accident |
439 | Microwave-Assisted Synthesis of 2,5-Piperazinediones under Solvent-Free Conditions | Abstract The synthesis of a prototype trisubstituted piperazinedione combinatorial library of 1,000 compounds has been achieved from three precursor sets — two sets of ten α-amino acids and one set of ten aldehydes. A sodium triacetoxyborohydride-mediated reductive alkylation was crucial to the success of the multi-step synthesis on resin. This protocol represents a new method to augment compound files rapidly with novel heterocyclic entities for high-speed screening. | Direct synthesis of 2,4,5-trisubstituted imidazoles from alcohols and α-hydroxyketones by microwave | Abstract Mercuric oxide mediated one-pot synthesis of substituted piperazines via oxidative diamination of olefins with N-protected ethylene diamine has been reported. Among the various conditions tried, mercuric(II)oxide/tetrafluoroboric acid gave good to excellent yields of the desired products. A series of piperazines have been synthesized and characterized by NMR and mass spectroscopy methods. | Abstract A simple and convenient one-pot multicomponent reaction (MCR) has been developed for the synthesis of highly functionalized piperidines catalyzed by molecular iodine. This strategy demonstrated five-component reactions of 1,3-dicarbonyl compounds, amines and aromatic aldehydes in methanol using 10 mol % of iodine at room temperature. This methodology provides an alternative approach for easy access of highly and fully substituted piperidines in moderate to good yields using three readily available starting materials. Notably, this method is mild, cheap, straight forward, applicable to broad range of substrates and environmentally friendly as compared to the existing methods. Synthetic and mechanistic studies are presented here. | Abstract A simple, efficient, and general method has been developed for the synthesis of 1-aminophosphonates from 1-hydroxyphosphonates under solvent–free conditions using microwave irradiation. 1-Aminophosphonates were obtained in high yield and mild conditions by reaction of diethyl 1-hydroxyalkylphosphonates with amines in the presence of acidic alumina. | A simple and efficient procedure for the synthesis of substituted 2-amino-2-chromenes employing one-pot three-component condensation reaction of aromatic aldehydes with malononitrile and activated phenols has been developed by using the odorless and easy to work piperazine in the absence of solvents under microwave irradiation. The present method is operationally simple and offers several advantages such as high yields, short reaction time, and simple workup. | Abstract Microwave irradiation accelerates the bromination of 1, 4-quinones and coumarins with (i) bromine adsorbed on neutral alumina in “dry media” and (ii) with iodine monobromide in acetic acid as compared to the reactions run at room temperature. Bromination takes place selectively at active quinonoid position in 1,4-quinones and at α, β-double bond in coumarins. | A new efficient synthesis of a wide range of ethers, e.g. (III), (V), (VII), is presented, involving the microwave-assisted reaction of tosylhydrazones with aromatic and aliphatic alcohols. | 439 | A general, efficient and environmentally friendly procedure for the synthesis of 2,5-piperazinediones is described, involving the microwave irradiation of N-Boc dipeptide esters. | Design, Synthesis and Biological Evaluation of Novel Piperazine Derivatives as CCR5 Antagonists | Asymmetric synthesis of 2-substituted piperidin-3-ols | Microwave enhanced solution synthesis of 1,4-benzodiazepin-5-ones | N-Arylation of DABCO with Diaryliodonium Salts: General Synthesis of N-Aryl-DABCO Salts as Precursors for 1,4-Disubstituted Piperazines. | Efficient copper-catalyzed cross-coupling of 1-Boc-piperazine with aryl iodides and its application in the synthesis of trazodone | Lactic Acid: An Efficient and Green Catalyst for the One-Pot Five-Components Synthesis of Highly Substituted Piperidines | Synthesis of 1, 4‐bis(3′‐bromopropionyl)‐piperazine‐2, 3‐14C via piperazine‐2, 3‐14C | The process for producing alkylated aryl piperazine, and alkylated aryl piperidine compounds including novel intermediates |
440 | Is the moose population endangered? | Is the deer endangered? | Are beavers endangered or extinct? | Are the beavers endangered? | How many animals are endangered today in canada? | There are roughly 1,000,000 moose living in the world right now. They live in the northern parts of the world and through the use of landbridges could access most of the globe. We are coming for you Australia, and you shall be seen as a bastion of survival until my moose militia rains down hell upon you. There will be no survivors, only me a my personal battalion. | Are mule deer endangered? | How many endangered animals are there in Canada? | What are camel endangered from? | 440 | Which type of moose are endangered? | Are there endangered animals in canada? | Are there endangered animals in canada? | Are there endangered animals in canada? | How are beavers endangered? | How are the arctic foxes endangered? | Is the snow white tiger an endangered animal? | You can control all the moose, anywhere in the globe | are there moose in vermont? |
441 | MA RIP) and it is still writing beautifully - the ink is dark and smooth and it ... | Didn't work well. Blobs of ink came out randomly that I wasn't able to control, so it just made a mess. When it did write , the color isn't very smooth either. Threw it away. | Meh.... The ink is nice but it bleeds all too well on to the next page and doesn't dry as fast | This pen was broken when I received it -- the clip on the cap was loose and the top of it was cracked. Other than these issues, the pen itself writes smoothly and holds a substantial amount of ink (easily enough for 2-3 days of regular writing use). | I really love this ink. It doesn't cake or dry out. Also the color is what I saw on the screen. | The ink runs out soooo fast. It's only been a week and a half and haven't written too much and it already ran out on one of the pens. Very disappointed because I really like the idea of all in one :( | I really like the way they write but after a few weeks the pens seem to dry out and won't write. | The ink is dark and writes smoothly. My favorite part is that this pink isn't super skinny but it feels very light, not heavy. | Came dried out. Barely any ink. I couldn't even get through one letter without the ink drying out, straight out of the package. | 441 | I have had this pen now for 10 years (bought at Pearl in Central Square in Cambridge, MA RIP) and it is still writing beautifully - the ink is dark and smooth and it has somehow never dried out. This is with periods of heavy use (including drafting and filling in plans for architectural school) and a year or two of non-use. When will the ink finally run dry?!! I still use it a couple times of day now. | now my favorite pen will seemingly never run out of ink | I ordered these pens for fine drawing. Two of them have already dried out ... | Strange life span of pens | When your pen runs out of ink, why is it that even with a fresh pen, you still can't successfully write on the area of the paper that you were writing on when the first pen ran out? | What's the longevity like for XP-Pen display tablets? | how long does a capital note last | My pen dies suddenly after around 2-3 minutes. | Ink doesn't last long |
442 | Hello! Im from europe and i met some usa soldiers when i was a kid im wondering if i can share pics i took with them here cause the name is visable? | I'm pretty sure this guy is from one of those military sites but I can't seem to find which one or his name for the life of me
Here's an image to help
He is on the right
Thanks in advance
| The National Infantry Museum and Soldier Center is a museum located in Columbus, Georgia, just outside the Maneuver Center of Excellence at Fort Benning. The 190,000-square-foot museum opened in June 2009.
The museum chronicles the history of the United States Army infantryman from the American Revolution to Afghanistan. It exhibits artifacts from all eras of American history and contains interactive multimedia exhibits. The National Infantry Museum emphasizes the values that are meant to define the infantryman, as well as the nation: loyalty, duty, respect, selfless service, honor, integrity, and personal courage.
In addition to galleries, the National Infantry Museum and Soldier Center also consists of:
Officer Candidate School (OCS) Hall of Honor
OCS Hall of Fame
Ranger Hall of Honor
Ranger Hall of Fame
DownRange Combat Simulators
The Fife and Drum Restaurant
Giant Screen Theater
Heritage Walk
Inouye Parade Field
Memorial Walk of Honor
Vietnam Memorial Plaza
Global War on Terrorism Memorial
The Soldier Store Gift Shop
World War II Company Street.
Until April 2008, the museum was housed in an old Army hospital on Fort Benning. Space and conditions for the museum’s collection was inadequate. In 1998, the 501(c)(3) National Infantry Foundation [1] was formed to plan, raise funds for and to operate a new museum. The National Infantry Museum Foundation has since formed a formal partnership with the Army to manage the facility and its contents. The National Infantry Museum does not receive federal, state or city funding. Through its lease agreement with the National Infantry Museum Foundation, the Army reimburses the foundation for approximately 30 percent of the museum’s annual operating expenses. There is no admission fee. The museum relies on donations, memberships and revenue-generating attractions such as the Giant Screen Theater, combat simulators, Fife and Drum Restaurant, Soldier Store and event rentals to cover operating expenses.
The museum is located on a 155-acre campus adjacent to Fort Benning. The campus includes Inouye Field, sprinkled with soil from the battlegrounds of Yorktown, Antietam, Soissons, Normandy, Corregidor, Korea, Vietnam, Iraq, and Afghanistan, and a 2,100-seat stadium which hosts graduations of Army trainees. The graduations are open to the public.
World War II Company Street is a collection of seven buildings constructed at Fort Benning during the ramp-up to World War II. They have been furnished as they were in the 1940s and are open for tours most days. The buildings include a chapel, barracks, mess hall, orderly room, supply room, and the sleeping quarters and headquarters building used by Gen. George Patton prior to his deployment to North Africa in 1942.
The Vietnam Memorial Plaza contains a ¾-scale replica of the Vietnam Wall on the Mall in Washington, D.C.
The Global War on Terrorism Memorial(under construction Summer 2017) includes the names of 6,800 Soldiers, Sailors, Airmen and Marines killed in service since 9/11. A 13-foot steel beam pulled from the wreckage of the World Trade Center and donated to the museum by New York City firefighters is featured in the design of the memorial.
The museum received a Thea Award for excellence from the Themed Entertainment Association in 2011, USA Today’s 2016, 2020 and 2021 Readers’ Choice Award for Best Free Museum, and TripAdvisor’s Hall of Fame recognition for continued excellence.
Gallery
Notes and references
Putnam, Walter, "Powell salutes soldiers at museum opening", Military Times (Associated Press), August 2, 2009.
Williams, Chuck, "Colin Powell speaks at Infantry Museum grand opening", Columbus Ledger-Enquirer, June 20, 2009.
External links
National Infantry Museum historical marker
Military and war museums in Georgia (U.S. state)
Museums in Columbus, Georgia
United States Army museums | John Hines (Australian soldier) John "Barney" Hines (1878–1958) was a British-born Australian soldier of World War I, known for his prowess at collecting "souvenirs" from German soldiers. Hines was the subject of a famous photo taken by Frank Hurley that depicted him surrounded by German military equipment and money he had looted during the Battle of Polygon Wood in September 1917. This image is among the best-known Australian photographs of the war. Born in Liverpool, England, in 1878, Hines served in the British Army and Royal Navy, and worked in several occupations. He arrived in Australia in 1915 and volunteered | John Hines (Australian soldier) John "Barney" Hines (1878–1958) was a British-born Australian soldier of World War I, known for his prowess at collecting "souvenirs" from German soldiers. Hines was the subject of a famous photo taken by Frank Hurley that depicted him surrounded by German military equipment and money he had looted during the Battle of Polygon Wood in September 1917. This image is among the best-known Australian photographs of the war. Born in Liverpool, England, in 1878, Hines served in the British Army and Royal Navy, and worked in several occupations. He arrived in Australia in 1915 and volunteered | U.S. soldiers throughout Iraq were in high spirits after President George W. Bush made a surprise, morale-boosting visit to Baghdad on Thanksgiving Day. NPR's Peter Kenyon reports. | Can you post a picture on wikianswers? | I ordered these for my high schooler for a project on WWI. We were both shocked that when we released these prisoners of war from their plastic encampment, some were on top of their fellow soldier in very compromising positions. So much for don't ask, don't tell. | Hello,
Does anyone know a local photographer who can do headshot photos for a LinkedIn profile? | 442 | I just wanna say its always fun meeting you guys i met quite a bit of USA soldiers in my town since i live close to the army base. I almost met some SEALS they wanted to visit my school but then corona hit and it all closed down lol. | Tell me about your first meeting! | Meeting for the first time!!! | The strangely epic folks of the Wednesday Gamenight Meetup will be gathering yet again tonight! Come join us! | SD folks who have been to a SMART meeting in person, what was it like and would you recommend? | Anybody ever been stationed at Shaw AFB, SC? | Should we have a daily sticky thread for impromptu meetups? | Pre-Back to School Meet-up, Anyone? | Has anyone here flown to visit their SO before going to boot camp/basic, during DEP? |
443 | when did lakeshore general hospital open | Lakeridge Health Oshawa Lakeridge Health Oshawa, formerly Oshawa General Hospital, is a hospital located in Oshawa, Ontario, Canada. It was founded in August 1910 in a two-story building, and major additions were made in the 1920s (surgical and maternity wings). "G" Wing opened in 1970 on the occasion of the hospital's 60th anniversary. In 1998 the hospital, along with Memorial Hospital Bowmanville, North Durham Health Services, and Whitby General Hospital were placed under the administration of the Lakeridge Health Corporation. The hospital was renamed and, with the closing of Whitby General as a full-service hospital, was given health responsibility over | of that facility; Maynard Hospital became history In the 1970s, important structural changes were made to the hospital's organization and in October 1975, the governing bodies of Seattle General Hospital and The Doctors and Swedish Hospitals eventually announced that they would merge. This was formally recognized on May 5, 1978 when they merged into the Swedish Medical Centre, leading to the closure of the Seattle General facility, which moved to the Seattle Doctors Pavilion in June 1980. Seattle General Hospital Seattle General Hospital and School for Nurses was a hospital and nursing school in the U.S. state of Washington. It | Panangad, Kochi "V.P.S Lakeshore Hospital" is located about a kilometer away from periphery,hence health is never a problem around here. <br> VPS Lakeshore is a multi super-speciality Hospital in Kochi, Kerala. Lakeshore Hospital and Research Centre Ltd was registered as a public limited company in 1996 and started functioning as a multi-specialty hospital in January 2003. The hospital is accredited by the National Accreditation Board for Hospitals & Healthcare Providers (NABH) and has 43 intensive care units and 10 operation theatres. It is one of the largest tertiary care hospital in the state and is managed by VPS Healthcare, which took over | What happened to Manhattan General Hospital? | department, Imaging department, outpatient registration, outpatient lobby, gift shop, laboratory, recovery room, and business office. As of 2011, Massac Memorial Hospital employees 22 full-time registered nurses, 12 full-time licenses practical nurses, 1 part-time physician, 25 part-time registered nurses, and 4 part-time licensed practical nurses. Massac Memorial Hospital Massac Memorial Hospital is a 25-bed general medical and surgical hospital located in Metropolis, Illinois, United States. In 2011, the hospital had 1,002 admissions, 10,031 emergency department visits, and 25,365 outpatient visits. The hospital has cardiopulmonary, cardiac rehabilitation, laboratory, physical therapy, speech therapy, occupational therapy, radiology, surgery, sleep medicine, and transitional care departments. | Brown remained operational for more than a year after the war, when the army finally closed it and transferred the last remaining patients to other sites. Brown General Hospital Brown General Hospital was a military medical facility erected by the Union Army in Louisville, Kentucky, during the American Civil War. It was the largest of six general military hospitals scattered throughout the city. Army surgeons administered the hospital, aided by civilian agencies such as the United States Sanitary Commission and the U.S. Christian Commission. The sprawling hospital was located near the Belknap campus of the University of Louisville, not far | Middlesex Hospital Middlesex Hospital was a teaching hospital located in the Fitzrovia area of London, England. First opened as the Middlesex Infirmary in 1745 on Windmill Street, it was moved in 1757 to Mortimer Street where it remained until it was finally closed in 2005. Its staff and services were transferred to various sites within the University College London Hospitals NHS Trust. The Middlesex Hospital Medical School, with a history dating back to 1746, merged with the medical school of University College London in 1987. The first Middlesex Hospital, which was named after the county of Middlesex, opened as the | demolished and the site used for housing. Chepstow Community Hospital Chepstow Community Hospital in Chepstow, Monmouthshire, Wales accepted its first patients on 26 February 2000 having been developed under the United Kingdom Government's Private Finance Initiative. It was officially opened on 27 October by Rt Hon Rhodri Morgan AM MP, the First Minister for Wales. The 84-bed hospital provides primary, community and secondary care delivered from one location. As well as hospital services the building also houses two GP practices. It was built and will be operated for 25 years by Kintra Ltd for a capital cost of approx £10m, | 443 | Hospital opened its doors in 1965. It is part of the "Centre de santé et de services sociaux de l'Ouest-de-l'Île" (West-Island Health and Social Service Centre). Lakeshore General Hospital The Lakeshore General Hospital (Hôpital général du Lakeshore) (LGH) is a Canadian acute care institution located in Pointe-Claire, Quebec, a suburban municipality near Montreal, Quebec. The hospital employs 1,200 employees and contains 257 beds, and serves an estimated population of 377,000 in the West Island region of Montreal. The LGH is situated close to major highway arteries such as Highways 13, 20, 40, and 520 and is often called upon to | what is the name of the hospital in lake edward, canada | how many sub-acute care beds are in lake taylor | what hospital is in lake charles louisiana | where is john g lake healing rooms located | what county is lakeland health located in | where is montreal general hospital located today | how many doctors work at henry mayo hospital | what hospital is located in maplewood minnesota |
444 | What is the distance between sacramento and washington DC? | How far is it from sacramento to DC? | What is the driving distance from Sacramento to Seattle? | How much distance between washington dc iad washington state? | Distance between trenton and washington DC? | What is the driving distance between Asheville NC and Washington DC? | How many miles from Washington Dc and Williamsburg VA? | What longitude is washington dc? | How long is los angeles from washington dc? | 444 | What is the mileage between Sacramento Ca and Washington DC? | What is the distance from sacramento to dc? | What is the distance between washington state and washington DC? | What is the mileage between alexandria louisian and washington dc? | How far is Washington State from San Francisco? | What is the distance from Los Angeles California to Seattle Washington? | What is the distance between American Samoa and Washington DC? | What is the distance between Washington DC to chicago? | What is the driving distance between washington DC and asheville? |
445 | what was the nationality of the miners hired by jonathan belcher | assistance. Denniston miners and mine managers, like Blackball miners, included former workmates, relatives and friends belonging to the same generation of immigrants, particularly those arriving between 1875 and 1885”. According to Wood: “The tragedy helped to break down some old world differences and establish a West Coast identity especially in the mining community. The bodies of all 65 miners from the Brunner mine were eventually accounted for, including a Mr. John Roberts and three of his sons who were all working that day. Brunner Mine disaster The Brunner Mine disaster happened at 9:30am on Thursday 26 March 1896, when an | of Liverpool. In 1867 there were 13 collieries in the district of Ashton-in-Makerfield. Others followed including Bryn Hall Colliery, owned by Edward Frederick Crippin, the Mains and Park Lane Collieries. Park Colliery and some of those open in 1867 (e.g. Garswood Hall) remained productive until the 1950s. A number of Ashton's coal miners made a significant impact on modern British history, including: Stephen Walsh M.P.; William Kenealy, V.C. and Lance-Corporal in the 1st Lancashire Fusiliers; and Joe Gormley, President of the National Union of Mineworkers in the 1970s and 1980s. In the late 19th century, the district was described by | Brunner Mine And, of course, "The West Coast had the added attraction of gold mining and it seems that the employment opportunity offered by coal mining was in the difficult economic times not one to be turned down". In March 1896 an explosion deep in the mine killed all 65 miners inside, and was labelled the worst mining disaster in New Zealand’s history. It seemed most likely that the explosion was caused by firedamp, a common hazard in coal mines, where a pocket of methane gas is accidentally ignited and explodes. <br> Today all that visibly remains of the mine and its | The Miner In "The Miner", the 19-year-old protagonist decides to flee his hometown of Tokyo after his relationship falls apart. He encounters a grotesque figure who specializes in recruiting cheap labour, and is persuaded to work in a copper mine. The story follows his journey towards and descent into the mine. The protagonist's perceptions and later reflections are described in great detail, such that a "split-second of visual clarity" is accorded three pages of analysis. The protagonist does not get along with the other "animalistic" miners, but eventually meets an educated individual who is, like himself, fleeing from a failed | details the method in which the Belekeri Port was the anchor point of export of illegally mined iron ore. The report mentions the involvement of private companies – Shree Mallikarjun Shipping Pvt Ltd (SMSPL), Adani Enterprises Ltd, Salgaonkar Mining Industries Pvt Ltd, Dream Logistics Company India Pvt.Ltd and Raj Mahal Silks in large scale illegal exports of ore. Belekeri port scam The Belekeri port scam relates to 3.5 million tons of confiscated iron ore that was exported illegally from Belekeri port near Karwar in Karnataka. After Deputy Conservator of Forests R. Gokul seized the ore and the high court refused | Belinga Belinga is a location in Gabon with as yet unexploited iron ore deposits. These ore deposits extend into neighbouring Cameroon and Congo. The Belinga iron reserves were discovered in 1895. They are estimated to hold about one billion tons of iron ore. Iron ore mining was expected to start in 2011 but due to a lack of financing the project is currently on hold. Because of slowness in getting the project started, the Chinese rights may be in 2012 given to Australia's BHP Billiton. A 237 km long railway branch line is proposed branching from Booue to enable these | George Tosh to a Canadian locomotive, two years earlier – but it was certainly a first in Britain, and pre-empted the London & North Western Railway's developments of the technology. Tosh was also amongst the first railway engineers in the country to introduce coal-burning (rather than coke) fireboxes (ten of the M&CR's locomotives had been converted to burn coal by February 1859) and fitted the first steel-tyred wheels to British locomotives. Most of his engines had domeless boilers. Nineteen locomotives of various wheel arrangements were provided during his superintentency. He was married and had at least seven children; at least one of | Deep Navigation Colliery a party of 15 men to the continent to investigate European systems of working practice. As a result of their report, a sum of £8,000 was authorised to build new baths for the miners. The new Treharris baths were constructed by Nicholls & Nicholls of Gloucester, that could accommodate 1,824 men at a time, who were each provided with two lockers: one for clean and the other for dirty clothes. Officially opened on 1 November 1933 by Ocean Coal Company director Thomas Evans O.B.E. of Pentyrch, the baths were the first such facility in South Wales. Having constructed a new | 445 | Jonathan Belcher copper in the colonies, necessitating the costly shipment of ores to England. He eventually established a technically illegal smelting operation. (The Simsbury site, later used by the state as a prison, is now a National Historic Landmark.) Upon the accession of King George I in 1714, Andrew Belcher sent Jonathan to London, seeking to capitalize on the existing connection to the new king. During this trip Belcher engaged in recruiting for his properties in Connecticut. In addition to hiring an experienced metal refiner in England, he also recruited German miners; the area near the Simsbury mine became known as "Hanover" | when was the belfry pulled down in king's chapel gibraltar | who is the name of belgrave square in london | where was prince george william baptized | when did george washington send his army to bedford pennsylvania | who was the captain of the adventure in 1792 | who built king alfred's tower in 1760 | where did the duke of buckingham land in 1460 | beledweyne is the capital of which country |
446 | what was reo speedwagon's best selling album | It should be noted that REM have many great songs. Listening to this CD as I write this review I must state there are only 3 songs out of the 9 that get my attention, they are Orange Crush, I remember California and the 11th which is untitled.
A band like REM have been around for a long time and cannot come out with chart topping songs in every album. What REM tried to do here was be experimental in their choice of style and the rythmns. I applaud REM's methodology but in this CD that played out against them.
I will state that it is overall a strong CD with a more pop feel to it. | Built for Speed is the third studio album by Australian recording artist Adam Brand. The album was released in January 2002 and peaked at number 24 on the ARIA charts. It was certified platinum in 2009.
Track listing
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Weekly charts
Year-end charts
Certifications
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2002 albums
Adam Brand (musician) albums
Mushroom Records albums | Beautiful Rewind is the seventh studio album by electronic musician Four Tet. It was released on 14 October 2013, by his own record label, Text Records. The song "Kool FM" appears in Grand Theft Auto V on the radio station Worldwide FM.
Release
The album was announced on 22 July 2013. Kieran Hebden hinted that the release of the album would be low-key, stating on Twitter that there would be "no pre order, no youtube trailers, no itunes stream, no spotify, no amazon deal, no charts, no bit coin deal, no last minute rick rubin [referring to Rubin's enlistment by Kanye West to perform last-minute alterations to his recently released album Yeezus]."
Reception
Upon its release, Beautiful Rewind received some critical acclaim. At Metacritic, which assigns a weighted average score out of 100 to reviews and ratings from mainstream critics, the album has received a metascore of 79, based on 18 reviews, indicating "generally favorable reviews."
Killian Fox, writing for The Observer, described the album as "an (almost) unexpected pleasure". Fact magazine's Tom Lea offered the opinion that "Beautiful Rewind feels like Four Tet coming full circle" before commenting that "no matter how grubby it gets, at the heart of it will always be one fact: Four Tet's forte is making Beautiful Music." AllMusic reviewer Andy Kellman stated that "while the album does seem rather patched together with a lack of focus... there's an irrefutable charm to the restlessness." In her review for Consequence of Sound, Paula Mejia was disappointed that "the tracks don't necessarily resonate as did the memorable, emotive singles that won over listeners' hearts to begin with on catalogue highlight Rounds".
Track listing
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2013 albums
Four Tet albums
Text Records albums
Albums produced by Kieran Hebden | This is a solid representation of Fastway. I normally find these packages to be lacking because they, without exception, leave out a number of worthy songs because the record companies want you to go buy all the albums anyway. That said, some of the Fastway cds are now out of print. As of July 2012 I can get "Fastway" (1983) & "Waiting for the Roar" (1985), their 1st & 3rd cds reissued, but if I want their 2nd cd "All Fired Up" (1984) I have to pay for the out of print original or get a 2 cd package of their first 2 albums. I find that those 2 cd packages are lacking due to the omission of most or all of the original album art.
If you feel the same as I do then "collection" may be the way to go. You also get a couple of songs from the "Trick or Treat" (1986) soundtrack but nothing from the last 2 Fastway cds "On Target" (1988) & "Bad Bad Girls" (1990). I do feel they could've added a couple more tracks from the "Trick or Treat" soundtrack but maybe they figured that's easy to get anyway. | The Oracle (Godsmack album) DVD including two bonus tracks including the hit "Whiskey Hangover". "The Oracle" has been praised by fans as well as critics as a comeback for Godsmack after their fourth studio album. So far, the album has received a score of 60 out of 100 from Metacritic based on some "mixed or average reviews". "The Oracle" is credited for being the first album by a hard rock band to ever reach number one in 2010, selling 117,500 copies in its first week of release, becoming Godsmack's third consecutive album to do so, and making Godsmack the seventh rock band to ever | Manchester’s The Slow Readers Club have announced their fourth album, 'The Joy Of The Return'. Available to pre-order now on Magenta Vinyl:
| This album is by far the best of all of REM's efforts! The first album after Green, which launched them into the big time, this is full of hits. Losing My Religion, Near Wild Heaven, Shiny Happy People, Belong, and Me In Honey really shine! If you liked Green, you'll love Out Of Time. | The ReAktion having a rock band came from Simon "Nowis" Rojas Hoces and Leonardo Signorelli. They were schoolmate during 97' in Santiago, Chile and became close friends listening intensely to rock music such as; Slipknot, Messhuggah, Alice in Chains, Nirvana and more harmonic rock; The Beatles, Radiohead, Smashing Pumpkins, looking a way to put their musical influences, lifestyle and career together. After playing in different rock bands they came up with "The Reaction" together with Phillip Monypenny in bass and Diego Sagredo in guitar, they both came with musical influences based on Rammstein, Nine Inch Nails, Marilyn Manson, Atari Teenage Riot and | 446 | REO Speedwagon REO Speedwagon (originally styled as R.E.O. Speedwagon) is an American rock band from Champaign, Illinois. Formed in 1967, the band cultivated a following during the 1970s and achieved significant commercial success throughout the 1980s. "Hi Infidelity" (1980) contained four US Top 40 hits and is the group's best-selling album, with over ten million copies sold. Over the course of its career, the band has sold more than 40 million records and has charted thirteen Top 40 hits, including the number ones "Keep On Loving You" and "Can't Fight This Feeling". REO Speedwagon's mainstream popularity waned in the late | what is the title of reo speedwagon's latest album | when was the band retox formed | when did matt o ree band start | reina canaan is a member of which japanese band | What does the initials of the rock band REM stand for? | edward reekers is the lead singer of which dutch vocal group | when did the group h.o.t. split up | who had a uk number one hit in 2006 with 'rewind' |
447 | viswanathan raghunathan is the former president of which bank | In which country is viswanathan anand based for most part of the year? | G. Viswanathan Govindasamy Viswanathan, is the founder and chancellor of Vellore Institute of Technology in India, was born on 8 December 1938 in a remote village in the south Indian state of Tamil Nadu. He served as a Member of Parliament and as a Member of the Legislative Assembly of Tamil Nadu. He was elected to the Tamil Nadu legislative assembly as an Anna Dravida Munnetra Kazhagam candidate from Anaicut constituency in the 1980 election and from Arcot constituency in 1991 election. G. Viswanathan was the sixth child to Thiru. Govindasamy and Tmt. Shrimathi, born on 8 December 1938 in | Raghunathabhyudayam The Raghunāthābhyudayam (or "Raghunāthā-bhyudayam", "Raghunāthābhyudaya", "Ragunatha Abhyudaya") by Rāmabhadrāmbā, one of the wives of the Thanjavur Nayak king Raghunatha Nayak (r. 1600-34), is a Sanskrit "mahākāvya" in twelve cantos. It was designed to valorise Raghunatha, situating his career as a type of the life of Rāma-Viṣṇu-Kṛṣṇa. The first few cantos of the poem invoke Raghunatha, seeking his patronage and assistance, and praise his generosity, piety, and intellect. Canto 4 presents Raghunatha's ancestry and the subsequent cantos discuss his early life and military successes. He succeeds his father Achuthappa Nayak in canto 8 and continues with his military exploits. The | Shaktikanta Das India by the ACC on 11December2018 for a period of threeyears, replacing Urjit Patel who had resigned the day before. Das' appointment as RBI governor received mostly positive responses, with DBS Bank's head of markets for India at Singapore, Ashish Vaidya, saying that previous governors of the RBI with an IAS background, such as Y. Venugopal Reddy and Duvvuri Subbarao, had been successful and that Das' "cordial relations with the government [...] [would] likely help policy negotiations." S. S. Mundra, a former deputy governor of the RBI, said Das was a "good and balanced choice" for RBI governorship and had | Siddique Hossain is a Bangladesh Awami League politician and the former Member of Parliament of Rangpur-8.
Career
Hossain was elected to parliament from Rangpur-8 as a Bangladesh Awami League candidate in 1973.
References
Awami League politicians
Living people
1st Jatiya Sangsad members
Year of birth missing (living people) | Waman Meshram Waman Chindhuji Meshram is the national president of BAMCEF, a non-political, non-agitational, and non-religious organization of employees which represents oppressed and exploited communities in the Indian society. BAMCEF is an acronym for "The All India Backward and Minority Communities Employees Federation". Waman Meshram became national president of BAMCEF in March 2000. Waman Meshram was born in Ramgaon village which is located in Darwha Tehsil of Yavatmal district in Maharashtra, India. Waman Meshram took his primary and secondary school education in Darwha. After this he went to Aurangabad for further education. Waman Meshram started contemplating to join public life | Roberts Orya He thereafter joined Lobi Bank of Nigeria Ltd in January 1994 and later became the Managing Director and Executive Chairman until January 1998 when he was reassigned to Premier Commercial Bank Plc as an Executive Director. Orya's last appointment was as an Executive Director in charge of Capital Markets, Financial Advisory Services and Research & Strategy with Afribank Capital Ltd. He held the position until his appointment on 14 August 2009, as the Managing Director/Chief Executive Officer of the Nigerian Export-Import Bank. Since his appointment less than three years ago, he has fundamentally transformed and refocused the bank and returned | Krishnan Raghunath is an Indian diplomat who served as the Foreign Secretary of India in the late 1990s. He previously served as the Indian High Commissioner to Bangladesh as well as Ambassador to Russia, Nigeria and the Philippines.
Biography
He graduated from the prestigious Madras Christian College. K. Raghunath went to school at La Martiniere Boys' College in Lucknow, India.
He joined the Indian Foreign service in 1962. He married, Sunny, in 1975.
In June 1967, Chinese authorities expelled Krishnan Raghunath and Vijai Padmanab two Indian diplomats on charges of espionage. India retaliated by expelling two members of the Chinese embassy in New Delhi.
From 1978 to April 1979 he was counsellor in the Indian Embassy in Moscow.
He became Foreign Secretary on 1 July 1997 when he took over from Salman Haidar.
In 2001 he became the Indian ambassador to Russia, taking over from Mr S.K. Lambah.
References
La Martinière College, Lucknow alumni
Living people
Indian diplomats
Ambassadors of India to Russia
Indian Foreign Secretaries
High Commissioners of India to Bangladesh
Madras Christian College alumni
1939 births | 447 | Viswanathan Raghunathan Dr. V. Raghunathan (born 1954) is an academic, author, columnist, hobbyist and a CEO. He is currently, Director, Schulich School of Business (York University, Toronto), India (Hyderabad Campus). Since 2013, for three consecutive years, Dr. Raghunathan has been featured in the list of top 50 thinkers in management across India and the Indian disapora. Since 2005, he is also the CEO of GMR Varalakshmi Foundation. Earlier he was President, ING Vysya Bank (2001-2004) and Managing Director, GMR Industries Ltd (2007-2008), GMR Group. He has been an Adjunct Professor at the Bocconi University, Milan, Italy, since 1990, and Schulich | when was bipan chandra appointed as national research professor | Nikil Viswanathan | where is srikanth balachandran working | viswanathan kumaran was listed in which list of top scientists from asia | when was raghavan seetharaman appointed ceo of doha bank | who was the founder of vishal sangh in india | who is the brand ambassador of shiva rajkumar | g viswanathan is the founder of which institute in india |
448 | What kind of habitat does the leopard gecko live in? | What type of habitats do snow leopards live in? | What habitat does a golden retriever live in? | What do amur leopards live in? | What are baby leopard geckos called? | What is a common habitat for a non domestic ferret? | What type of habitat does a snake live? | Where the clouded leopards habitat? | How do panthers live in their environment? | 448 | What is the leopard gecko's natural habitat? | Do crested geckos like the outdoors? | Does the Snow Leopard share a habitat with anther animal? | where does a jaguar live? | im really worried about my female leopard gecko. | How often do you play with a leopard gecko? | Where is the ferret natural habitat? | What is the largest exo terra terraium an adult crested gecko can live in without having to provide multiple food sources. | Looking for very active, small (a bit less commonly kept) gecko species? |
449 | We continued the complex ecological studies in the Lower Volga Region that we started in 2014 in order to investigate the current state of floodplain terrestrial ecosystems and to describe the tendencies in the transformation of the natural environment and the responses of terrestrial ecosystems to changing climate conditions and the influence of anthropogenic factors. The study was conducted with the original two-step method for the estimation of disorders in terrestrial ecosystems after changes in the water content of studied areas. The first step included the determination of possible changes in the water content based on an analysis of long-term annual changes in climatic and hydrological data. The second step included the estimation of disorders in the ecosystems and landscapes based on various biological parameters. We determined the main factors of changes in the abiotic (hydrological and climatic) components of ecosystems in the Lower Volga Region influencing the transformation of landscapes. We described the main causes of changes in a separate component of terrestrial ecosystems related to the increased water flow rate of the winter low-water period and winter runoff, which contribute to a higher level of gleying throughout the entire soil profile and to the degradation of floodplain ecosystems (along with climatic changes). | This paper aims to present the current state of the floodplain forests on the right hand bank of the river Danube around the villages Rusovce and Cunovo. The stands belong to the association Fraxino pannonicae - Ulmetum Soo in Aszod 1936 corr. Soo 1963. They represent fragments of hardwood floodplain forests with typical flora and fauna for such areas. They are important for water management, soil protection, environment protection and landscaping. This paper presents phytocoenology analysis combined with Ellenberg's bio-indication approach and species diversity calculations. | AbstractAs a consequence of the climate change, localized torrential rainfall has increased in intensity and frequency in Northeast Asia. This work aims at studying the effect of rainfall intensity on benthic macroinvertebrate communities in a mid-sized mountain stream in Korea, where the summer monsoon flood is prevalent. Sampling was conducted along reaches in Gapyeong stream (42 km in length), central Korean Peninsula, from a period of July 2008–October 2009. The sampling collection was carried out within 48 h after every major rain occasion and rainfall intensity was quantified by using the total rainfall amount for three days. Both water velocity and Froude number were found to increase with respect to the increase of rainfall at all study sites. As a consequence, the habitat environment and benthic macroinvertebrate communities were apparently affected by the rainfall intensity along the stream reaches. The species richness and abundance of benthic macroinvertebrates were significantly decreased at ... | Abstract The paper focuses on future changes in the short-term rainfall intensities in the western region of Slovakia. The analysis was performed for 4 climatological stations, namely: Malacky, Myj... | RECENT CHANGES OF ANNUAL FLOW DISTRIBUTION OF THE VOLGA BASIN RIVERS | The study analyzes hydrological hazards which should be considered during the planning and implementation of the strategy of long-term socioeconomic development of the Russian Arctic. The results are presented of the analysis of multiyear dynamics of the maximum and minimum streamflow for the Russian Arctic rivers and for the largest rivers in the Arctic Ocean basin and their changes under the current climate warming. The examples of significant negative socioeconomic consequences of hydrological hazards are considered. A high importance of hydrological conditions on navigable waterways for the delivery of most goods to the Arctic region is noted. |
Proglacial areas, defined as the areas left free from glaciers since the Little Ice Age, are open-air laboratories to study the effects of climate change on high mountain environments. Their different abiotic features (i.e. geodiversity) depend mainly on the bedrock characteristics, the type of glaciers acting in the areas and the morphometry of their hydrographic basins, which influence the geomorphic dynamics (i.e., geomorphodiversity). From this, it could derive a different response of glacier forefields to deglaciation and particular evolutionary trends. Hydrological elements and dynamics are particularly variable (i.e. hydrogeodiversity), especially in terms of proglacial lakes diversification, having effects down-valley, even far from the strict proglacial area, and also in term of potential natural hazards. Moreover, geodiversity of proglacial areas may have implications on other types of "diversity". After the glacier retreat, glacier forefields are, in fact, characterized by soils development and vegetation settlement. In particular, soils characterized by different ages and by different degree of development coexist over short distances (i.e. pedodiversity), functioning also as a support for living organisms. Biotic components gradually colonize such areas, from the pioneer to the late-successional species, bringing varied species along the proglacial plains (i.e. biodiversity). All these aspects can be discussed in the perspective of the abiotic ecosystem services (i.e. regulating, supporting, provisioning, and cultural) provided by glacier forefields. Regulating services are related to both atmospheric and terrestrial processes, including natural hazard regulation. Supporting services deal mainly with habitat provision and soils development. Provisioning services include both material (freshwater, building materials) and immaterial (i.e. tourism) resources. Finally, cultural services, that are the most numerous, take into account, among the others, the spiritual and historical meaning, the geohistorical importance for the Earth Sciences development, the educational and geotourism-related opportunities, and the landscape benefit effects. Considering all these aspects, and the intense dynamics proglacial areas are affected by, which will be illustrated through examples mainly from the European Alps, it emerges the importance of a careful monitoring and management of such areas, hopefully through an even more holistic approach.
Università degli Studi di Milano, Earth Science Department A. Desio, Milan, Italy ([email protected]; [email protected]; [email protected]; [email protected]) Università degli Studi di Torino, Earth Science Department, Turin, Italy ([email protected]) CNR-IRPI, Turin, Italy ([email protected]) CNR-IGAG, Milan, Italy ([email protected]) Institute of Geography and Sustainability and Interdisciplinary Centre for Mountain Research, Bramois, Switzerland ([email protected])1
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The Mediterranean region is a hotspot of biodiversity characterized by a warm, dry, and rapidly fluctuating climate 1,2 . This region is particularly threatened by a rapid increase in both the intensity and length of heatwaves and drought periods 3 . North Africa, in particular, is severely affected by drought 4 , threatening the persistence of wetland integrity and freshwater biodiversity 5 . Although local biodiversity is adapted to cope with drought and heatwave episodes 6 , it is unclear whether species plasticity and adaptive mechanisms are effective enough to cope with the rapid rate of environmental fluctuations 7,8 . In addition, species that depend on permanent water such as many lotic species are most likely more at risk of escaping habitat loss 9,10 and so require particular attention.
Free-flowing rivers are characterized by a gradient in physicochemical conditions that govern a biotic gradient of vertebrates and invertebrates from upstream to downstream 11 . However, most rivers worldwide are disturbed by dams, which have consequences for hydrology 12 , sediment loads 13 , fish and invertebrate migration 14 , genetic diversity of populations 15 , and other ecological processes 16 . The combined effects of river damming and drought, which is regular in a hot climate 17 , might exacerbate the ecological impacts of climate change and may lead to the extinction of aquatic fauna 18 . For instance, in southwestern Portugal, where climate is warm and dry, the construction of the Alcántara dam caused an increase in the duration and magnitude of the drought in the Tagus basin downstream 17 with potential consequences for the riparian woody vegetation 19 and fish communities 20 .
In North Africa, there are both temporary and permanent rivers. In dry years, permanent rivers can undergo a great decline in water level and flow with changes in physical and chemical aspects of the riverine habitat. Given
Results
Climate variability. The average annual temperature (Tm) across the Seybouse watershed showed a significant increase during 1980-2018, with a temporal slope of 0.03 °C year −1 (LM: t = 5.64, P < 0.0001, R 2 = 0.46) (Fig. 2a). During the decade 2009-2018, increase in Tm was 0.04 °C year −1 , but it was not significant (t = 1.58, P = 0.15, R 2 = 0.23). The analysis of the annual temperature extremes (minimum [Tmin] and maximum [Tmax]) for the same decade showed that Tmin did not change significantly (slope = 0.004 °C year −1 , t = 0.17, P = 0.86, R 2 ~ 0.00), whereas Tmax showed a marginal increase (slope = 0.07 °C year −1 , t = 2.11, P = 0.06, R 2 = 0.28). Annual precipitation across the Seybouse watershed showed high year-to-year fluctuations but no overall significant pattern during 1980-2018 (t = − 0.51, P = 0.61, R 2 ~ 0.00) (Fig. 2b). However, between 2009 and 2018 there was a temporal decline of annual precipitation of − 10.5 mm year −1 (t = − 2.75, P = 0.02, R 2 = 0.49). Figure 2c shows 30 years (1970-2000) and were obtained from WorldClim v2 77 . HFI ranges from 0 (low human influence) to 100 (very high human influence) 79 . Red circles are historical records of C. exul. Inverted triangle indicates the Bouhamdane dam. The maps were generated using the software R 4.0.2 (https:// www.r-proje ct. org/) and the package raster (https:// rspat ial. org/ raster). Dam water management. Dam maximum water level showed a temporal decline between 2011 and 2018 ( Fig. 3a), following a quadratic pattern (LM: quadratic effect, t = − 4.92, P < 0.0001; Table S1). The rapid decline started in 2016 when the water depth declined by 9.8% from March 2016 (360.6 m) to June 2018 (325.1 m). Although the water discharge for irrigation decreased in recent years compared to previous decades (LM: quadratic effect, t = − 4.02, P = 0.0004) (Fig. 3b), discharges for human consumption increased continuously (LM: slope = 0.37 hm 3 year −1 , t = 7.02, P = 0.0001), albeit at a slower rate in the last decade (Fig. 3c). ) showed a significant increase (P < 0.05). Four (TH, Ca 2+ , Fe 2+ , and NH 4 + ) showed a marginally significant increase (P = 0.05-0.07), whereas the other variables did not show a significant pattern, albeit most of the non-significant increases (e.g. Conductivity, Mg 2+ , Cl − , and RS, P = 0.12-0.16) were probably due to the low sample size (7 years of data).
Damselfly distribution response. The largest number of subpopulations of C. exul in the Seybouse River was recorded in 2011 (N = 8; Table S2, Fig. S3a, b). Of these populations, three were permanent subpopulations during 2011-2016, whereas four were transitory (disappeared and reappeared). In 2018, the number of subpopulations in the river declined to one transitory subpopulation, although previously extirpated since 2012 (Fig. S3c). All three permanent subpopulations, where reproduction commonly occurred, disappeared in 2018 ( Fig. 4a,b). Thus, during the period 2011-2018, there was a loss of 0.85 subpopulations per year.
A combination of all sites from the entire watershed shows that C. exul lived in areas where the average human footprint index (HFI: a metric that varies between 0 and 100 and indicates human influence) within 2 km-radius hexagon was 55.5 ± 14.9 with a minimum of 23.4 and a maximum of 75.6. This value corresponds to the 83 percentile of the HFI occurring in the main watercourses of the watershed which had a mean HFI of 38.2 ± 17.4 (range 21-93).
Adult abundance and recapture rate. Since the water level of the reproductive sites of C. exul fluctuated regularly within a season (Fig. 5), mainly due to dam water discharges and water pumping for irrigation, we investigated the effect of water-level fluctuation on the abundance and detection (recapture) of the species. Based on the temporal pattern of daily abundance in the reproductive season of 2011 at El Fedjoudj P, the number of adult (all and only mature individuals) C. exul showed a quadratic pattern with water depth (Table S3). The largest number of adult C. exul individuals was recorded on days when water depth was intermediate (0.2-0.7 m), while a relatively lower number was noted on days with low water depth (< 20 cm), and lowest numbers were recorded during floods (> 1 m) (Fig. 6a). A great reduction (drought) or increase (flooding) in water level resulted in the disappearance of the preferred reproductive substrates. Drought and subsequent water level drop resulted in bank vegetation being far from the water, while in turn, flooding led to submergence of the oviposition host plants. Even if a number of individuals was recorded during drought, they were not showing reproductive behavior.
Our analysis included 313 marked adults, of which 56.8% were recaptured at least once. The average ± SD number of recaptures was 2.78 ± 2.46 with a median of 2 and a range of 1-19 (Fig. S4). Capture-mark-recapture data showed that the best model for the recapture probability was a quadratic effect of water depth and sex, with lowest recapture probabilities recorded in days with very low or very high water levels, whereas the highest Table S4). Given our sampling was extensive, the absence of both males and females indicated a potential dispersal out of the sampling area due to hydrological changes. Males had a larger recapture probability than females.
Discussion
Our study showed that climate change could have an interactive effect with dam water management, exacerbating drought intensity, and ultimately affecting aquatic organisms. Specifically, the results showed that the prolonged severe drought changed water chemistry, including an uptake in the level of pollutants, and reduced the volume of dam water discharge released into the river yet maintaining water discharges for human consumption. This greatly affected the river water level and flow, and consequently, the distribution of the endangered North African endemic damselfly, C. exul. We highlight the need for action plans that account for climate change scenarios, dam water management plans, wetland bathymetry and hydrology, irrigation requirements, and human disturbance 39,40 to determine the future impacts of climate change on freshwater ecosystems and for managing biodiversity effectively.
Climate change and dam water management. North Africa is one of the driest and warmest regions in the world, which has also undergone severe climate change 4 . Our results showed that the thermal and hydric conditions fluctuated during 2009-2018, showing warmer conditions and more severe drought in later years of the study period. In this region, most freshwater ecosystems are present in the coastal area where communities are limited in the south by the Sahara Desert, and to the north by the Mediterranean Sea, limiting the extent of potential refuges for species. This means that particular attention should be given to the impact of climate change on freshwater communities 41,42 where species physiology, behavior, and distribution are particularly sensitive to environmental changes 41,42 . Increased temperatures, together with reduced precipitation and changes in the 44 , reduced physical space, while also increasing vulnerability to competition and predation 45 , and weakened habitat connectivity 46 which under extreme conditions may lead to drought and local extirpations 47 . Here, we show the consequences of the combined effect of anthropogenic control of river water flow and climate change, leading to increased magnitude and duration of drought, which is expected to increase in frequency in the following years due to climate change 17 .
The results show that the water discharges declined in 2017, stopping completely in 2018 after extreme drought events, leading to a huge reduction in river water flow (river width and water depth). The decision to stop water discharges was the result of a great decline in the dam water supply for human consumption. The river was further exploited for agriculture with water pumps, leading to a complete drought in some parts of the river. This reduction in water quantity and a constant input of agricultural run-off, sewage and industrial discharges reduced water quality for aquatic fauna, crops, and livestock 48 . The predicted increase in water consumption due to human population growth and irrigation is likely to intensify hydrological drought in North Africa 49 . Future models should include all these components to predict future climate effects on the local lotic fauna 50 .
Water physicochemical properties. The temporal pattern of water chemistry showed that many parameters, including indicators of pollution, increased during the drought event. Phosphate increased substantially to levels that foster eutrophication (> 0.1 mg/L) and other adverse environmental effects 51 . Ammonia, which is toxic to aquatic fauna at high concentrations 52 , has also increased in recent years. These changes in water chemistry are probably due to drought which reduces water levels and increases the concentration of nutrients and toxic compounds in the water. The data were used only to infer potential temporal changes in water chemistry and quality in the dam, rather than the levels encountered downstream where C. exul occurs. For instance, phosphate, ammonia, and sulfate levels recorded in raw water before treatment in 2013 were > 60, ~ 50, and ~ 3 times lower than those recorded at Medjez Amar (Seybouse River, upstream) in the same year 53 , respectively. Such increases are due to sewage from high human populations, intensive land use by agriculture, and industrial pollution. Therefore, drought not only affects the physical aspects of the water but also its chemistry, making the environmental conditions of lotic habitats very stressful and pushing the aquatic fauna beyond their physiological tolerance.
Impacts on Calopteryx exul populations. The severe drought of 2014-2017 has probably caused directly and/or indirectly the extirpation of different populations of the endangered damselfly C. exul because it coincided with the rapid loss of populations. The distribution of C. exul in the Seybouse River has rapidly declined over the past decade 34 . Its rapid local extirpation rate in the region now suggests that the species should locally be raised to the category of Critically Endangered in accordance with IUCN criteria B2 and E, which take into account the range size, severity of habitat fragmentation, and local extinction probability over the decade. The current study shows that subpopulations that have shown the most resilience in the past decade 34 were extirpated in 2018, and the remaining populations colonized sites that have experienced extirpation in the past. It is important to point out that the species could have found refuges elsewhere in the watershed, which warrants further investigations in future monitoring. Nevertheless, the future persistence of the species in the Seybouse River is threatened by future drought events, as well as by dam water management for human needs.
Since C. exul is highly dependent on flowing water during its entire life cycle, drought per se (excluding other interactive effects) could have fitness consequences for all life stages. First, eggs are typically laid inside floating leaves near the bank of the watercourse 34 . The reduction in water levels in dry conditions causes desiccation of the plant and eventual mortality of eggs. Second, larvae that typically occur in relatively fast-flowing water are also highly affected by drought as the water velocity and physical aquatic space decline. Although larvae can find refuge in humid areas, a prolonged drought could cause mortality due to predation or desiccation and other non-lethal effects (depleted energy reserves and reduced body size). Third, even if adults are adapted to disperse in the case of drought, and seek more suitable sites to reproduce or hospitable refuge sites to retreat to but not reproduce 54 , drought can have great consequences for the species' population dynamics. The life span of mature adults is short (< 1 month), and so the species is in a 'race against time' , where every day of the reproductive season counts in the overall population dynamics of the species. During an extended drought, suitable reproductive habitats become scarce or even disappear, with females laying only a small fraction of their clutch, if any at all, before they die. This affects the initial population size (number of viable eggs) and, consequently, the size of the emerging population in the next year or the second emergence season of the same year 55 .
Unfortunately, the reproductive season of C. exul overlaps with both the dry season and the increased need for agricultural water pumping. The Seybouse River is bordered by agricultural lands of various crops and fruits, some of which are very demanding on water supply (e.g. watermelons, melons, and tomatoes). This means that there is an already existing high water use for irrigation, which reduces water levels of the river and streams. In dry years, water quantity in the river is reduced but the water pumping demands remain constant, which increases the effects on hydrology and causes cascading effects on aquatic and terrestrial communities 56 . After an extremely dry period and a major decline in dam water quantity threatening water security, the dam stops all water discharges and the river is exploited by farmers until it becomes completely dry. While Bouhamdane Dam occurs in one of the tributaries of Seybouse River, the other main tributary (Cherf River) also has a dam (Foum El-Khanga Dam) with a capacity of 157 million m 3 . This dam is 15 km away from the remaining C. exul population of the Cherf River near Ain Makhlouf, which exists in a highly degraded site due to the construction of a bridge. Therefore, all known subpopulations of the Seybouse watershed are threatened by the combined effect of dam and drought. www.nature.com/scientificreports/ Besides drought, flooding due to dam water discharges changes the water flow dynamics and pushes C. exul to the other extreme of inhospitable hydrological conditions. Flooding may drift larvae to unsuitable habitats where food availability and water quality are low and mortality risks are high 57,58 . Flooding often changes the community structure of the bank vegetation and reduces the availability of oviposition sites. Because the species is selective of habitat characteristics such as water flow and bank vegetation 33 , males do not show reproductive behavior (territoriality) and females stop oviposition during high water, because water velocity exceeds the optimal conditions. Future models for the prediction of the population dynamics of the species should take into account not only drought but also flooding events as important factors that influence demographic parameters of all life stages.
Capture-mark-recapture. Further evidence that the species is sensitive to hydrological regimes is shown by analysis of capture-mark-recapture data. There is a noticeable relationship between the recapture probability of the adults and stream water depth. In both sexes, the recapture rate declined greatly when the water levels were very low (drought) or very high (flooding). This suggests that the species prefers intermediate water depth (also correlated with intermediate water velocity), probably because it confers the optimal conditions for oviposition and survival of eggs and larvae 59 . In non-optimal conditions, C. exul appears to disperse in search of other potential sites, and to escape adverse local environmental change, similar to other species of Calopterygidae [60][61][62] . Previous studies on the species have shown that habitat improvement (due to increased availability of reproductive territories) increases the recapture probability, the local population size of the adults, and the number of reproductive events 34,63 . The sexual difference in the average recapture rate of C. exul is common in odonates, and it is due to behavioral differences where males are territorial, more conspicuous, and permanently near the water, whereas females are more cryptic and may leave the water after oviposition 64 . Nevertheless, both sexes responded similarly to variation in stream levels due to dam water management. It is unclear whether the new patches colonized after habitat quality degradation (drought or flooding) are only temporarily suitable for reproduction or whether the deposited eggs will survive to emergence.
Management perspectives.
Drought is a common event in the Mediterranean climate, with future increases in both frequency and duration imminent 65 . Severe drought could exacerbate the impact of other disturbances such as pollution and water abstraction for irrigation. Effective measures are required for the management of water supplies for consumption and irrigation in conjunction with the conservation of vertebrates and invertebrates of the rivers 66 . We discuss a suite of actions that could improve the conservation status of C. exul and increase the resilience of lotic biodiversity against extreme weather events.
The creation of an artificial ditch system that could be used both for irrigation and refuge for the lotic fauna in case of an extreme drought could attenuate the impact of drought on lotic ecosystems 67 . In South Africa, some Cape endemic species occupy artificial reservoirs during severe drought events and use them as refuges until the favorable conditions return in their reproductive areas 68 . This escape behavior is typical of drought-adapted lotic endemics that are dependent on perennial rivers and streams to reproduce and maintain viable populations. Ideally, a network of artificial watercourses at the watershed scale (conservation corridors) allows the species to disperse, escape drought, and potentially find suitable patches to establish populations 69 . The success of such artificial sites will likely increase if these sites support some attractive habitat elements such as bank vegetation for perching, and floating leaves to guard oviposition sites. For instance, C. exul adults could be lured to specific areas by providing oviposition sites (floating leaves of plants) 34,63 , which could be displaced back to their original sites when the water level return to normal.
In North Africa, lotic habitats receive much less conservation attention than do lentic habitats. Recognizing the ecological and socioeconomic importance of lotic ecosystems is an essential goal for creating protected areas in local watersheds 70 . In the case of C. exul, and in view of the loss of several of its subpopulations these last few years, one approach to save it from extinction would be to designate it a protected area where there is a reduced riparian disturbance. Such habitat protection is likely also to have an umbrella effect, benefitting other threatened endemic dragonflies, such as Gomphus lucasii Selys, 1849, as well as a wide spectrum of invertebrates and vertebrates.
While C. exul might have some resilience to drought, the pumping of water from the river and streams is a supplementary pressure that could push the species to the limits of its resilience by extending the drought period and adversely affecting the larvae out of the water. Watercourse modification is another element that changes water velocity and modifies the integrity of the habitat for aquatic macroinvertebrates in general 71 . Strict regulations are required to limit the amount of water pumped out of natural habitats. Furthermore, alternative water sources for irrigation, such as artificial ditch systems that direct dam water appropriately, are key to solving the problem.
Given that most of the watershed is occupied by agricultural lands, establishing new conservation and restoration measures that promote eco-friendly farming practices and more natural landscape configuration should be at the forefront of the strategic conservation plan 72 . Shifting farming practices that rely heavily on fertilizers and pesticides to more organic farming will most likely reduce the toxic run-off into rivers and streams, improve water quality at reproductive sites, ameliorate habitat quality in foraging sites, and improve food quality (uncontaminated prey) 73,74 . Since the agricultural lands in the watershed are highly simplified monocultures with very low habitat complexity, the addition of habitat elements such as trees, shrubs, and mixture of crop species alongside native grasslands will improve the landscape complexity, maintain biodiversity, and increase ecosystem resilience 75 . Such a shift to diversified agroecosystems should be implemented gradually following awarenessraising among the public and other stakeholders, and subsequently policy directions. www.nature.com/scientificreports/ Instigation of a regular long-term monitoring program at the watershed scale would be crucial for the assessment of population trends, species sensitivity to environmental and anthropogenic perturbation, and the success of newly implemented management plans. In addition, even if conservation authorities will not devote much attention and resources to watershed conservation, a well-planned community science initiative could play a considerable role in data collection, public education, and conservation advocacy. Ultimately, effective management of riverine ecosystems as well as future climate impacts requires a multidisciplinary collaborative network involving scientists from a variety of disciplines-ecology, climatology, hydrology, sociology, economics. Unfortunately, we are currently far from having such an established network along the Seybouse River, despite the urgency of the situation.
Methods
Study site. The study was conducted in the Seybouse River, located in northeastern Algeria (Fig. 1). The river flows into the Mediterranean Sea and is formed by the confluence of oued Bouhamdane and oued Cherf at Medjez Amar, 10 km west of Guelma city.
The Bouhamdane Dam is located in the Hammam Debagh province, 23 km west of Guelma, Algeria (36°27′40″N, 7°14′15″E). The dam is on the Bouhamdame River 7 km upstream from the origin of the Seybouse River (36°26′35″N, 7°18′39″E) and restricts the flow of the Bouhamdame River, which feeds into the Seybouse River. The surface area of the dam is 1070 km 2 with a maximum water level of 370 m. It has a capacity of 220 hm 3 and a velocity of 2240 m 3 s −1 . It provides drinking water for different provinces (Guelma, Ben Djerrah, Medjez Amar, Hammam Debagh, and Ain Hessainia) which require an estimated 9 hm 3 year −176 . In this region, the wet season (mild moist winters) spans October to April/May, and the dry season (hot and dry summers) June to September, coinciding with the reproductive season of most aquatic insects, including odonates 35 Climate and dam data. To characterize the regional climatic conditions in the Seybouse watershed, annual average temperature (Tm) and annual precipitation (P) data (averaged across 1970-2000 and the Seybouse watershed) obtained from WorldClim v2 with 2.5-min spatial resolution were used 77 . Historical monthly weather data for 1980-2018 78 were averaged across years (summed in the case of precipitation) and the Seybouse watershed to assess the temporal trends of Tm and P. To determine the human influence on the watershed and C. exul localities, the global human footprint index (HFI) with a spatial resolution of 1 km 2 was used 79 . HFI is a multivariate metric that includes population density, human land use, infrastructure disturbance (e.g. buildings, night-time artificial lights), and human access (e.g. road, railroads). To evaluate the intensity of human influence in localities where C. exul lives, and across the water system of the Seybouse watershed, the average HFI at 4 km 2 -hexagon grids was calculated. One-month values of Standardized Precipitation Evapotranspiration Index (SPEI), which is a metric that measures drought severity (intensity and duration), were obtained for the study region (36.25°N, 7.25°E) from the global SPEI database 80 . One-month SPEI was used (instead of 3-and 6-month periods) to avoid temporal autocorrelation. A threshold value of − 1.5 was used as an indicator of a significant drought 81 . To assess temporal changes in dam water levels and discharges, data on the monthly maximum water level and monthly quantity of water discharge of Bouhamdane Dam for the period of 2011-2018 were acquired from the dam management executives. In addition, to determine whether the physicochemical properties of the water changed during the drought period, we tracked temporal trends in water physicochemical characteristics of the raw incoming water to the Bouhamdane Dam before treatment using the average annual values of 16 parameters surveyed during 2012-2018, namely, pH, turbidity, conductivity, Total alkalinity (TAC), Total hardness (TH), Bicarbonate ( Sampling methods. Surveys were conducted on the subpopulations of C. exul in the reproductive season of 2017 and 2018. The occurrence of adult individuals (teneral, immature, and mature) were recorded using a 200 m-transect along the bank of the water course. In addition, we used species occurrence data from sites as early as 2011, when the number of subpopulations in the Seybouse was the highest 34 . Each subpopulation was visited at least three times during the reproductive season. For this paper, we assess only subpopulations that occur in the Bouhamdane and Seybouse Rivers because the latter are sensitive to Bouhamdane Dam's water management.
In the reproductive season of 2011, an extensive capture-mark-recapture scheme was carried out in the Seybouse River upstream to estimate population size, assess emergence patterns, describe the reproductive behavior, and understand dispersal habits of the species 35,55,82 . During 28 April-29 May 2011, daily captures of C. exul adults were made in a channel of the river (El Fejdoudj P; 36°28′21″N, 7°22′15″E) across a transect of 100 m during the morning (09:00-12:00). After capturing the damselflies with hand nets, individual alphanumeric codes were marked on a wing with a permanent marker. The marking took only a few seconds and the insect was released immediately in the same location. Daily recaptures (resightings) were carried out. The total number of adults (males and females) was also recorded across the transect. Since the species is morphologically and behaviorally conspicuous, it is highly likely that all individuals at the site were recorded. As the water depth of the study site fluctuates from one day to another (Fig. 5), water depth was daily estimated from a specific location at the center of the waterbed of the studied channel using a graded stick. The fluctuation of water level at the center of the waterbed is reflective of the general changes in the hydrology of the watercourse. We tested for the correlation of this hydrological fluctuation with the variation in recapture rate using the capture history of 313 marked individuals. It is important to note that variation in water depth at a specific location is also an Scientific Reports | (2021) 11:7725 | https://doi.org/10.1038/s41598-021-86383-z www.nature.com/scientificreports/ indicator of a change in water flow; an important environmental feature that highly influences the likelihood that a Calopteryx species reproduces at a given site 59 .
Statistical analyses.
All analyses were carried out using R 3.2.3 83 . The yearly changes in dam water level were analyzed with polynomial regression. Linear regressions were used to assess the yearly changes of water discharges, average, minimum and maximum of annual temperature, and annual precipitation. Capture-markrecapture data were analyzed using a Cormack-Jolly-Seber (CJS) model with the RMark package 84 . The assumptions of the model were checked with the function release.gof which provides tests that assess the independence and transience (TEST2, TEST3, and TOTAL) 85 . All tests showed non-significance (P > 0. 50), suggesting that the data meet the requirements for the CJS model 86 . Since the recapture probability (p) was the variable of interest, the survival probability (Phi) was fixed at one, and tested for the effect of individual (sex) and environmental covariates (water depth [WD]). Candidate models were selected starting with a simple model including the linear effect of one covariate (sex and water depth) to a more complex model including polynomial terms of the water depth (WD + WD 2 ), and the additive effects of the covariates (WD + WD 2 + sex). The models were first computed, then model selection based on the AICc (corrected Akaike information criterion) was performed.
The effect of water depth on the number of adults across the watercourse was analyzed using a negative binomial regression. Values are mean ± SD.
Figure 1 .
1Some environmental characteristics and geographic location of the historical records of Calopteryx exul populations in the Seybouse watershed, Northeast Algeria. (A) Average annual temperature, (B) annual precipitation, and (C) human footprint index (HFI). Temperature and precipitation data represent an average across
Figure 2 .
2Variability in climatic conditions during 1980-2018 in the Seybouse watershed, northeastern Algeria. (a) Temporal pattern of the annual average (solid line), minimum (lower dashed line), and maximum (upper dashed line) temperature. The last ten years are highlighted in red (2009-2018). The blue line is the linear regression and the grey ribbon is the standard error. (b) Temporal pattern of the annual precipitation. The last ten years are highlighted in red (2009-2018). The blue line is a loess regression (grey ribbon is the standard error), indicating a decline in the last years. (c) Monthly values of Standardised Precipitation Evapotranspiration Index (SPEI) for the study region. Positive values (wet period) are in blue and negative values (dry periods) are in red. The dashed horizontal line is set to − 1.5, indicating severe drought. The dashed vertical line indicates the start of the last decade (2009). The black line is a loess regression, indicating a decline in SPEI during the last decade (a drier period).
Figure 3 .
3Temporal pattern of water management at the Bouhamdane Dam (Guelma, Algeria). (a) Monthly values of the maximum water level during 2011-2018. The blue line is a quadratic regression and the grey ribbon is the standard error. (b) Estimated discharge quantity for irrigation during 1990-2018. The blue line is a loess regression and the grey ribbon is the standard error. The last ten years are highlighted in red (2009-2018). (c) Estimated discharge quantity for human consumption during 1990-2018. The blue line is a linear regression and grey ribbon is the standard error. The last ten years are highlighted in red (2009-2018). discharge for irrigation were cyclic events occurring during spring, summer, and autumn (Fig. S1); a period during which the species emerged and reproduced. On average (2011-2018), the largest quantities of releases were recorded in July (6.83 ± 2.87 hm 3 ) and August (5.89 ± 2.82 hm 3 ), while intermediate quantities were released in late spring (May: 3.54 ± 3.64 hm 3 ), early summer (June: 4.65 ± 3.23 hm 3 ), and autumn (September: 4.46 ± 2.83 hm 3 , October: 2.83 ± 2.32 hm 3 ). Between 2011 and 2016, the peak monthly release ranged from 7.2 hm 3 in July 2010 to 9.1 hm 3 in June 2016, and in 2017, there was a small discharge in May (0.26 hm 3 ) and June (1.74 hm 3 ), and no discharges in 2018, leading to a prolonged drought in the Seybouse River.Chemical parameters.To determine whether the physicochemical characteristics of the raw water before treatment showed a temporal pattern during the drought period, 16 environmental variables of the river water (incoming to the Bouhamdane Dam) were assessed during 2012-2018(Fig. S2). Three parameters (TAC,
Figure 4 .
4Reproduction of Calopteryx exul in the Seybouse River in 2011 (a) and a huge drop in water level of the river in 2018. Note that the patches of long-leaf pondweed (Potamogeton nodosus), a preferred oviposition site for the species, which typically float on the water surface are dry due to water abstraction (recorded at intermediate water levels(Fig. 6b;
Figure 5 .
5Variability of stream water level where the endangered Calopteryx exul reproduced, Seybouse River, northeastern Algeria. (a) stream nearly dry, (b) intermediate water level (preferred level of the damselfly), and (c) flooded stream. Note that the three states of the stream were recorded within a single week (Credit: Rassim Khelifa).
Figure 6 .
6The effect of stream water depth on the number of adult individuals (a) and the recapture probability of marked male (blue) and female adults (red) of Calopteryx exul (b). (a) The fitted line is a negative binomial regression for all individuals (blue) and mature individuals only (red). (b) The fitted line is a prediction of a Cormack-Jolly-Seber model for capture-mark-recapture including a constant survival.
. Climatic and anthropogenic characteristics of the Seybouse watershed are presented inFig. 1.
https://doi.org/10.1038/s41598-021-86383-z www.nature.com/scientificreports/Scientific Reports
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© The Author(s) 2021
Data and materials availabilityData were deposited in a GitHub repository: https:// github. com/ rassi mkhel ifa/ Data.AcknowledgementsWe thank the two reviewers for their helpful comments and suggestions. We are thankful to all colleagues who helped in the field surveys. Special thanks to John S. Richardson for his useful input. We also thank the managers of Bouhamdane Dam (Hammam Debagh) for sharing data. The authors declare no conflict of interest. R.K. was funded by the Swiss National Science Foundation (P2ZHP2_175028).Author contributionsCompeting interestsThe authors declare no competing interests.Additional informationSupplementary InformationThe online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-021-86383-z.Correspondence and requests for materials should be addressed to R.K.Reprints and permissions information is available at www.nature.com/reprints.Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 InternationalLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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| The Arctic hydrologic cycle is intensifying, as evidenced by increased rates of precipitation, evapotranspiration, and riverine discharge. However, the controls on water fluxes from terrestrial to aquatic systems in upland Arctic landscapes are poorly understood. Upland landscapes account for 1/3rd of the Arctic land surface and are often drained by zero-order geomorphic flowpath features called water tracks. Previous work in the region attributed rapid runoff response at larger stream orders to water tracks, but models suggest water tracks are hydrologically disconnected from the surrounding hillslope. To better understand the role of water tracks in upland landscapes, we investigated the surface and subsurface hydrologic responses of six water tracks and their hillslope watersheds to natural patterns of rainfall, soil thaw, and drainage. Between storms, both water track discharge and the water table in the hillslope watersheds exhibited diel fluctuations that, when lagged by five hours, were temporally correlated with peak evapotranspiration rate. Water track soils remained saturated for more of the summer season than soils in their surrounding hillslope watersheds. When rainfall occurred, the subsurface response was nearly instantaneous, but the water tracks took significantly longer than the hillslopes to respond to rainfall, and longer than the responses previously observed in nearby larger order Arctic streams. There was also evidence for antecedent soil water storage conditions controlling the magnitude of runoff response. Based on these observations, we used a broken stick model to test the hypothesis that runoff production in response to individual storms was primarily controlled by rainfall amount and antecedent water storage conditions near the water track outlet. We found that the relative importance of the two factors varied by site and that water tracks with similar watershed geometries and at similar landscape positions had similar rainfall-runoff model relationships. Thus, the response of terrestrial water fluxes in the upland Arctic to climate change depends on the non-linear interactions between rainfall patterns and subsurface water storage capacity on hillslopes. Predicting these interactions across the landscape remains an important challenge. | 449 | In the basin of the Lower Volga, the main trends of climate change and their impact on the dynamics of ecosystems are identified. The results were obtained by analysis of longitudinal meteorological characteristics (average, maximum, and minimum air temperature and total seasonal and year precipitation) and the establishment of expected changes in the land ecosystems and landscapes. The basic directions of the aggregate impact of changes of the humid-temperature regime in the basin of the Lower Volga are identified, and the general directions of the dynamics of terrestrial ecosystems in this regard are stated. | Evaluation of climate on the Tibetan Plateau using ERA-Interim reanalysis and gridded observations during the period 1979–2012 | what is the most important climate-forming factor in bulgaria | Trends in sub-annual climate variability since the Little Ice Age in western Europe | Analysis of Ecosystem Services of Wetlands along the Bulgarian Section of the Danube River | Does eastern europe have a milder climate than russia and the cis countries? | Seasonal and interannual variability of the sea surface temperature field in the Adriatic Sea is analyzed from the low-resolution advanced very high resolution radiometer data. The spatial resolution of 18 km allowed analysis of only basin and subbasin scale features. Average monthly and seasonal sea surface temperature fields for the entire studied period (1984–1992) are discussed. The analysis shows the absence of any permanent sea surface thermal features in the Adriatic Sea. The south Adriatic sea surface temperature minimum presumably associated to the cyclonic gyre, previously considered as one of the permanent features, appears to be recurrent, being prominent only in late autumn and early winter, i.e., in the preconditioning and a deepwater formation phases. The major Ionian water inflow is documented in autumn while the thermal signature of the western surface outflow of Adriatic water appears most prominent in winter. The variability of the basin-wide thermal pattern in the Adriatic reveals four distinct seasons, which is different from both the eastern and western Mediterranean, where only two major patterns are recognized. A prominent interannual signal occurs in a northward extension of the warm water plume along the eastern coast, which in some years reaches the northernmost corner of the Adriatic, while in other situations it remains trapped in the south Adriatic cyclonic gyre. The surface thermal signature of the south Adriatic gyre also varies on an interannual timescale, and it was weak or completely absent during the period 1984–1986 while it was rather prominent in the period 1987–1992. A constant trend of sea surface temperature decrease in the center of the south Adriatic gyre and in the northernmost corner of the Adriatic was evidenced over the studied period. | Ecosystem response in temperature fronts in the northeastern Arabian Sea | Climate change analysis in the last 50 years in Xifeng City |
450 | Ms. Cherry shows talent for weaving believable fantasy worlds | Mysterious, dark, full of political intrigue and meticulously researched, Rayne Hall brings this Bronze Age world to life right before the reader's eyes with unique, fascinating characters, vivid detail, and a complex, compelling plot. She puts her expertise on writing fantasy, magical systems, and fight scenes to the test, leaving readers unable to resist turning the page. | Recently my non-Redditor pal's girlfriend (an artist) gifted him a personalized Legendary. He's a rabid Hearthstone fan so it totally floored him. I think it came out great, thought this crowd might appreciate it.
| Derpin aroond the internet as usual, when I chanced upon the concept art of Overwhored (an Adult RPG-Maker Game)
One of the art pieces was done by Mindwipe, and featured a dwarf woman getting it
So yeah, Dwarf Princess aud. Princess coz, idk
And impreg coz impreg is AWESOME
Also BBW, coz Dwarven women in fantasy are built that way
Pretty much it, yeahh
As usual, feel free to make something not *exactly* like it, or something completely different
Hey, thanks :D | Fiddler's Fantasy: Martin, Gil, and Akira wrap up their conversation and performance with Gil and Akira's singular version of the "Carmen" Fantasy, combining the arrangements of Pablo de Sarasate, Franz Waxman, and Jeno Hubay into a singular performance. (NPR) | Final Fantasy: The Spirits Within "technical milestone" while conceding that its "nuts and bolts" story lacked "the intelligence and daring of, say, Steven Spielberg's "A.I."" He noted that while he did not once feel convinced Aki Ross was an actual human being, she was "lifelike", stating her creators "dare us to admire their craft. If Aki is not as real as a human actress, she's about as human as a Playmate who has been retouched to glossy perfection." He also expressed a desire for the film to succeed in hopes of seeing more films made in its image, though he was skeptical of its ability | We address the multilingual and semantic upgrades of two digital catalogues of motifs and types in folk-literature: the Thompson’s Motif-Index of Folk-Literature (TMI) and the Aarne-Thompson-Uther ... | I have heard about manim in YouTube (3Blue1Brown) when I was learning Linear Algebra for my school and was fascinated by the animations and felt in love with it. Later on I found out that it was open-source 🤩 and wanted to contribute.
So what’s your story and how is it going for you ? | I am writing a fantasy novel and I wanted to get some opinions on including real objects or substances in fictional worlds.
I was just writing a scene with two characters in a tent and began to describe the tent walls as being made of hemp when it struck me that perhaps this wasn't correct. Why should hemp exist in another world? It got me thinking that why should a great many other things exist - water, oranges, cotton, horses?
But then again, I feel if I did an entire overhaul of this world, I'd spend far too much time explaining what every little thing is, and then comparing it to familiar items in the real world, which would then arguably take the reader out of the story and out of the narrator's voice since, well, how could the character know to compare their object to one they don't know exists?
How do you feel about including real-world things in fantasy worlds? Do you overhaul? If so, how? Do you pick and choose? I'm interested to know what other people do to make their worlds both fantastical and relatable to the reader. | 450 | Courtesy of Love Romances
This was an average debut book, with a unique futuristic story line. Ms. Cherry shows talent for weaving believable fantasy worlds.
Planet Tigron is home to an amazing race of beings, the Great Djinn, known for their magickal powers and their sexual prowess. The Imperial bloodline is in danger of being wiped out. Their only hope lies in Prince Tarrant-Arragon, the Tiger Prince and the "terror of the dodecahedrons," marrying and impregnating the last remaining virgin of pure Djinn blood to cement his family's line for another generation. There is only one small problem though... she despises him, sight unseen.
Djinni-Vera has lived her life on Earth, safe on a planet away from her enemy, Tarrant-Arragon. There on Earth she waits... until the day comes she is "of age" to marry her betrothed, J-J, the biggest rival of the Tiger Prince, who also happens to be in disguise as Tarrant-Arragon's best friend. Djinni is one of the Saurian White Knights, sworn to destroy the Imperial Prince at any cost and restore balance to the empire.
Tarrant-Arragon goes to Earth in disguise, where he captures Djinni and begins his plan to seduce her and impregnate her before she discovers he is really her most hated enemy, and an enemy who is known for his own unique brand of "justice." He cannot reveal his true self to her, and as he falls in love, he hopes her growing feelings for him are enough to overcome the great deceits he has staged against her. Can he win the affection of his lady love? What will she do when she discovers her beloved "Tigger" is really the Tiger Prince in disguise?
This should have been a very good story that would captivate readers from beginning to end, but it fell flat, at least for this reviewer. The story is well developed, almost too well developed. There are too many sub-plots going on at once to really keep from losing the reader. This reviewer had to keep going back and re-reading parts to try and make sense of what was happening in some scenes. However, it was interesting to see how the game of seduction is compared to a game of chess, which is an underlying theme to the entire story.
Tarrant-Arragon and Djinni are intriguing characters, or Djinni was at least. Tarrant Arragon was too obsessed with sex throughout the whole book. Granted, the key plot focuses on the fact he must get Djinni pregnant, but he spends too much time talking about how he is going to seduce her and how skilled he is at love-making, to actually do much of anything... in or out of the bedroom. Several other characters were incredibly well done, this reviewer's favorite character being Grievous the Earthling, who becomes Tarrant-Arragon's loyal "second." Unfortunately so many characters were introduced in to the story with such similar names that it got way too confusing. There is a family tree in the front of the book to explain the relationships between all the players, but there are just too many people to keep track of, even though they all play key roles in different plot lines. This reviewer kept forgetting who was who and how they were related to others in the story. J-J was an interesting villain throughout, and it would be interesting to see how he comes across in future books... and it is evident there is more planned in the series.
Ms. Cherry certainly wins points for originality in her story. Her worlds are beautifully rendered, vividly drawn so that readers will see and feel the scenery and events unfolding throughout the entire book. The majority of futuristic romance fans will likely enjoy this story and be eager for more to come from the pen of this "Dorchester's New Voice in Romance" finalist.
Kelley A. Hartsell, November 2004. All rights reserved. | Tata masterfully entwines the action and suspense of a terrorist attack with the love, compassion | what is the title of ferenc máté’s first novel | I'm madly in love with darshan | Whats the deal with Karametra's Acolyte? Where's the love at? | Not Romantic Maria has done another wonderful job with a character I simply find ludicrous | An Intimate View Into the Heart of Aikido | where does neela come from in love is a battlefield | True romance like Aokana's Asuka's route |
451 | An Improved Algorithm for Iris Location | A Fast Method for Iris Localization | Iris location is a crucial step in iris recognition.Taking into consideration the fact that in the inner of pupil,there would have some light spots because of reflection,this paper improved the commonly used coarse location method.It utilized the gray histogram of iris graphics,firstly according to the nature of gray level distribution of pupil to compute the binarization threshold,adopting the method of average of the center of chords to coarsely estimate the center and radius of pupil,so as to decrease the searching range and computation time in fine location.After that,the inner boundary(pupil) was fine located using the algorithm of circular detection in the binary graphic.Compared to the previous algorithms,this method could reduce the error of locating within the pupil.Experiments have shown the applicability and effectivity of this algorithm. | Improved Iris Verification System | Adaptive noise reduction and code matching for IRIS pattern recognition system | A new iris segmentation method for recognition | A novel methodology for the interoperability evaluation of an iris segmentation algorithm | Iris recognition is one of the most accurate identity verification systems. Since its initial introduction by J. Daughman, many methods have been proposed to enhance the performance. We present an overview of the latest research on iris recognition by categorizing the research in four groups outlined as localization, segmentation, coding and recognition. We present the latest developments explaining advances to solve problems existing at each of iris recognition stages. ::: ::: ::: ::: Key words: Biometrics, identity verification, iris recognition, pattern recognition, iris segmentation, iris localization and iris code. | Iris recognition technology recognizes a human based on his/her iris pattern. However, the accuracy of the iris recognition technology depends on accurate iris localization. Localizing a pupil region in the presence of other low-intensity regions, such as hairs, eyebrows, and eyelashes, is a challenging task. This study proposes an iris localization technique that includes a localizing pupillary boundary in a sub-image by using an integral projection function and two-dimensional shape properties (e.g., area, geometry, and circularity). The limbic boundary is localized using gradients and an error distance transform, and the boundary is regularized with active contours. Experimental results obtained from public databases show the superiority of the proposed technique over contemporary methods. | 451 | Biometric technologies have shown much more impor- tance in various application. Among them, iris recognition is considered as one of the most reliable and accurate tech- nologies. As the first step of iris recognition, the location of iris will affect the performance of the whole system. This paper proposes an improved algorithm to locate iris and eyelids. Morphological operation is applied to remove eye- lashes in process of iris boundary location. And optimal step length is calculated to reduce search time. Experimen- tal results demonstrate that the proposed iris location algo- rithm is able to achieve a good performance with accuracy more than 97.6%. | A review on advances in iris recognition methods | Improved Iris Location Algorithm | Iris Segmentation and Recognition | A Brief Research and Lookback of the Development of Iris Recognition | Preliminary study on iris recognition system: Tissues of body organs in iridology | This paper briefly reviewed the research background of iris recognition and its development.Gave a survey of existing automatic iris recognition methods,mainly aiming at the latest progress.Concluded and distilled key factors of research difficulties as suggestion to future research. | The richness of the iris texture and its variability across individuals make it a useful biometric trait for personal authentication. One of the key stages in classical iris recognition is the normalization process, where the annular iris region is mapped to a dimensionless pseudo-polar coordinate system. This process results in a rectangular structure that can be used to compensate for differences in scale and variations in pupil size. Most iris recognition methods in the literature adopt linear sampling in the radial and angular directions when performing iris normalization. In this paper, a biomechanical model of the iris is used to define a novel nonlinear normalization scheme that improves iris recognition accuracy under different degrees of pupil dilation. The proposed biomechanical model is used to predict the radial displacement of any point in the iris at a given dilation level, and this information is incorporated in the normalization process. Experimental results on the WVU pupil light reflex database (WVU-PLR) indicate the efficacy of the proposed technique, especially when matching iris images with large differences in pupil size. | A novel and adaptive iris recognition method |
452 | Do you have to have all your music on one computer when you have a zune? | are immediately needed. In his review of the Zune, Paul Thurrott argued that "a song on Zune typically costs 79 Microsoft Points, which, yes, is about 99 cents. But it seems to be less because it's just 79 Points." Walter Mossberg also noted that "to buy even a single 99 cent song from the Zune store, you have to purchase blocks of "points" from Microsoft, in increments of at least $5. You can’t just click and have the 99 cents deducted from a credit card, as you can with iTunes. So, even if you are buying only one song, you | are immediately needed. In his review of the Zune, Paul Thurrott argued that "a song on Zune typically costs 79 Microsoft Points, which, yes, is about 99 cents. But it seems to be less because it's just 79 Points." Walter Mossberg also noted that "to buy even a single 99 cent song from the Zune store, you have to purchase blocks of "points" from Microsoft, in increments of at least $5. You can’t just click and have the 99 cents deducted from a credit card, as you can with iTunes. So, even if you are buying only one song, you | In 2008, when I was shopping around for an MP3 player, everyone was saying go to ipod or iPad. However, on a whim, I tried the Zune and was OUTSTANDING! I only had the regular one at the time, but I bought the Zune HD Touch, the 120 gig. I carry it everywhere. I highly recommend Rhapsody and Tunein for cellphones and computers. | I have wiped my Zune multiple times on purpose to test it. I have 5153 songs in one folder that I am syncing. I format the Zune then sync. Every folder has album art. I start the sync, and every time, the results of the sync are different. All the variables are the same, the results differ EVERY TIME. Here are my findings:
If you have edited your music tags, the Zune will not find the album art the majority of the time, even when the artwork is in the folder of the music. Say you burned a Jimmy Buffett CD and the CDDB used found Jimmy as COUNTRY, so you change it to Caribbean or something else, since the Zune searches pretty much the same CDDB's as everyone else, it will not upload the information or Album art because it does not recognize Jimmy Buffett as Caribbean music under the genre tag. Many of my music is not tagged with the same genre as what it is found on the CDDB's of the world (Porcupine Tree is NOT metal, Miles Davis is NOT Smooth Jazz, etc...). For some reason, it will grab the album art from the directory, sometimes it won't, but I am pretty sure it is because of differences in the tags.
Song totals are not very consistant. I have 5153 songs in one folder with artist and album sub folders. First sync after formating, 4123, second, 2841, third 4875, fourth 5153 (yeah!), fifth 4356. WHAT IS WITH THAT? Every time I get a different sync total. My iPod gets it, the Zune does not.
Sync times are REDICULOUS IF you have files with over 192 bit rate. Every time I do a sync, I kill all running software to let it do it's thing with full processing power. I monitor some of the files that get converted. A certain Tool CD can take 1 minute to convert down from 320 to 192, or it could take over an hour. Conversion of songs is BRUTAL. I don't understand this concept. Most people that want a high capacity player are music nutcases and want quality, not quantity. I want to hear every nuance in Even Less by Porcupine Tree, including the talking in the background, but my ears can tell the difference between 320 and 192, and I don't want 192. Microsoft needs to understand this and change it so some people can stop the conversion.
I think a little more testing and researching the marketplace would have helped Microsoft with the Zune. Hopefully a firmware/software update will fix all of the above, but I doubt Microsoft will be in much hurry. So for now, my Zune goes back on the shelf and my iPod becomes my go to player again. Sadly, I am afraid. | How do photos on your ipod from another computer? | Is ipod better then the zune? | love my zune i had a 30gb got it stolen out off my car so i went out and got a 80gb love it a freind has ipod a zune is much better than the ipod and the car adapter is better than enything ipod sells its worth the money i take it every were i go cell phone and my zune don't leave home with out it | How should I go about that ? I want the jukebox to play all music disc constantly one after another. | 452 | How do you put zune music on computer? | How do you move music from ares to zune? | Where do you go to put music on a mp3 player? | How can you take a wav file and download it to Zune software to make a WMA file out of it and put it on the player? | How do i put music from windows media player onto my mp3 player? | How do you put songs onto an itouch from a computer? | How can you put music on your memory stick? | Does not support Zune | How do you transfer songs off of iTunes onto a CD? |
453 | Promoting independent learning by curriculum design and assessment in a taught postgraduate | Research shows that many students are unprepared for the transition from school or college to university and that the more we can help prepare students for this change, the more likely they will be to succeed and achieve at university. A recent study (Independent Learning; Students’ Perspectives and Experiences, 2015) found that not only are students unaware of the expectations of them to be independent at university, but also of the value of being independent as a life skill. This work has formed the basis and rationale for developing an innovative platform to engage students. Independent Learners’ Toolkit is an online game experience that will help raise students’ awareness of the independent nature of university life. | This paper analyses the importance and present situation of independent English study in our country,with the teaching practice for years,the auther raised from such aspects as the building targets,the building principles and the building plans on the construction of independent English study,the thinking,pos- sibilities and practical. | Investigating the capacity of self and peer assessment activities to engage students and promote learning | SUMMARY A number of case studies in education show that self assessment is possible and is a potentially valuable aid in teaching and learning. However, many of these case studies seem to be dealing with small groups of advanced and fairly well motivated students and so do not necessarily apply to large groups sizes and lower levels of higher education. This paper looks at the case for self‐assessment and argues that self‐assessment is a valuable teaching and learning aid but, that students need to develop skill in self‐assessment. The paper discusses some learning tasks which may develop this skill and argues that these need to be put into a coordinated framework. The paper also examines some of the problems of self‐assessment. This paper was produced as a result of trying to introduce an element of self‐assessment in a level 1 maths and statistics unit for an HND in business and finance. | should be made while keeping integrity of the overall class. Independent study Independent study is a form of education offered by many high schools, colleges, and other educational institutions. It is sometimes referred to as "directed study", and is an educational activity undertaken by an individual with little to no supervision. Typically a student and professor or teacher agree upon a topic for the student to research with guidance from the instructor for an agreed upon amount of credits. Independent studies provide a way for well-motivated students to pursue a topic of interest that does not necessarily fit into a | Independent enquiry study Independent Enquiry Study (IES; Traditional Chinese: 獨立專題探究), which is adopted as the school-based assessment (SBA), counting as 20% of students’ total result in Liberal Studies and sharing one-third of teaching hours, is a compulsory public examination component of the Hong Kong Diploma of Secondary Education (HKDSE) offered by Hong Kong Examinations and Assessment Authority (HKEAA) since 2009. Students are requested to complete an individual project in written (between 1,500 and 4,000 words) or non-written form and either in Chinese or English based on the Liberal Studies Curriculum. With the aims of enhancing students to be ‘independent’ and | I'm envisioning a freely accessible public website that anyone can log in to to take courses (similar to Coursera), but with the following changes:
* Instead of videos, courses are fully interactive modules with graphics, text, voice read-along, links (if a viewer is confused about any aspect of the material, they can click on it for more examples and in-depth explanation), fully worked out examples with annotation and explanation, interactive 'lab'-like components, online tests, etc.
* Option to send questions and suggestions to the course creator to tell them where it could be improved, and these course creators would be on standby for the first year of the course's release during which they will continuously add to and update content according to feedback they receive
* Students' progress would be monitored and should they fall behind, the educational system will loop in their parents to resolve the issue.
And at the end of it all students can sit for a proctored exam to earn a certificate indicating they've learned the material. Teachers would of course be reimbursed for providing this content and helped to transition to a new field.
It would not:
* Would not cover high-level college courses since the number of students would be too few to warrant it and/or changes in the field happen too quickly.
* Would not cover middle and elementary level because students wouldn't necessarily be mature enough to study on their own.
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The reasons I support this is are:
* It would be far cheaper than hiring teachers each year, in each school, for hundreds of thousands of schools (the content may take $500,000 to create per course but only needs to be created once each).
* Anyone who wanted to could use it as an immersive, interactive way to learn a topic of interest.
* The education these courses provide would be far superior to any provided by a single teacher since it would be based on feedback from numerous people, be a collaborative effort by multiple content creators, and they would be held responsible for the quality of their work. Compared to the current system, where there's not much that can be done to either accurately evaluate or improve teaching ability or replace underperforming teachers.
* It gives everyone equal access to education for these courses. This makes it fair for the students, facilitates standardized testing, and makes grades comparable across the country.
* It makes competing for good high schools and colleges (such as moving to the required school zone) less of a concern since the educational quality is consistent.
* It can be done without attending school, and can be done at any time. This also means that parents wouldn't have to send their children to school.
* It allows the teachers to then progress to doing something more useful to society (than just rehashing the same thing over and over), like post-graduate research.
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> *Hello, users of CMV! This is a footnote from your moderators. We'd just like to remind you of a couple of things. Firstly, please remember to* ***read through our rules]( *If you see a comment that has broken one, it is more effective to report it than downvote it. Speaking of which,* ***[downvotes don't change views](#wiki_upvoting.2Fdownvoting)****! If you are thinking about submitting a CMV yourself, please have a look through our* ***[popular topics wiki]( *first. Any questions or concerns? Feel free to* ***[message us***. *Happy CMVing!* | In the School of Business and Finance at Sheffield Hallam University attention has been given to issues which arise as undergraduates seek to cope with the demands of exercising increased responsibility for the management of their learning. Recent research has focused on the views of tutors concerning the degree of support they provide in respect to student self-managed learning. Findings reveal a number of different ‘philosophies’ which range from ‘maximalism’ to ‘minimalism’. While some variety can be justified, it is by no means unproblematic. Hence, it is necessary to give due cognisance to its implications for the student learning experience in the planning and delivery of courses and to ensure that students are made aware of the nature, and reasons for the adoption, of a particular philosophy. | 453 | The purpose of this paper is to consider the need for and importance of independent learning in an emergent profession, radiography. The paper then evaluates whether the revali- dated postgraduate programme in magnetic resonance imaging (MRI) encourages independent learning by its curriculum design, delivery and assessment. Evidence regarding the promotion of independent learning was gained from staff, mentors, learners and external examiners. Fur- thermore the scientific presentations and posters that the learners contributed in the profes- sional conference arena during or subsequent to programme completion were seen as indicators of independent learning, as this work lay beyond the remit of the course. Based on the above, the ethos of the programme to promote independent learning has been successful. | Current situation and improvements in the clinical practice for interns majoring in medical imaging | independent enquiry study is a compulsory component of which diploma | Discussion of the PBL teaching of medical imaging postgraduate on sectional anatomy | Background: Teaching general practice in a university setting is still challenging. In our department we have developed a teaching format with content from a previous lecture-style-teaching into an interactive small group format taught by frontline general practitioners (GPs). The "GP learning stations" introduce students to the skills and attributes of a GP working in primary care in a university setting. Our main objective was to understand whether the teaching format had proven itself sustainable in a university setting over eight years. Furthermore, we wanted to better understand the role of the GP as a medical educator.Methods: More than eight years of experience in organizational and staff expenses were collected and analyzed. In addition, the grade point average of the students' evaluation was calculated and their free text answers were categorized and evaluated descriptively. During two teach-the-teacher seminars attending GPs were asked why they teach and if they feel equipped to teach the format.Results:The initially high organizational and staff expenses were significantly reduced. The recruitment of GPs, their didactic contribution, and their joint creation of content went smoothly throughout the whole period. A total of 495 students participated in the regular evaluation. The analysis yielded a grade point average of 1.9, on a scale from 1 = very good to 6 = insufficient. In the free text answers students praised the educators, the format and the practical relevance. The interactive transfer of the content, the didactic competence of the educators and the spatial environment were viewed critically. Reasons for GPs to teach were the joy to pass on knowledge and experience, and to make the work of GPs more attractive to students. Most GPs felt prepared to teach through their experience as a physician although some felt unprepared to teach through their lack of didactic knowledge.Conclusion:Despite reducing the costs of the format, a grade point average of 1.9 could be achieved in the long term. This supports the teaching concept of learning stations and its "mixture of discussion, scientific background and role play, combined with (…) experiences and exciting individual cases from (GPs) everyday life", hopefully making general practice more attractive to the students. | Abstract Purpose Integrating interactive three-dimensional post-processing software into undergraduate radiology teaching might be a promising approach to synergistically improve both visual-spatial ability and radiological skills, thereby reducing students’ deficiencies in image interpretation. The purpose of this study was to test our hypothesis that a hands-on radiology course for medical students using interactive three-dimensional image post-processing software improves radiological knowledge, diagnostic skills and visual-spatial ability. Materials and methods A hands-on radiology course was developed using interactive three-dimensional image post-processing software. The course consisted of seven seminars held on a weekly basis. The 25 participating fourth- and fifth-year medical students learnt to systematically analyse cross-sectional imaging data and correlated the two-dimensional images with three-dimensional reconstructions. They were instructed by experienced radiologists and collegiate tutors. The improvement in radiological knowledge, diagnostic skills and visual-spatial ability was assessed immediately before and after the course by multiple-choice tests comprising 64 questions each. Wilcoxon signed rank test for paired samples was applied. Results The total number of correctly answered questions improved from 36.9 ± 4.8 to 49.5 ± 5.4 ( p p p p = 0.001), and visual-spatial ability by 11.3% ( p Conclusion The integration of interactive three-dimensional image post-processing software into undergraduate radiology education effectively improves radiological reasoning, diagnostic skills and visual-spatial ability, and thereby even diagnostic skills for imaging modalities not included in the course. | Residents have many questions concerning their training, education, and future, especially in a professional atmosphere that is considered difficult due to increasing financial and political restrictions in the health care system. To objectify this matter, the young radiologist section (YRS) created an online survey.Two hundred and eleven Belgian residents and recently graduated radiologists were invited to answer this electronically based questionnaire within two years after the end of training. The survey was anonymous. Fifty-one questions had to be answered with a scaled response system from 1 to 5 corresponding to an opinion, ranging from "completely disagree" to "completely agree". Questions were divided into five categories: perception of the quality of the residency, reporting system, attitude towards scientific research, career opportunities, and view on the Belgian Society of Radiology.Results showed that residents and recently graduated radiologists are not undividedly satisfied about their training. There is a clear need for information and communication on the curriculum, goals, and expectations of the training. Young radiologists perceive there are differences in the quality of training across Belgian universities; therefore, a need for uniformisation in the residency is expressed. In that view, exchange between universities seems to be an opportunity, and a global interuniversity evaluation at the end of training should be considered.Furthermore, the balance between workload on one hand and education and study on the other remains a juggling act. There is a clear need to create better conditions for scientific work to create awareness to novel evolutions and to prepare colleagues for an academic career. More than 90 per cent of participants responded positively to a subspecialisation in the last year(s) of training, which corresponds to the demands of the European training model in radiology.In conclusion, this survey among residents and recently graduated radiologists showed several points of attention in the current Belgian radiology training. The YRS emphasizes that the radiology residency should be adapted to the quickly evolving landscape of today's health care system.Competing InterestsThe authors declare that they have no competing interests. | Centre for Independent Studies | Student-facilitated radiology-pathology correlation conferences: an experiential educational tool to teach multidisciplinary patient care. |
454 | the buttons for this case are loose and makes the ... | One thing to note is that the side buttons are a bit hard to reach from your thumb. You have to slightly move your thumb up to press them. I prefer if my thumb rests closer to the buttons so you don't have to re-position to actually depress them. | My buttons never lined up. Yes this post is very late, I purchased this a year ago and was never able to properly use the case. I always have to remove my phone from the case to access the power button. | The case was fine but when putting the phone in it or taking it out, the case pressed buttons as it moved through it. | The blue rubber around it is loose but the hard pink case Inside kept phone protected. Of course you need the blue part to push the buttons | Buttons are a bit sticky and some of the rubber on the body is peeling off, but it works fine | Can't use buttons on phone due to it not lining up with them. Doe not fit into the belt carrier well and is hard to get out. | The case never stays on ! Its hard to access the buttons . Waste of money and time !! | the buttons stick out really far so you get get some accidental volume ups and down. | 454 | the buttons for this case are loose and makes the phone look bigger than it really is, not sure how i feel about this case. its also hard to put the phone on silent mode because the slits are too deep. 2/5 stars | This would be my favorite phone case ever if the buttons weren't like pressing cement | The case fits well. Buttons are a little bit ... | Love this case I never realized how well the buttons | Quality case looks and feels just amazing. But the buttons are extremely stiff | I am very disappointed with this case as the edges cover buttons making ... | Case is very bulky and finding the buttons in the ... | No frills case for your iphone 5c, covers some buttons | Buttons break off easily, but protects phone well |
455 | Common house spiders could be vectors of Community-Acquired Methicillin-Resistant Staphylococcus aureus | Are spiders similar to snakes? | How could you kill spiders? | Spider-pathogenic fungi within Hypocreales (Ascomycota): their current nomenclature, diversity, and distribution | As a warrior, I seem to always get overwhelmed and immobilized by the web attack performed by the spiders, the bloody spiders.
Is there any way of defending against it? Shield wall doesn't seem to work against it. | Spider bite not feed on humans and typically bites occur as a defense mechanism. This can occur from unintentional contact or trapping of the spider. Most spiders have fangs too small to penetrate human skin. Most bites by species large enough for their bites to be noticeable will have no serious medical consequences. Medically significant spider venoms include various combinations and concentrations of necrotic agents, neurotoxins, and pharmacologically active compounds such as serotonin. Worldwide only two spider venoms have impact on humans—those of the widow and recluse spiders. Unlike snake and scorpion envenomation, widow and recluse species bites rarely have fatal consequences. | What is the spiders' predators? | I see the little fucks running around my apartment all the time. I never knew Ohio had such spiders til this year. | Spiders of the Kansas Ecological Reserves | 455 | Common House Spiders Are Not Likely Vectors of Community-Acquired Methicillin-Resistant Staphylococcus aureus Infections | small house spiders as "pets"? | Methicillin resistant Staphylococcus aureus (MRSA) as a community strain. | What does it mean if you have spiders in your house? | what do spiders eat in general | Are spiders afraid of conkers because they hate the colour brown? | DAE have a LOT of brown recluse spiders in their apartment/house? | why do ctenizidae spiders need to be protected | Anybody have any orb-weaver spiders in their garden? |
456 | NASA new camera for every blue marble? | Abstract : This document describes a new CCD camera controller adapted to Schmidt telescopes. This report contains the following sections: Generalities; Implementation of a multi CCD camera; Controller boards design requirements; Controller design; Electronic components selection; Current status; Test system; Future readout system; Performance of a 9 wide CCD camera in sky surveillance; plus Appendix 1: Schematics of the controller Appendix 2: Data sheets of the all the major components. | Just bought a Carson RP-100 and I'm inquiring on what celestial objects I'll be able to see. Some of the reviews say that the moon, Venus, Saturn and Jupiters moons are able to view. Will I be able to see Andromeda or any nebulas (nebulae?)? Anything else that I should try and view? Thanks so much for any advice for this noob!
Carson RP-100 specs via link | Where is the world's largest refracting telescope located? | Hello everyone,
Maybe some of you have been taking very nice photos / videos of the blood moon tonite? Could you maybe share them ? Unfortunately I have a rather bad camera on my cellphone. If you find any material in the media I'd appreciate a heads up too. :=) | In the framework of the Near-Earth Objects (NEOs) observational research programme undertaken at the Department of Physics and Astronomy of Catania University, a CCD camera was developed to be used at the 41/61-cm Schmidt telescope of M.G. Fracastoro station. The camera, equipped with BVRI Johnson filters and mounting a bare, front-illuminated 2048×2048 Kodak KAF-4202 CCD with 9 μm pixel-size, was realised at the Catania Astrophysical Observatory Laboratory for Detectors (COLD). The optimisation of the operating conditions of both electronics and cryogenics and the CCD characterisation are presented. | WorldView-4 WorldView-4, previously known as GeoEye-2, is a third generation commercial Earth observation satellite launched on 11 November 2016. The spacecraft is operated by DigitalGlobe. With a maximum resolution of , "WorldView-4" provides similar imagery as "WorldView-3", the highest resolution commercially available at the time of its launch. Work on "GeoEye-2" began in October 2007 when commercial imagery company GeoEye selected ITT Corporation to begin work on long lead-time items for the satellite camera system. In March 2010, an initial contract for construction of the spacecraft was awarded to Lockheed Martin Space Systems, which previously built the "Ikonos" imaging satellite. | The Air Force space surveillance telescope. | Bought this for a recent trip to England. It was perfect for capturing bigger pictures of the castles and architecture. Cant wait to whip it out on more vacations.
Great to get as a gift for travelers. | 456 | So like NASA never release many globes from one "camera". Each time it's different colours and styles and they pretend it's a new camera, so like a camera just gets used once and then they put another satellite for the next globe photo. That's just funny. They must have money to burn because everyone in America is riding around in Ferraris and they've started putting illegal immigrants on welfare instead because they have too much money at NASA.
Meanwhile India hired all their staff from the slums and didn't even put a camera on their rockets to save budgets and the prime minister Modi sells tea on the streets daily to pay for their moon mission. | how many countries participated in india is global photography challenge | where is nasa 360 being filmed at | how many sony saturns were sold worldwide | Controversies and Speed Cameras: Lessons Learnt Internationally | Why is the Google camera so awful at panoramas? | how many countries participate in wildlife photographer of the year | Well, seems VR180 camera are going away | where are the 48 cameras located in the world |
457 | Howard Gordon | Leo Gordon many changes were later instituted on the series, such as the marshal's office and Long Branch Saloon looking markedly different and the relationship between Matt Dillon and Kitty being subtly more formal as well, so the episode was buried deep in the season in the hope that viewers would not notice, which apparently worked. Gordon was often cast to make the most of his 6'2" (189 cm) height, intense features, deep menacing voice, and icy stare. He had radiant light blue eyes. One of his earliest films was "Riot in Cell Block 11", shot at Folsom prison. Other notable roles | Who did gordon merchant start out? | What is dwight howard's number? | How many times has dwight howard been in the olmpics? | Lewis Howard Latimer WHAT did he ivent? | Is howard carter important? | Bob Edwards talks with Roy Johnson, creator of a new magazine geared toward African Americans. | Birth date of gordon merchant? | 457 | Howard Gordon (born March 31, 1961) is an American television writer and producer.
He is well known for his work on the Fox action series 24 alongside the Showtime thriller Homeland, which he co-developed with Alex Gansa and Gideon Raff, and for the FX political drama Tyrant, which he co-developed with Craig Wright. He also produced the NBC science fiction thriller Awake.
Life and career
Gordon was born to a Reform Jewish family in Queens, New York City and graduated from Roslyn High School. After graduating from Princeton with a major in creative writing in 1984, Gordon came to Los Angeles with fellow filmmaker Alex Gansa to pursue a career in writing for television. Both broke into the industry with single episodes of ABC's Spenser: For Hire. Their Spenser work turned industry heads, and the pair joined the series Beauty and the Beast as staff writers, and were later named producers.
In 1990, the Gansa-Gordon team was signed to a two-year deal with Witt-Thomas Productions, during which they produced several pilots. One was an ABC project called Country Estates, which caught the attention of producer Chris Carter.
Soon after, Carter invited Gordon and Gansa to join The X-Files as supervising producers; Gordon wrote or co-wrote several scripts each season, before departing from the series in 1997 to pursue other projects.
After co-writing one episode of Buffy the Vampire Slayer, Gordon created his own show, the short-lived Strange World in 1999. Strange World went to seed 13 episodes in, but Gordon and Strange World writer Tim Minear's services were quickly snapped up by Buffy creator Joss Whedon on another project: Angel. After two years with Angel, Gordon jumped ship in 2001 for FOX's successful 24, where he would write several episodes in Seasons 1 & 2, then crafted the entire story arcs for Seasons 3 and 4. Gordon temporarily left 24 in the middle of the 2004 season to re-join Minear, this time as co-creator of another FOX series, The Inside. Despite The Inside 's cancellation and short run, talk circulated of including the two Minear-Gordon series, Strange World and The Inside, on a special DVD set sometime in 2006.
Beginning in 2006, Gordon became 24'''s showrunner, a title he held through its final season. The successful deal led up with his continuing deal at Fox. In 2019, after a stint at Fox through Teakwood Lane Productions, he signed a deal with Sony.
Gordon is also the author of the Gideon Davis novels.
Homeland
In 2010, after finishing 24, Gordon began co-developing (along with Gideon Raff and Alex Gansa) the thriller Homeland for Showtime. Based on the Israeli series Prisoners of War, it centers on a woman (Claire Danes) who works for the CIA and is convinced a recently returned American prisoner of war (Damian Lewis) has been turned by al-Qaeda. The show premiered Sunday, October 2, 2011, at 10/9 central. It has been met with major critical acclaim and maintained a steady viewership rating throughout its first season. Showtime premiered its fourth season on October 5, 2014.
In 2012, he won the Primetime Emmy Award for Outstanding Writing for a Drama Series for writing the "Pilot" of Homeland and the series itself won the Primetime Emmy Award for Outstanding Drama Series.
Awake
In 2011, Gordon signed on to NBC's new Kyle Killen fantasy pilot Awake as an executive producer. When NBC picked the project up to series status, Gordon added writer and showrunner to his occupational duties on the show. The series only ran from March 1 to May 24, 2012 before it was cancelled.
Second Chance
Most recently, 2015 would see him working as an executive producer on the horror-drama series Second Chance for Fox Television Network. The pilot for Second Chance is based on a script written by Rand Ravich, who also worked as an executive producer on the series.
NovelsGideon's War (also published in the UK as The Obelisk) - 2011Hard Target (also published in the UK as The Chamber'') - 2012
References
External links
Howard Gordon Bio at Icebox.com
1961 births
American male screenwriters
Television producers from New York City
American television writers
Homeland (TV series)
Jewish American screenwriters
Living people
People from Queens, New York
Primetime Emmy Award winners
Princeton University alumni
Showrunners
American male television writers
Screenwriters from New York (state)
Roslyn High School alumni | who presented the tv series 'gordon's great escape' | who played gordon brown in the deal | how many days did alex gordon play on baseball tonight | what was the first leo gordon movie | what is gordon's job in mosquito's | what was the name of the film in which gordon collingridge played a miner | Miles Gordon Technology | where does the character gordon lish appear in |
458 | A thought on Helena Tarsis. | Everyone seems to love a feisty, unconventional heroine, but sometimes I feel that these heroines are "forced." Not in this case! Helena is very unconventional, but there is more than enough motivation making her into the person she is. She is strong and there are plenty of reasons for her becoming a strong heroine. This book is well written. It flows well. I highly recommend it! | Head of the Class her young age, was in high school and the IHP class because of her advanced intellect. Arts student Simone Foster (Khrystyne Haje) was a quiet, sensitive redhead with a particular fondness for poetry. A notable development in the show was the relationship between Simone and Eric Mardian (Brian Robbins), an aspiring writer and, outwardly, the most unlikely member of the IHP - Eric wore black leather, drove a motorcycle, acted tough, and ostensibly disliked anything academic (to Dr. Samuels's delight, he was the only one in the class not on the academic team, although he would never leave the IHP). | The casting is perfect and all characters resemble their older and future selves. And the woman looks more like Martha to me. Though I know it doesn't make sense | Any one think they have an idea who it might be? think its a character we know or if she's yet to be introduced? | Interesting story, but the cast, except for Helen Mirren, is not very strong. The context could be made more informative and textured, although certain episodes certainly convey the feeling of persecution and dread. | I'm playing AC4 for the first time, and I don't get the point of Anne Bonny. She was a little background character in a few cutscenes in Nassau, then a cutscene with her and Mary Read, and now she's suddenly a main character and I'm supposed to care about her. How? She just popped up out of nowhere but it feels like the writers thought she had been a beloved main character all along. | ... she doesn't seem to make sense as an actual person (to me). She feels like a collection of characteristics. But Stevenson has surrounded her with an array of wonderful people and a fun village -- and, as usual, pokes gentle fun at the ridiculous social dilemmas people get themselves into.
This book is considerably better than Miss Buncle's Book (the first in this trilogy) and makes a great intro to the excellent 3rd book, The Two Mrs. Abbots. | In her representative work Wide Sargasso Sea,Jean Rhys deconstructs the speechlessness of the "Other" and thus endows them with discourse right.Among others,Christophine,the black nanny of Antoinette,not only casts a threatening voice for those patriarchal as well as colonial authorities in order to help the heroine and her mother to live through difficulties,but she also spares no efforts to fight for her own rights and liberation.The post-colonial feminism is applied in the paper to interpret Christophine's counter-discourse against colonists and patriarchy authority,thus shedding a new light on this minor character and dual outsider. | 458 | I legitimately believe that Helena Tarsis could control the Anthem of Creation.
thought 1. The sword she wielded. the story goes that she fought a monster, and made it into a sword. That "monster" was called Fulminous.
There's a line in the game where faye is talking about this story, i forget what you say. but i vividly remember faye saying that the heart of rage "is a monster." simply meaning that it's a big ass storm.
the story of helena and the mountain states that: Fulminous lived atop a mountain, from there he sent giants and terrible storms. which sounds a lot to me like an interpretation of a cataclysm that spawned titans and storms from atop a mountain.
she "fought the monster". she then "pried his thunderous voice from his throat and forged it into a sword... the voice of heaven to sing a song of triumph."
so my thought now is this, Tarsis beat a cataclysm. and at the heart of it was the relic responsible, a relic named the Fulminous. (just like the relic at the heart of rage is called the Centotaph)
that bad-ass bitch... forged a sword from, possibly, an active shaper relic.
Thought 2. in "Tarsis and the Sword of thunder" she used the sword on an Urgoth horde. as Arden Vassa says in the main story "She unleashed the voice of the heavens and the mountains bowed to her will"
conclusion: Helena tarsis wielded an active shaper relic, and used it to command the anthem of creation itself. | What famous Greek her fought monsters and killed the nine headed hydra? | So Lore-wise, what is monstrosity in a planeswalker duel? | in greek mythology, who was the queen of messenia who was murdered by | [Monster Concept] Selena, the Dark Spirit Goddess. | Why did Helena Ravenclaw want Tom Riddle to destroy the Diadem? | Enyo, Greek goddess of blood and destructive war | Were did hades rule? | Tempest shouts and Powerful aura |
459 | Some regions have 0 base tax that shouldn't, how do I change this? |
Introduction
Tax revenue in countries in the Latin American and Caribbean (LAC) region remains low compared with developed economies. In 2015, the average tax-to-GDP ratio was 22.8 percent for the entire region and up to 34.3 percent on average for OECD countries (OECD et al., 2017). These differences can be partially explained by the lower contribution of income taxes to total tax revenue. In 2015, taxes on income and profits represented 27 percent of total tax revenue in the LAC region compared to 33.7 percent in OECD countries. Moreover, in 2014, the share of personal income tax was extremely low in LAC countries compared to personal income tax in OECD countries, at 8.7 and 24 percent, respectively. The literature has pointed to three main drivers explaining the region's modest personal income tax contribution: high levels of informality (OECD et al., 2021), the generosity of thresholds for which income is exempted from tax payments, and the presence of generous tax deductions (IDB, 2013). For instance, the income needed to reach the personal income tax brackets represents 0.24 of the income per capita in OECD countries and 1.4 times the income per capita in Latin American Countries. The income needed to reach the highest tax threshold represents 2.38 and 9.1 times of the income per capita in OECD countries and Latin American countries, respectively. Additionally, it is estimated that the region of Latin America sacrifices about 1 percent of GDP in personal income tax revenues due to tax expenditures (IDB, 2013).
The aim of this paper is twofold. First, it provides a comprehensive assessment of the financial cost informal workers would incur if they entered formal employment in five countries in the Andean region: Bolivia, Colombia, Ecuador, Peru, and Venezuela. Financial disincentives to formal employment are measured by formalization tax rates (FTRs), which capture the percentage of earnings in informality that would be lost due to increased social insurance contributions and income tax payments or benefit withdrawal upon entry to formal employment. Then, it analyzes the extent to which formalizing informal workers would contribute to increase fiscal capacity in the region-at a time when fiscal budgets are under pressure-and assesses the distributional implications of counterfactual entries to formal employment. The analysis makes use of multi-country tax-benefit microsimulation models based on nationally representative household survey data: COLMOD (Colombia), ECUA-MOD (Ecuador), PERUMOD (Peru), and LATINMOD (Bolivia and Venezuela). The models have been developed within the EUROMOD framework to ensure cross-country comparability through data and modeling language harmonization (Sutherland & Figari, 2013). The countries under analysis share a number of similar features. They had similar standards of living proxied by GDP per capita in the year under analysis (i.e., 2015). They are all commodity exporters and characterized by volatile revenues and growth. Andean countries have the highest informality rates in the LAC region.
Our results show a wide variation in financial disincentives to enter formal employment implied by the tax-benefit system, with FTRs ranging between 8.5 and 42 percent (in Venezuela and Colombia, respectively). These rates are particularly high for self-employed informal workers with low earnings in all five countries and 1 3
Financial disincentives to formal employment and tax-benefit… are mainly driven by high costs associated with social insurance contribution payments for this group, due to the requirement that social insurance contributions are paid at least on the basis of the national minimum wage. Our simulations of counterfactual entries to formal employment show that the potential to increase social insurance contributions revenue would be substantial under a fully formalized economy. The additional revenue from personal income tax would be high in Bolivia and Venezuela, whereas the effect of a fully formalized economy would be limited in this respect in Colombia, Ecuador, and Peru. Interestingly, our results show that a formalization of informal workers with the highest probability of being formal (i.e., 10 percent of the total informal workers) would enable to capture a substantial share of the additional tax revenue lost due to informality. This group of workers generally face low FTRs, and their potential entry to formal employment would have a positive effect in inequality reduction due to an increased redistributive effect of personal income tax.
The work herein contributes to the literature on potential factors influencing workers' decisions to enter formal employment, with a strong focus on the role of the design of the tax-benefit system as a whole. From a policy perspective, understanding the incentives to formal employment inherent to this system is essential to implement formalization strategies aimed at increasing fiscal capacity and providing long-term social protection to workers. Cross-country comparative analysis offers the additional advantage of learning from the design of different tax-benefit policies to consider potential reforms aimed at creating incentives to formalization. Our work also contributes to the literature discussing the link between informality and low tax revenue. In particular, contrary to the general idea that informality constrains tax collection (OECD et al., 2021), our results show that the additional tax revenue from personal income tax would be marginal even under a scenario of a fully formalized labor force, due to the current design of personal income tax in the countries under study.
The paper is organized as follows. Section 2 provides a brief overview of informal employment in the five Andean countries. Section 3 presents the models and data used in the analysis. Section 4 presents the results of our empirical analysis, and Sect. 5 concludes with a discussion of policy implications.
Informal employment in Latin America
This section provides an overview of the definition and causes of labor informality, followed by a review of the extent to which labor markets in the countries under study are affected by the presence of informal employment.
Brief review of informal employment
High and persistent labor informality has been a major problem for developing countries, especially those in the LAC region, where, on average, 60 percent of the labor force works in the informal sector (IDB, 2018). 1 In the countries under analysis in particular, informal employment accounts for 70 percent of total employment on average. 2 A similar phenomenon is observed in terms of firm informality (La Porta & Shleifer, 2014), 3 which accounts for half of the economic activity. The incidence of informality is one of the most persistent, negative, and worrisome characteristics of the labor markets in the LAC region, where about 140 million of the 263 million workers work in the informal market.
To study labor informality, it is important to understand the origin of its definition. The concept first appeared in 1972 in a publication describing the employment situation in Kenya (Guerguil, 1988;ILO, 1972), 4 and since then, there have been many other published definitions (see Perry et al., 2007). In general, all definitions can be summarized in two main approaches used by the International Labour Organization. The first is the productivity view, which defines informality according to the characteristics of the firm-usually the size-where the individual works. Small firms are considered to be of low productivity and therefore part of the informal sector. 5 The second is the legalistic view, which characterizes informal workers as those without access to the social security or pension systems (Saavedra & Chong, 1999). 6 This paper uses the legalistic view-non-affiliation to any type of social security regime-to define labor informality, as information about affiliation to social security is reported in the data used in the analysis.
The causes of labor informality are widely argued in the pertinent literature. Some works claim that informal firms provide refuge for the poor against excessive government regulations (De Soto, 2000), while other authors look at the informal employment activity as a way of avoiding taxes and regulations, for both workers and firms (Levy, 2008). Another point of view is that informality is correlated 2 Based on the average for 2018 (see the IDB Database: Labor Markets and Social Security Information System, available at https:// www. iadb. org/ en/ sector/ social-inves tment/ sims/ home). 3 Different perspectives can be applied to the study of firm informality. One of the most common approaches relates informality to the size of the firm (in terms of workers), while others relate it to the incorporation of a firm as a legal entity; see Levy (2018) for a discussion on firm informality in Mexico. Another perspective comes from La Porta and Shleifer (2008), who define informal firms as those that are not registered with the government. 4 In the LAC region, the concept of the informal sector was first promoted by the Regional Employment Program in Latin America and the Caribbean (PREALC) of the International Labor Organization. 5 La Porta and Shleifer (2008) analyze the size and productivity of formal and informal firms in poor countries, finding that an average formal firm employs 126 people, while an average informal firm employs only 4. 6 These two views of labor informality can overlap but do not necessarily cover the same set of workers in the informal sector (Gasparini & Tornarolli, 2009
3
Financial disincentives to formal employment and tax-benefit… with poverty (Harris & Todaro, 1970;Rauch, 1991). These studies show differences between the formal and informal firms, where formal entrepreneurs usually have higher education levels, run larger businesses, and are able to pay taxes and adhere to government regulation, with the benefits of increasing customers, raising capital, and accessing public goods, among others. In contrast, informal entrepreneurs tend to have lower education levels, run smaller businesses, and have less productivity.
Regardless of the causes, in general, informality and low productivity are closely associated and put both workers and firms in a vicious circle that is difficult to overcome without a comprehensive public policy strategy. In such vicious circle, low productivity workers, usually self-employed, earn too little to even consider formalization as a viable option. Complementarily, if the benefits of operating in the formal sector are not perceived as valuable, incentives to formalization are further reduced. In this scenario, active formalization policies should follow a holistic approach, reducing labor costs to foster demand of low-income workers, increasing the perceived benefits in the eyes of employees and firms, and boosting labor productivity with reforms in sectors such as education, health, infrastructure, and innovation.
Informal employment in the countries under study
Since 2007, LAC countries have experienced decreasing rates of labor informality (Salazar-Xirinachs & Chacaltana, 2018). However, the evolution shows heterogeneity among countries due to the implementation of different formalization policies (ILO, 2014). Among the countries under study, Ecuador has had a marked reduction in informality (14 percentage points between 2007 and 2018) as a result of active formalization policies and employment surveillance, whereas decreases in Bolivia, Colombia, and Peru have been lower (about 6 percentage points in the same period) ( Table 1). Venezuela's evolution is harder to portray due to a lack of data for the most recent years. Figure 1 provides additional information about the characteristics of informal employment in Bolivia, Colombia, Ecuador, and Peru in 2018, specifically comparing firm size, gender ratio, and quintiles of labor income. As expected, informality was higher among employees of small firms, especially in Peru and Bolivia, while in larger firms, incidences were between 5 and 20 percent. Informality was higher among female workers in Bolivia and Peru, equal in Colombia, and lower in Ecuador. Finally, informality was negatively associated with labor income, as expected. Informality was strikingly high in the first quintile of labor income, ranging between 80 and 100 percent, compared to 30-60 percent in the top quintile of the distribution of labor income. Informality (% of workers not contribuƟng to social security) Financial disincentives to formal employment and tax-benefit…
QuinƟles of labor income
Methodology
The analysis makes use of tax-benefit microsimulation models for Latin American countries, based on nationally representative household survey data. The models are harmonized computer programs performing the computation of taxes and social contribution paid, and benefits received, by each household in the underlying data depending on its income and demographic characteristics.
Data and microsimulation models
Data Our results are based on nationally representative household survey data from Bolivia, Colombia, Ecuador, Peru, and Venezuela. The data sources used in the analysis are summarized in Table 3. All surveys contain detailed information on household and personal characteristics, employment, earnings, income from capital and property, private transfers, cash transfers, pensions, and expenditures. Income concepts have been harmonized in all datasets to achieve comparability in the simulation results (see . Importantly, all surveys contain information about affiliation to social security, which we use to define informal employment. 7 More precisely, informal employment is defined as work without affiliation to social security. 8 Table 2 provides information about informality rates for employees and selfemployed workers in our data. Informal employment is strikingly high among selfemployed workers in all countries, and particularly so in Bolivia, Colombia and Venezuela, where informality rates are above 85 percent. Informal employment is less prevalent among employees but still high, ranging between 38.8 percent in Colombia and 58.6 percent in Venezuela. Figure 11 in Appendix B presents the distribution of earnings for formal (blue distributions) and informal (gray distributions) workers, distinguishing between employees and the self-employed. In all countries, the earnings distributions of The surveys used in the analysis are the official sources to track the evolution of household incomes and expenditures in each country and information about non-affiliation to social security is consistent with official labor force surveys. 8 Note that in Ecuador and Venezuela self-employed social insurance contributions are not mandatory, therefore, the denomination of "informal workers" for self-employed non-affiliated to social security might not be totally fitting. However, the overall aim of the paper still holds, as we are interested in assessing the costs these workers would face if they were affiliated to social security.
formal workers are shifted to the right compared with those of informal workers, for both employees and the self-employed. The only exceptions are for selfemployed workers in Ecuador and Venezuela, where the distributions of informal and formal workers broadly overlap with very similar median incomes (dashed vertical lines). The results for Ecuador and Venezuela are consistent with the design of self-employed social insurance contributions, which are not mandatory for this category of workers. The Figure further shows that there is a high proportion of self-employed informal workers with earnings below the minimum wage (vertical red line), ranging from 28 percent in Venezuela up to 71 percent in Colombia. The proportion of informal employees with earnings below the minimum wage ranges from 9 percent in Venezuela to 54 percent in Colombia. As discussed below, our approach to measure formalization costs for workers, and the budgetary and distributional effects of formalization, consists of simulating transitions from informal to formal employment in our data. Our selected sample for the simulated transitions is composed of all informal workers aged between 18 and 60, excluding full-time students or retirees and those earning less than US$1.9 per day. The latter restriction is imposed to consider only informal workers who might potentially enter formal employment and is in line with the literature on subsistence self-employment in the developing world, where low-productivity workers engage in informal self-employment activities to ensure a minimum level of subsistence, due to the lack of better alternatives (Margolis, 2014). Table 12 in Appendix B presents descriptive statistics for our sample of analysis.
Tax-benefit models Our analysis makes use of harmonized, tax-benefit microsimulation models for Latin American countries based on nationally representative household survey data: COLMOD (Colombia), ECUAMOD (Ecuador), PERUMOD (Peru), and LATINMOD (Bolivia and Venezuela). 9 Tax-benefit microsimulation combines country-specific coded policy rules with representative household microdata to simulate direct taxes, social insurance contributions, and cash transfers for the household population in each country. More precisely, information about market incomes and sociodemographic characteristics of households from the microdata is taken as input in the models for the simulation of tax-benefit instruments, following as closely as possible the policy rules of each instrument according to the national legislation. All models are static in the sense that tax-benefit simulations abstract from individuals' behavioral reactions and no adjustments are made for changes in the population composition over time. Table 3 summarizes the information about 1 3
Financial disincentives to formal employment and tax-benefit… LATINMOD-Venezuela the microsimulation models and data used in the analysis. 10 Appendix A provides a formal presentation of the tax-benefit microsimulation modeling approach. The present analysis uses 2015 tax-benefit policies (as on June 30th) as the starting point in all five countries (i.e., social insurance contributions, personal income tax and benefits in force on 30 June 2015 are simulated for each household in the data). When the data year does not match the policy year, market incomes and nonsimulated tax-benefit variables are adjusted to 2015 levels using source-specific updating factors .
Scope of the simulations and assumptions Our analysis focuses on the concept of disposable income, defined as market income minus direct taxes and social insurance contributions plus cash benefits and pensions. 11 In all five countries, the main policy components of disposable income have been simulated, including employee and self-employed social insurance contributions, personal income tax, and the main cash transfer programs of each country. 12 Due to data limitations, some tax-benefit instruments cannot be simulated and are included directly from the data as part of disposable income. For example, contributory benefits such as public pensions and severance payments cannot be simulated due to the lack of data on contributions; disability benefits, due to insufficient information on the severity of the disability; and property taxes and motor vehicle taxes, due to the absence of information on value in both cases. With the exception of contributory pensions, all other non-simulated instruments represent a minor part of disposable income in the countries under study. Appendix A summarizes the main parameters of the tax-benefit instruments simulated in the models.
To account for the presence of informal employment in the analysis of the Andean countries, we use a harmonized approach to simulate social insurance contributions and personal income tax payments under partial compliance. More precisely, in all countries, employee and self-employed social insurance contributions are simulated only for workers reporting affiliations to social security in the survey. In Peru, only health insurance contributions are simulated for the self-employed, assuming that these individuals do not contribute to a pension fund (neither public nor private). In Venezuela, voluntary self-employed contributions are simulated for 1 3 Financial disincentives to formal employment and tax-benefit… those individuals reporting affiliations to social security, but it is assumed that they pay the minimum (based on the minimum wage) independently from their income levels. 13 For the simulation of personal income tax, we follow a similar approach and assume that only workers affiliated to social security pay taxes. In countries such as Ecuador and Venezuela, where social insurance contributions are voluntary for the self-employed, our assumption could be considered stringent, as some of the self-employed workers not affiliated to social security could in fact be paying income tax. In Bolivia, this assumption is relaxed for the self-employed, where personal income tax is simulated also for those registered in the general or simplified tax regimes.
Measuring financial disincentives to formal employment implied by the tax-benefit system
To quantify the financial cost of formalization, we follow Jara and Rodriguez (2019) and perform counterfactual simulations consisting of moving informal workers in the data into formal employment and comparing their household disposable income before and after the transition. As previously mentioned, transitions to formal employment are simulated for all those in the dataset between the ages of 18 and 60 currently working and reporting non-affiliation to social security (i.e., informal workers), excluding full-time students or retirees and those with very low earnings (i.e., below US$1.9 per day). More formally, our approach to simulate transitions from informal to formal employment consists of the following steps. First, household disposable income is calculated for all informal workers in the dataset before any transition is simulated. Then, for each informal worker in the household, we impose affiliation to social security in the data and assume that their earnings remain the same under the new status of formal workers. 14 Finally, our tax-benefit models simulate the amount of social insurance contributions and personal income tax the worker would be liable to pay according to the legislation in force in 2015, as well as his or her corresponding household disposable income under formalization. We simulate transitions to formal employment for each informal worker in household member separately, assuming that the status of any other informal workers remains unchanged.
This simulation makes it possible to analyze budgetary and distributional effects of entries to formality and quantify the financial incentives to formalization implied by the tax-benefit system, which we measure with FTR (Koettl, 2013;Koettl & Weber, 2012;Weber, 2015). We follow Koettl and Weber (2012) and define FTR as the proportion of earnings in informal employment that would be taxed away after entry to formality. More precisely, we define FTR of individual i as:
where x w i represents worker i 's earnings in informal employment, y h,i represents household disposable income for worker i in informal employment, and y ′ h,i represents the counterfactual household disposable income for worker i when they are moved to formal employment.
Different from previous studies that use hypothetical data to measure the burden of formalization implied by the tax-benefit system in European countries (Koettl, 2013;Koettl & Weber, 2012;Weber, 2015), our models make it possible to calculate these indicators using household survey data from LAC countries . As such, we are able characterize the distribution of FTRs across populations and sub-populations, as well as select different categories of individuals for specific transitions into formal employment.
Caveats Our approach assumes that earnings of informal workers remain the same upon entry to formal employment for the calculation of FTRs. This assumption is made to obtain an FTR indicator which reflects the financial disincentives to enter formal employment implied by the design of the tax-benefit system, given the observed characteristics of the population. As suggested by Jara and Rodriguez (2019), assuming changes in earnings upon entry to formal employment would not only require imputing potential earnings for informal workers but also redefining the concept of FTR to measure the proportion of changes in earnings (rather than the proportion of earnings) that would be taxed away upon entry to formal employment. Under the latter approach, FTRs would capture the combined effect of changes in earnings and the design of tax-benefit policies, which would need to be disentangled. Moreover, FTRs incorporating changes in earnings might be sensitive to the method to impute earnings. For this reason, we opt for the original definition of FTRs, which captures the financial disincentives to formal employment implied by the tax-benefit system only.
However, it is important to note a number of caveats related to our definition of FTR. First, our analysis measures FTRs from the perspective of workers, as payroll taxes paid by employers are not factored into the financial disincentives of informal employees to enter formal work. Integrating payroll taxes as a cost for employees would increase their FTR. Even if they are not integrated into FTRs, it is important to bear in mind that higher costs for employers (i.e., additional payroll tax payments) might result in a reduction in wages for salaried workers, potentially increasing the formalization costs of informal employees. Second, our FTR indicator is a 'short-term' measure of financial disincentives implied by the tax-benefit system, in the sense that we assume that social insurance contributions are considered a cost
(1) FTR i = y h,i − y � h,i x w i 1 3
Financial disincentives to formal employment and tax-benefit… in the short term. However, in the medium and long term, social insurance contributions give entitlement to the public health system and public pensions. These expected benefits are not taken into account in our FTR indicators. Third, our analysis assumes tax compliance upon entry to formal employment, in the sense that social insurance contributions and personal income tax is calculated on the basis of the totality of earnings reported by informal workers in the survey (i.e., no tax avoidance or tax evasion). Finally, our FTR indicators focus on the financial disincentives implied by the tax-benefit system. However, overall incentives to enter formal employment might be influenced by a large number of factors including, for instance, gender role attitudes and labor market constraints. Note that some of the caveats become more relevant in the second part of the empirical analysis, where we assess the budgetary and distribution effects of potential entries to formal employment (Sect. 4.2). We come back to these points when we discuss the results.
Empirical results
This section presents the results of our evaluation in two steps. First, it provides a comprehensive comparative analysis of the distribution and composition of FTR, as well as the variation of financial disincentives to formal employment across population subgroups in each of the five countries under analysis. Second, we simulate a number of counterfactual distributions where a fraction of informal workers would enter formal employment and assess the implications of such entries on personal income tax and social insurance contributions revenue, the number of taxpayers, and social insurance contribution payers, and income inequality.
Financial disincentives to formal employment
In this section, we use household representative data to calculate financial incentives to formal employment, allowing us to characterize the distribution of FTR at the population level of informal workers in each of the five countries and compare indicators across different subgroups. We focus on the contribution of different taxbenefit components to FTR. Based on this analysis and the heterogeneity in the data, we assess whether particular population subgroups face higher disincentives to enter formal employment. In particular, we distinguish between salaried employees and self-employed informal workers who have presented contrasting patterns . We also distinguish informal employees in firms of different sizes (i.e., micro, small, medium, and large). As they might face the lowest financial disincentives to formality in large firms, informal employees in those firms are also more likely to make the transition to formality. To broaden the analysis, we study the distribution of FTR across economic activities.
Distribution of FTRs
Figure 2 presents mean and median FTRs, as well as the inter-quartile range between the 25th and 75th percentile of FTR. The results show a large variation in financial incentives to enter formal employment across countries. The highest average FTR is in Colombia, where 42 percent of earnings in informality would be taxed away upon entry to formal employment as a result of increased social insurance contributions and tax payments or reduced benefits, and the lowest (8.5 percent) is in Venezuela.
In between are Ecuador with 18.3 percent, Bolivia with 23.1 percent, and Peru with 23.9 percent.
The results further show that countries with higher values of average FTR are also characterized by a more dispersed distribution, depicted by the inter-quartile range. Venezuela shows very little variation of FTRs, while in Colombia inter-quartiles range between 11.4 percent (25th percentile) and 52.7 percent (75th percentile). In all countries, although to a lesser extent in Venezuela, the distribution of FTR is skewed to the right, with means higher than the median.
The differences in the distribution of FTR across countries can be explained by differences in: (i) the composition of the informal population and (ii) the design of tax-benefit policies. The following section provides further insights into the role of tax-benefit policies and population characteristics by analyzing the contribution of different policy instruments on FTRs across populations at different income levels.
Decomposition of FTRs
The distribution of FTR is determined by the design of specific tax-benefit instruments. We would expect that social insurance contributions contribute the most to FTR, as entry to formal employment implies affiliation to social security. Minimum and maximum social insurance contribution payments, or the presence of different contribution rates for specific categories of workers, would affect the distribution of FTRs. Personal income tax would also influence financial disincentives to formal employment. The extent to which it would contribute to FTRs depends on parameters such as the level of the exempted threshold, the structure of the tax schedule, Financial disincentives to formal employment and tax-benefit… and the presence of tax deductions. Given the generosity of the design of the personal income tax in the countries under analysis, we do not expect it to have a large contribution to the FTR and to the decisions regarding potential formalization. Finally, cash transfers would also affect FTRs if entitlement to the benefit is linked to (non-)affiliation to social security.
In addition to the design of tax-benefit policies, the characteristics of the population in each country will also determine the distribution of FTRs. For instance, countries where informal workers' earnings are particularly low would present higher FTRs if social insurance contributions require minimum payments above the level of their earnings. An overrepresentation of self-employed workers in informality would also induce higher FTRs if the social insurance contribution rate is higher for this group compared to the rate for formal employees. Figure 3 presents a decomposition of mean FTR. To account for the role of individual instruments at different points of the income distribution, the figure provides the decomposition across four socio-economic categories: poor, vulnerable middle class, consolidated middle class, and rich. The categories are defined based on household disposable income per capita and the income thresholds specified by the Inter-American Development Bank (IDB, 2020) included in the Appendix of this paper (Table 13).
Our results show that in all countries except Venezuela, mean FTR decreases with income and social insurance contributions contribute the most to FTR. Financial disincentives to formal employment are particularly high for poor informal workers in Colombia, with FTR of 65 percent. This reflects mainly lower average Authors' calculations based on microsimulation models. Note Socio-economic categories defined based on household disposable income per capita and the income thresholds specified by IDB (2020) earnings in this population group compared to earnings for the same group in other Andean countries, 15 as well as relatively high self-employed contribution rates of 28.5 or 30.5 percent. 16 In general, direct taxes play only a minor role in determining FTR. In Colombia, Ecuador, and Peru, direct taxes contribute to FTR of wealthy informal workers only. Direct taxes represent 1.4, 4.2, and 3.2 percentage points of average FTR for Colombia, Ecuador, and Peru, respectively. In Venezuela, they play a larger role than social insurance contributions for the rich, contributing with 11.8 percentage points to their average FTR (15.7 percent) and, to a lesser extent, for the consolidated middle class. In the case of Venezuela, the misalignment between the evolution of prices and wages and the parameters of the personal income tax (the Unidad Tributaria) make its structure similar to that in more developed countries in terms of progressivity, which explains the larger contribution of taxes to FTR. In fact, it is due to the role of direct taxes that FTRs are U-shaped in income, rather than presenting the decreasing pattern observed in other countries.
Bolivia is the only country where direct taxes contribute to FTR throughout the income distribution. In Bolivia, personal income tax is part of the Régimen Complementario del Impuesto al Valor Agregado (RC-IVA), which allows the value added tax (VAT) paid on purchases to be deducted from the income tax liability. Since the parameters of the standard and VAT deductions are the same across the income distribution, the income tax is not progressive. On the contrary, it slightly favors individuals with higher incomes whose VAT purchases are higher. Another reason is the tax on self-employed workers, where no exempted income threshold applies. The contribution of direct taxes remains, however, modest in all five countries, which is due to two main characteristics of design of personal income tax in LAC countries, in which the Andean region is not an exception. In all countries, except Bolivia, personal income tax is characterized by the presence of high non-taxable thresholds, meaning that in the event of entering formal employment, most informal workers would not fall into the tax brackets that would make them liable to pay income taxes. Moreover, deductions from personal expenditures can be made from taxable income, which reduce the volume of taxpayers. Finally, the contribution of cash transfers to FTRs is extremely limited (or null) because eligibility to social benefits in these countries is based on composite welfare indexes (i.e., proxy means-testing), which do not directly depend on affiliation to social security. 17
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Financial disincentives to formal employment and tax-benefit…
Heterogeneity across population subgroups
The previous section pointed to the interaction between the design of tax-benefit policies and the characteristics of the population-namely income-in determining FTR. This section exploits the advantage of using representative household survey data to assess whether indicators of FTR vary across different population subgroups, such as by gender, age, skill level, employment status, region, and economic activity. Figure 4 compares the distribution of FTR distinguishing between informal employees and informal self-employed workers. This comparison is interesting because the prevalence of self-employment among informal workers varies across countries (see Table 12 in the Appendix) and, at the same time, the rules of social insurance contributions are specific to each of these employment statuses. Our results show that the wide variation in the overall distribution of FTR across the five countries (see Fig. 2) is mainly driven by the distribution of FTR of the self-employed. In fact, mean FTRs for employees vary minimally across countries, ranging between 7.3 percent in Venezuela and 16.8 percent in Peru. On the contrary, important differences are observed in terms of financial disincentives to formal employment for self-employed workers, who face on average higher FTRs than employees in all countries. FTRs are particularly high for self-employed workers in Colombia, for whom 57.9 percent of their earnings in informality would be taxed away upon entry to formal employment. In Ecuador, Bolivia, and Peru, selfemployed workers face similar FTRs, ranging between 26 and 30 percent on average. Venezuela is the only country where the FTR of the self-employed remains low (12 percent). Looking at the distribution of FTR for employees versus self-employed workers, there is a wide variation in terms of inter-quartile ranges, driven mainly by the dispersion of FTR observed for the self-employed.
The large financial disincentives to formal employment observed for selfemployed workers in Colombia can be explained by three factors. First, the proportion of informal self-employed workers is larger than in other countries. Self-employed workers account for 64.6 percent of our sample of informal workers in Colombia (see Table 12 in the appendix). Only Bolivia shows a higher prevalence of self-employed informal workers (69.6), whereas the levels are much lower in Ecuador (40.2) and Venezuela (25.9). Second, labor income is low in Colombia compared to the other Andean countries. According to our estimates, monthly labor income is US$209 in Colombia compared to US$360 in Peru, US$392 in Bolivia, and US$478 in Ecuador. Finally, as explained earlier, Colombia presents higher selfemployed contribution rates than the other countries under analysis. In any case, the pattern of high FTR among the self-employed is expected since these workers tend to earn less than their salaried counterparts and their earnings are more volatile.
To explore more deeply the differences in financial disincentives to formal employment among employees, Fig. 5 presents mean FTRs by the size of the firm in which employees work in all countries except Venezuela (due to lack of data). In Bolivia, Colombia, Ecuador, and Peru, employees working in firms with 1-5 workers present higher FTR than employees in larger firms. The pattern is consistent with the discussion about the relationship between firm size and productivity, which might regroup low-skilled and low-paid workers. Colombia and Peru show a clear decreasing pattern of FTR by firm size. The gap in mean FTR of employees in small firms (1-5 workers) compared to big firms (more than 100 workers) equals 5 percentage points in Colombia and 3.7 percentage points in Peru. The relatively minor differences in FTR observed are explained by the fact that the design of employee social insurance contributions does not vary across firm size. The differences are mainly due to the varied composition of the workforce across firms.
In addition to differences in FTRs between salaried employees and the selfemployed workers-which are related to the composition of the labor market, wage structure, and design of social insurance contributions-there could be other patterns for population subgroups based on varying characteristics of gender, age, skill Financial disincentives to formal employment and tax-benefit… level, location, economic activity, and income. Table 4 presents mean FTR across different population subgroups.
A gender divide in financial disincentives to enter formal employment is observed in all five countries. Female informal workers present higher FTRs than their male counterparts. The gap in FTRs is the largest in Colombia, representing a 12.2 percentage point difference (49.7 percent versus 37.5 percent), followed closely by Ecuador, where female informal workers face an FTR 11.9 points higher than male informal workers (25.8 percent versus 13.9 percent). The differences in FTR between male and female workers are driven by the characteristics of these groups. On average, there is a higher prevalence of low-skilled, self-employed female workers, which most likely leads to lower incomes. Table 4 confirms that these characteristics are associated with higher levels of FTR. The results also show pronounced differences between workers in rural and urban areas. In all countries, except Ecuador, informal workers in rural areas face higher FTRs than those in urban areas. The gap is the largest in Colombia, where rural informal workers face an FTR almost twice as large as their urban counterparts (35.9 percent versus 60.4 percent). The gap is larger than 10 percentage points in Peru (34.4 percent versus 21.4 percent) and Bolivia (30.4 percent versus 19.9 percent). A contrasting pattern is observed in Ecuador, where rural informal workers face an FTR 6.3 points lower than their urban counterparts (14 percent versus 20.3 percent). The rural-urban pattern observed in Ecuador relates to the presence of Seguro Campesino in Ecuador, a social insurance regime for self-employed rural workers with lower contribution rates than the general regime. Under Seguro Campesino, the amount of social insurance contributions paid by members of this regime is equal to 2.5 percent of 22.5 percent of the minimum wage, compared to a 20.5 percent contribution rate on gross employment income for other categories of selfemployed workers.
Differences in financial disincentives to formal employment are also observed across economic activities. In Bolivia, Colombia, and Peru, mean FTRs are the largest in the agriculture and fishing sector. The results are consistent with the patterns observed between informal workers in rural and urban areas, as agriculture and fishing activities are mainly located in rural regions. Consistent with this pattern, mean FTRs for agriculture and fishing are the lowest in Ecuador, again mainly due to the presence of Seguro Campesino, which covers rural workers in these sectors of activity. Both the mining, manufacturing, and utilities sector and the retail, wholesale, hotels, and restaurants sector also present higher FTR than other industries, especially in Colombia. The lowest mean FTRs are observed in the construction sector in Colombia, and Peru. In Ecuador, the construction sector, along with the agriculture and fishing sector, presents low mean FTRs. In Bolivia, the construction sector follows that of financial intermediation, real estate, and business activities, in terms of the lowest mean FTRs. Finally, in Venezuela, the results do not show a clear pattern, with similar mean FTRs across sectors.
To analyze the association between individual characteristics and financial disincentives to formal employment, we regress mean FTRs on the set of characteristics discussed above. Table 5 shows the results.
To account for the role of earnings in determining the level of FTRs, in addition to the logarithm of earnings, we include a dummy variable identifying informal workers with very low earnings, i.e., below half of the minimum wage in each country. Our results show that having earnings below half of the minimum wage is positively correlated with higher FTRs, whereas the logarithm of earnings has a negative and significant effect capturing the fact that FTRs decrease with the level of earnings. The only exception to the latter is Venezuela, where the logarithm of earnings has a positive and significant effect, which is in line with the U-shaped form observed in Fig. 3 due to the contribution of personal income tax to FTRs at the top of the distribution in Venezuela. Our low-earnings dummy and the logarithm 1 3
Financial disincentives to formal employment and tax-benefit… of earnings account for a large share of the variation in FTRs, from 68 percent of the variation in Ecuador to 92 percent of the variation in Bolivia.
In line with the descriptive assessment of FTRs across population subgroups, being self-employed is significantly associated with higher FTRs. The effect of being a rural worker is negative and significant in Ecuador, in line with the discussion about the role of Seguro Campesino. On the contrary, after controlling for other variables, the coefficient for female informal workers has a positive and significant effect only in Venezuela, meaning that higher mean FTR for females (Table 4) are explained by differences in earnings and other covariates included in the regression.
Assessing the distributional and budgetary implications of formalization
Informality is usually discussed in the literature as a barrier to increase fiscal capacity and boost long-term growth in developing countries (see OECD et al., 2021). Yet, little is known about the budgetary and, in particular, distributional implications of potential entries to formal employment. In this section, we simulate a number of counterfactual distributions where a fraction of informal workers would enter formal employment. We then assess the implications of such entries on tax revenue, the number of taxpayers, and income inequality. The analysis is static, in the sense that it does not consider behavioral responses due to potential entries to formal employment, neither does it consider the fact that the structure of wages might change as a result of entries to formality. In this sense, the results should be interpreted as first round effects with the aim of providing insight into the effects of potential transitions from informal to formal employment, with a focus on the characteristics of the labor force and design of tax-benefits systems.
In contrast with the previous section, here we assume that upon entry to formal employment informal employees would earn at least the minimum wage (i.e., the firms where they are employed would comply with the minimum wage legislation). Earnings of self-employed informal workers are kept fixed upon entry to informal employment as they are not automatically entitled to receive the minimum wage (i.e., they are not employees working for firms) and their earnings usually fluctuate depending on their economic activity. Note, for instance, that an important share of the actual self-employed formal workers in the data has earnings below the minimum wage, ranging between 16 percent in Colombia and 47 percent in Ecuador ( Figure 11 in Appendix B).
It is important to acknowledge that the probability of entering formal employment is not uniformly distributed across all informal workers. As suggested in the previous section, some population subgroups face overly high FTRs and therefore, could be less likely to move to formal employment. From the demand-side, low-skilled workers could face difficulties finding work in the formal sector. For this reason, our approach works in two steps. First, we rank workers in informal employment by their probability of being formal. Then, we simulate counterfactual distributions where a fraction of informal workers would enter formal employment based on their probability of being formal.
The counterfactual simulations work as follows. First, the 10 percent of informal workers with the highest probability of being formal are moved into formal employment by means of tax-benefit microsimulation. Then, cumulatively, the next 10 percent of informal workers with the highest probability of being formal are moved to formality, until we assess the effect of moving all informal workers to formal 1 3 Financial disincentives to formal employment and tax-benefit… employment. For each of these counterfactual distributions, we assess the additional tax revenue and number of taxpayers due to transitions to formality, as well as the effect of such transitions on income inequality.
Probability of being in formal employment
We use a probit model to estimate the probability of being in formal employment. The dependent variable takes the value one if the person is in formal employment (is affiliated to social security) and zero otherwise. Variables traditionally used in the literature discussing the determinants of formality are used as regressors and include the following: gender (male), age and age squared, education (skill levels), marital status (married), number of children, rural area, employment status (self-employed), the logarithm of earnings and a dummy identifying individuals below the minimum wage. Table 6 presents the results of the estimation.
Our results show that earnings are a strong determinant of the probability of being formal. In all countries, the probability of being formal increases with the logarithm of earnings. Controlling for other variables, being male increases the probability of being formal in Bolivia and Colombia, but it decreases it in Ecuador, Peru, and Venezuela. The probability of being formal increases with age at a decreasing rate, except in Venezuela, where the coefficient of age squared is positive and not significant. The probability of being formal increases with education level, even after controlling for earnings.
In all five countries, being self-employed is associated with a lower probability of being formal. In terms of family characteristics, being married increases the probability of being formal, whereas the probability decreases with the number of children, probably capturing the fact that low income families where informality is more prevalent have a larger number of children on average. Finally, we find no particular pattern for rural area. Controlling for all other factors, living in a rural area increases the probability of being formal in Colombia and Ecuador, it decreases it in Peru and Venezuela, and it is not significant in Bolivia.
Budgetary implications of transitions to formal employment
Based on the coefficients estimated in Table 6, we predict the probability of being formal for our sample of informal workers under analysis. We then select the 10 percent with the highest probability of being formal and create groups, adding the next 10 percent of informal workers with the highest probability of being formal, until we cover the whole informal population. 18 As expected, those with the highest probability of being formal have, on average, higher education levels and earnings. Table 14 in the Appendix for the characteristics of the 10 percent with the highest probability of being formal.
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Financial disincentives to formal employment and tax-benefit… Table 7 and Fig. 6 present the results of our simulations. Table 7 presents tax revenue under the baseline scenario (official statistics) and additional tax revenue under our counterfactual where our whole sample of informal workers would enter formal employment. Panel A in Fig. 6 presents the simulated cumulative percentage of additional tax liability, starting from individuals with the highest probability of being formal until the whole sample of informal workers is covered. Results distinguish between personal income tax and social insurance contributions, as the pattern differs between these two instruments.
In terms of personal income tax, our counterfactual of a fully formalized economy would have varying effects across countries. In Bolivia and Venezuela, personal income tax revenue as a percent of GDP would more than double, from 0.22 to 0.77 percent (0.55 percentage points increase) and 0.80 to 2.1 percent (1.3 percentage points increase), respectively. The effect of a fully formalized labor force would be the smallest in Colombia, representing a 2.5 percent increase in personal income tax revenue with respect to the baseline scenario. In Ecuador and Peru, the effect would also be limited with an increase in personal income tax revenue of 15.5 percent and 7 percent, respectively.
Note that although the literature usually refers to informality as a barrier to increase fiscal capacity, our results show that this seems to be the case in Bolivia and Venezuela, but to a much lesser extent in Colombia, Ecuador, and Peru. Moreover, the potential tax revenue from personal income tax under a fully formalized economy remains low compared to tax revenue collected currently from this source in OECD countries, which represented on average 8.3 percent of GDP in 2015 (OECD, 2020). As discussed before, the high exempted thresholds and the presence of generous tax deductions-and in the case of Bolivia, the little progressivity and collection capacity of personal income tax-limit the potential to increase fiscal capacity as a result of entries to formal employment. Moreover, it is important to bear in mind that our analysis considers full tax compliance of informal workers upon entry to formal employment, meaning that they would declare the totality of their earnings for the calculation of personal income tax. However, tax avoidance and tax evasion are prevalent in the region, so the potential additional tax revenue under a fully formalized labor force might be lower if the possibility of avoiding or evading taxes was considered. Figure 6 shows that in all countries except Bolivia, a transition of the 10 percent with the highest probability of being formal would be enough to capture a large share of the additional personal income tax potential liability. In Colombia and Ecuador, over 80 percent of the additional personal income tax liability would be captured following a transition of the 10 percent with the highest probability of being formal. In Peru and Venezuela, the share of personal income tax liability captured by those with the highest probability would amount to 54.6 and 68 percent, respectively. In Bolivia, the pattern of the cumulative tax liability differs namely because of the design of personal income tax, which is proportional with a rate of 13 percent for salaried employees and 15.5 percent for the self-employed workers.
In terms of social insurance contributions, Bolivia, Peru, and Venezuela would see their revenue more than double under a fully formalized economy (Table 7). The effect would also be large in Ecuador, where social insurance contributions revenue 1 3
Financial disincentives to formal employment and tax-benefit… would increase by 79 percent. In Colombia, however, the effect would be smaller, representing an increase of 23 percent of social insurance contributions revenue. The pattern of the cumulative social insurance contributions liability by probability of being formal is similar in all countries (Panel A in Fig. 6), given that in all countries social insurance contributions are proportional to income. The share of the additional social insurance contributions revenues captured by those with the highest probability of being formal would range between 10 percent in Colombia and 20 percent in Venezuela. Table 8 and Panel B in Fig. 6 show the results of our simulations in terms of additional number of taxpayers. Under a fully formalized economy, the additional number of personal income tax payers would be the highest in Bolivia and Venezuela, representing 23.2 percent and 14.5 percent of the active population, respectively. The increase would be smaller in Colombia, Ecuador, and Peru, representing 0.34, 0.89, and 2.47 percent of the active population, respectively. The effect would be much larger in terms of social insurance contributions payers across countries, with the additional number of payers ranging between 36.5 (Ecuador) and 40.6 percent (Peru) of the active population. The pattern of cumulative number of taxpayers follows closely that of tax revenue (Fig. 6). Figure 7 presents the effect of potential entries to formal employment on income inequality. The figure depicts the percentage change in the Gini coefficient by the probability of being formal, where the zero percent line represents the baseline Gini coefficient in each country in 2015. 19 Our results show that an entry to formality of the 10 percent of informal workers with the highest probability of being formal would decrease inequality in all countries, although the decrease is small in Peru, where the Gini coefficient would remain broadly the same. The decrease on income inequality, as a result of this counterfactual transition, is driven by the fact that those with the highest probability of being formal are mainly high earners. Inequality drops as they enter formality, because this group of workers would start paying personal income tax (see effect on Fig. 6), which would improve the redistributive effect of the tax-benefit system. Note, however, that the increased redistributive effect might be lower if the possibility of tax avoidance or tax evasion was taken into account. As more individuals with a lower probability of being formal enter formality, income inequality starts increasing and surpasses the baseline Gini coefficient in 2015. In Colombia, the increase in income inequality under a fully formalized economy would be the largest, amounting to a 1.9 percentage points increase in the Gini coefficient (from 56.4 to 58.2 percent). The increase in income inequality as more informal workers (with a lower probability of being formal) enter formality is due to the fact that most self-employed informal workers are low earners and a move to formal employment would represent an additional cost in the form of social insurance contributions payments. As discussed in the previous section, the cost of entering formal employment (FTR) is the largest for the selfemployed and individuals at the bottom of the income distribution, meaning that inequality will increase, as self-employed workers with low earnings (which represent the majority of informal workers) would face social insurance contribution payments.
Distributional implications of transitions to formal employment
The only exceptions to the increasing pattern in inequality are Ecuador and Venezuela, where inequality would decrease even under the counterfactual of a fully formalized economy, although the decrease would still be larger for entries of groups with a higher probability of being formal. The contrasting pattern observed in Ecuador is explained by the fact that, contrary to other countries, the informal population is composed of a large share of employees who upon entry to formal employment are assumed to earn at least the minimum wage, which reduces income inequality. For Venezuela, the contrasting pattern is explained by the fact that the decrease in income inequality due to increased taxes paid by informal high earners (those with the highest probability) dominates the effect of increasing inequality due to social Financial disincentives to formal employment and tax-benefit… insurance contribution payments of informal low earners. As seen earlier in Fig. 3, financial costs of entering formal employment are in general low for informal workers at the bottom of the income distribution in Venezuela. However, a point of caution has to be made regarding the impact of the macroeconomic distortions in prices and wages in Venezuela that made the income tax payments particularly high for the consolidated middle class and the rich, as mentioned previously.
To confirm the point made about the pattern of inequality by probability of being formal and the costs of entering formal employment, Fig. 8 presents the pattern of FTR by the probability of being formal. Our results show that the FTRs are low for informal workers with the highest probability of being formal (around 10 percent). In all countries except Venezuela, FTRs increase as the probability of being formal decreases. In Venezuela, FTRs are broadly similar for workers with different probabilities of being formal, which is in line with the pattern observed in terms of inequality. Although FTRs are low for the 10 percent of informal workers with the highest probability of being formal, in absolute terms the cost of formalization might be high for these workers as pointed out by the fact that a large share of the additional tax revenue would be captured upon their entry to formal employment.
To further highlight the link between informal employment and inequality, we replicate the analysis in this section but rather than using the predicted probability of being formal to formalize informal workers, we simulate the effect of transitions to formal employment based only on the level of earnings of informal workers (from higher to lower). Figure 12 in Appendix B presents the simulated cumulative percentage of additional tax and social insurance contribution liability (Panel A) and cumulative percentage of additional tax and social insurance contribution payers. Figure 13 in Appendix B presents the effect of potential entries to formal employment by earnings deciles on income inequality. The results show that a potential entry of the 10 percent highest earning informal workers to formal employment would capture between 36 percent of the additional personal income tax revenue in Bolivia and up to 100 percent of the additional personal income tax revenue in Colombia. In Ecuador, Peru, and Venezuela, the proportion of the additional personal income tax captured by the formalization of the 10 percent highest informal earners would also be substantial, representing 99, 96.5, and 90.4 percent, respectively.
The distributional consequences of the formalization of the 10% highest earning informal workers would also be important. The Gini coefficient would drop between 0.18 percentage points in Peru to 1.76 percentage points in Venezuela. Note that inequality starts increasing (compared to the situation when only the 10 percent highest informal earners are formalized) as more informal workers with lower earnings would enter formal employment, however, a slight decrease in inequality is observed once the bottom of the informal earnings distribution has been formalized. This is explained by the fact that earnings of informal employees below the minimum wage have been raised to the level of the minimum wage upon formalization.
Reducing financial disincentives to formal employment: a hypothetical reform
The results of the previous sections show that informal workers with low earnings have a lower probability of being formal and face higher financial disincentives to enter formal employment. As previously discussed, high FTRs are mostly explained by the requirement that workers contribute at least on the basis of the minimum wage, which translates into a high cost of formalization for informal workers with low earnings (i.e., below the minimum wage). In this section, we therefore analyze the effects of a counterfactual policy reform in which the minimum contribution base is reduced to half of the minimum wage in all countries. The choice of this counterfactual is motivated by the fact that a large share of workers in informal employment have earnings below the minimum wage, in particular the self-employed. Figure 9 shows the distribution of FTR under our counterfactual scenario. Our results show a decrease in FTRs in all countries under our hypothetical reforms. Financial disincentives to formal employment and tax-benefit… Colombia would experience the largest reduction in average FTR of 47 percent (42-22.3 percent). The decrease would also be important in Ecuador (18.3-12.7 percent), Bolivia (23.1-17.9 percent), and to a lesser extent Venezuela (8.5-6.6 percent). Peru would experience the smallest drop in FTR, of 8 percent (23.8-22 percent) because the reform affects only employees, for whom the minimum wage serves as basis for the minimum contribution base, whereas self-employed workers pay fixed contribution amounts for health insurance depending on age. In addition, the reform would also reduce the variation of FTRs, depicted by the inter-quartile range, particularly in Colombia. Figure 10 shows the distribution of FTR by employment status under our counterfactual scenario. The largest decrease in financial disincentives to formal employment is observed for the self-employed, who have in general lower earnings than their salaried counterparts and incur higher formalization costs due to the requirement of contributing at least on the basis of the minimum wage. Colombia would experience the largest reduction in average FTR for the self-employed, from 57.9 to 29.7 percent, followed by Venezuela with a drop in FTR for the self-employed of 43 percent (12.1-6.9 percent). The decrease in FTR would also be important in Ecuador (26.1-16.9 percent) and to a lesser extent in Bolivia (27.1-20.5). Selfemployed workers in Peru would experience no change in their FTR as social insurance contributions are set as fixed amounts depending on age. The decrease in FTRs for employees is less sizeable in all countries ranging from a 10 percent decrease in Venezuela to a 30 percent decrease in Colombia.
Conclusions
Informality is deeply rooted in the economic systems of LAC countries, and the Andean countries discussed herein are not the exception. Working at the margin of the tax and social security systems compromises fiscal resources, which some countries urgently need. More importantly, however, it puts a limit on long-term growth and the perspectives of achieving convergence to more developed economies. Despite the governments' efforts to tackle both workers and firm informality, this phenomenon still accounts for two-thirds of the workforce and roughly half of the firms, although information on the latter is scarce.
In this paper, we attempt to make a contribution to the literature by examining the incentives that workers have to work formally or informally. More precisely, we use harmonized microsimulations models based on the information provided in official household surveys to construct indicators of financial (dis)incentives to formality implied by the design of the tax-benefit systems in Bolivia, Colombia, Ecuador, Peru, and Venezuela. Our indicators of FTRs capture the percentage of earnings in informality that would be lost due to increased social insurance contributions and income tax payments or benefit withdrawal upon entry to formal employment.
Our analysis provides a number of interesting results. First, we find that financial disincentives to formal employment are higher and more volatile among selfemployed workers, whereas salaried employees show lower and more stable formalization costs, which might make them more likely to make the transition to formality, especially those working in medium-sized and large firms. The higher FTRs of selfemployed workers, compared to their salaried counterparts, are explained by higher social insurance contribution rates applied to this group, and in particular by the requirement of paying social insurance contributions at least on the basis of the minimum wage, whereas self-employed workers belong to a broader spectrum of the income distribution, with many of them earning very low income, making formalization not financially viable.
Our results also show that the fiscal capacity of governments would improve following the potential entry of newly formalized workers who would start contributing to social security. On the other hand, given the generous design of the personal income tax systems in the Andean region, we estimate that its contribution to the fiscal space would only be marginal, even in a scenario of a fully formalized labor force. Interestingly, a potential formalization of the 10 percent of informal workers with the highest probability of becoming formal would harvest important fiscal gains, allowing to capture a substantial share of the additional tax revenue lost due to informality, with positive impacts on inequality reduction. FTRs for the 10 percent of informal workers with the highest probability of being formal are low. However, the fact that their potential formalization would capture a large share of additional tax revenue might reflect that they face high costs of formalization in absolute terms.
In terms of economic policy, there are several ways to achieve the formalization targets resulting from the simulations, ranging from making formality more attractive for workers and firms, designing audit strategies to reduce informality within formal firms, and training workers to improve labor productivity. Naturally, higher Financial disincentives to formal employment and tax-benefit… formalization rates might imply greater labor costs, which can induce behavioral changes of workers and firms. Thus, in implementing formalization strategies, governments need to evaluate these potential fiscal gains against increased hiring costs for the firms. In any case, there is ample evidence that formalization has positive effects in terms of salaries, social protection, productivity, and long-term growth (IDB, 2010;Carpio & Pagés, 2009;La Porta & Schleifer, 2008;Santa María & Rozo, 2008). The comparative perspective herein provides insights into strategies that Andean governments could adopt to reduce the financial burden to formalization of certain population groups. For instance, the Seguro Campesino in Ecuador offers social insurance coverage to self-employed workers in rural areas, with lower contribution rates than their counterparts in the general regime. Similar schemes could be implemented to target different categories of self-employed workers in the region, with the aim of reducing their financial disincentives to enter formal employment.
There are some avenues for further research related to the methodological caveats of our study. One consists of revising the assumption that earnings in informality remains the same upon entry into formal employment. Likely, the income perceived by employees in formality would be at least the minimum wage in the case of lower earners, and other informal workers might experience a change in earnings in the event of entering formal employment . Incorporating changes in earnings to measure financial disincentives to formal employment requires rethinking the definition of FTR, and developing a method to disentangle the effect of changes in earnings from the effect of the design of the tax-benefit systems in the FTR indicator. Moreover, our analysis has looked at FTRs only from the perspective of workers, assuming that the cost of payroll taxes is fully absorbed by employers. However, increased costs for employers might reduce wages, which would need to be factored into the cost of formalization of workers. It is also important to stress that our analysis focuses on the financial disincentives to formal employment implied by the tax-benefit system. However, the decision to enter formal employment is also affected by other factors. In particular, some categories of workers (e.g., low-productivity workers) might be limited in the job opportunities available for them, pointing to the need to further analyze this issue from the perspective of a labor supply model with demand-side constraints. Finally, we have examined financial incentives to formal employment from a short-term perspective in which social insurance contributions are considered as a cost. However, within a dynamic setting, it would be useful to study the future benefits of formalization (e.g., access to contributory old-age pensions). Related to this, an increase in formal employment might imply higher expenditures in terms of health coverage and public pension payments. In this sense, the long-term sustainability of the welfare system should be considered in parallel with the need to increase fiscal capacity in the region. All of these extensions represent promising directions for future research.
Appendix A: Tax-benefit microsimulation models for Latin America
Our analysis relies on a novel set of tax-benefit microsimulation models for Latin American countries: COLMOD for Colombia, ECUAMOD for Ecuador, PERUMOD for Peru, and LATINMOD for Bolivia and Venezuela. In a nutshell, tax-benefit microsimulation models represent a series of arithmetic equations which are applied to representative household surveys to compute taxes and social contribution paid, and benefits received by each household in the data depending on its income and demographic characteristics. All income concepts and sociodemographic characteristics in the surveys have been harmonized, and the models have been implemented in the EUROMOD software (Sutherland & Figari, 2013), following the same conventions for the simulation of taxes, social insurance contributions and benefits, to ensure cross-country comparability.
More formally, let the vector z denote labor market and sociodemographic characteristics of a given household, including information about affiliation to social security, and the vector x denote household market income. 20 Household market income is made of earnings (from employment and self-employment), capital income, income from property rent, etc., which are reported separately in the data. For completeness, let x w denote earnings and x −w denote other components of market income.
Household disposable income, y , is given by. y(z, x, p) = x + d (z, x, p),where d(z, x, p) is a compositive arithmetic function representing the sum of benefits received (positive values) and social insurance contributions and taxes paid (negative values) by the household, and p is a set of parameters of the tax-benefit system (e.g., benefit amounts, minimum thresholds for social insurance contributions, level of tax bands, etc.). Each tax-benefit instrument (i.e., social insurance contributions, taxes and benefits) is calculated according to the policy rules in place in each country, taking into account potential interactions between the different instruments (e.g., social insurance contributions usually need to be calculated first to deduct them from earnings to derive taxable income for the purposes of personal income tax simulations).
Tax-benefit microsimulation models allow us to simulate counterfactual distributions of household disposable income by changing the labor market, sociodemographic characteristics ( z ) or household market income in the data ( x ), or the parameters of the tax-benefit system ( p ). In our analysis, affiliation to social security is imposed to workers in informal employment, which is represented by a modified vector z ′ , resulting in a counterfactual distribution of disposable income:
y � z � , x, p = x + d � z � , x, p ,
The original distribution, y , and the counterfactual distribution y ′ are then used to assess the financial disincentives to enter formal work for each informal worker to whom affiliation to social security was imposed, following Eq. (1) in Sect. 3.2.
Scope of the simulations In all countries under analysis, the main policy components of disposable income have been simulated, including employee and selfemployed social insurance contributions, personal income tax, and the main cash transfer programs of each country. Financial disincentives to formal employment and tax-benefit… Venezuela in Venezuela. All these cash transfer programs are characterized by being proxy means-tested, meaning that eligibility to receive the benefit is assessed based on a composite welfare index (e.g., based on characteristics of the dwelling and the household) rather than household income. For this reason, potential transitions to formal employment do not automatically result in benefit withdrawals.
Tables 9, 10, 11 summarize the main parameters of simulated employee and self-employed social insurance contributions and personal income tax. We focus on the design of these three policy instruments as they play a role in the financial cost informal workers would incur upon entry to formal employment.
In terms of employee social insurance contributions (Table 9), there is a large variation in contribution rates from 6 percent in Venezuela to 12.71 percent plus an additional 10 percent on income above 21 minimum wages in Bolivia. In all countries, formal employees need to pay social insurance contributions at least on the basis of the minimum wage, whereas maximum levels of payment (i.e., ceiling) exist only in Colombia and Venezuela. Finally, employee social insurance contributions are deducted from labor income for the purpose of personal income tax payments in Bolivia, Colombia, and Ecuador.
In terms of self-employed social insurance contributions (Table 10), contribution rates vary between 13 percent and 30.5 percent in Bolivia, Colombia, Ecuador, and Venezuela. In Peru, fixed amounts between 0.18 and 0.29 times the minimum wage depending on age apply to health insurance contributions. In all countries except Peru, formal self-employed need to pay social insurance contributions at least on the basis of the minimum wage, whereas maximum levels of payment (i.e., ceiling) exist only in Colombia and Venezuela. Finally, self-employed social insurance contributions are deducted from labor income for the purpose of personal income tax payments in Bolivia, Colombia, and Ecuador.
In terms of personal income tax, in all countries individual taxation applies with the option of joint taxation in Venezuela. The level of the exempted threshold (i.e., lowest tax band limit) varies from 1.8 (Venezuela) to 4 (Colombia) annualized minimum wages. A larger variation is observed in terms of the threshold for the highest tax band, which reaches 7.1 (Venezuela) and 25.9 (Ecuador) annualized minimum wages. The highest tax rates are broadly similar across countries, ranging from 30 percent in Peru to 35 percent in Ecuador. Finally, it is worth noting that the number of tax deductions available in the design of personal income tax is mostly composed of expenditures in education, health, and housing. In Bolivia, personal income tax is part of the Régimen Complementario del Impuesto al Valor Agregado (RC-IVA), which allows the value added tax (VAT) paid on purchases to be deducted from the income tax liability, and a unique tax rate of 13 percent applies.
Validation Simulation results for the models used in the analysis have been validated against official statistics. The validation consists in comparing the aggregate number of recipients of simulated benefits and payers of simulated taxes and contributions, as well as the aggregate annual expenditure in social benefits and revenue from taxes and social insurance contributions, with external benchmarks. For validation results for Ecuador see , for Colombia see , for Bolivia and Venezuela see Arancibia et al. (2019), and for Peru see Torres and Chang (2021). Financial disincentives to formal employment and tax-benefit… Financial disincentives to formal employment and tax-benefit… Appendix B : Tables and figures See Tables 12, 13 and 14. (2020) This classification follows the international lines of the World Bank for extreme poverty and its multiples (1.6, 4, and 20 times, respectively). A household belongs to the vulnerable middle class if it lives with an income between US$5 and US$12.4 per day; and the consolidated middle class corresponds to a range of income between US$12.4 and US$62 per day. The definition of the thresholds that separate the vulnerable middle class from the consolidated middle class is based on the concept of economic security. According to Duryea and Robles (2017), the probability of falling back into poverty increases for incomes below US$12.4 per day, which supports the use of this threshold. The threshold of US$62 is supported by several studies and exercises that define socio-economic status based on self-reported information Financial disincentives to formal employment and tax-benefit… If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
Fig. 2
2Distribution
Fig. 3
3Mean FTR decomposition in Andean countries by socio-economic Category, 2015. Source
Fig. 4
4Distribution of FTR by employment Status, 2015. Source Authors' calculations based on microsimulation models. Note Countries are ordered by mean FTR of the self-employed
Fig. 5
5Mean FTR of employees by number of workers in the firm, 2015. Source Authors' calculations based on microsimulation models. Note Data for Venezuela are not available 1 3
Fig. 6
6Cumulative percentage of additional tax liability and additional taxpayers by probability of being formal. Source Authors' calculations based on microsimulation models
Fig. 7
7Change in the Gini coefficient by probability of being formal (in percentage points). Source Authors' calculations based on microsimulation models. Note The dashed horizontal line corresponds to the baseline level of inequality measured by the Gini coefficient: Bolivia: 48.4 percent, Colombia: 56.4 percent, Ecuador: 46.4 percent, Peru: 48.2 percent, and Venezuela: 47.
Fig. 8
8Formalization tax rates by probability of being formal. Source Authors' calculations based on microsimulation models 1 3
Fig. 9
9Distribution
Fig. 10
10Distribution of FTR by employment status, hypothetical reform scenario. Source Authors' calculations based on microsimulation models. Note Countries are ordered by mean FTR of the self-employed
11 Main Characteristics of personal income tax in the countries under analysis (2015). Source Authors' elaboration based on the 2015 legislation of personal income tax and the legal minimum wages in each country Tax bands are expressed in terms of annualized minimum wages in each
Fig. 11
11Earnings distribution of formal and informal workers. Note Formal (informal) earnings distributions are depicted in blue (gray). Blue (gray) dashed vertical lines represent median earnings in formal (informal) employment. Red vertical lines represent minimum wages in each country. Sources Authors' calculations based on household surveys (Color figure online) 1 3
Fig. 12
12Cumulative percentage of additional tax liability and additional taxpayers by earnings. Source Authors' calculations based on microsimulation models. Note Earnings deciles are based on earnings before the simulated transition to formal employment ▸ Fig. 13 Change in the Gini coefficient by earnings (in percentage points). Source Authors' calculations based on microsimulation models. Note The dashed horizontal line corresponds to the baseline level of inequality measured by the Gini coefficient: Bolivia: 48.4 percent, Colombia: 56.4 percent, Ecuador: 46.4 percent, Peru: 48.2 percent, and Venezuela: 47.0 percent). Earnings deciles are based on earnings before the simulated transition to formal employment are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.
).
Table 1
1Evolution of informal employment in the Andean region, 2007-18 (in percent of workers). Source Authors' elaboration using information from the IDB's labor market and social security information system, available at https:// www. iadb. org/ en/ sector/ social-inves tment/ sims/ home, which is based on official household surveysa 2015 data correspond to 2014
2007
2010
2015
2018
Bolivia
85.4
n.d
81.1
79.7
Colombia
66.8
68.5
62.3
61.1
Ecuador
72.8
64.8
53.4
58.4
Peru
84.0
82.8
79.0
78.2
Venezuela a
65.7
63.2
61.4
NA
Fig. 1Characteristics of informal employment, 2018 (in percent of workers). Source Authors' elaboration using information from the IDB's labor market and social security information system, available at https:// www. iadb. org/ en/ sector/ social-inves tment/ sims/ home, which is based on official household surveys. Note Data for Venezuela are not availableBolivia
Colombia
Ecuador
Peru
78
61
59
75
81
61
58
81
16
6
5
21
44
38
46
70
95
90
77
99
0
20
40
60
80
100
120
Bolivia
Colombia
Ecuador
P eru
Informality (% of workers not contribuƟng
to social security)
Men
Women
Large
Medium
Small
1 3
Table 2
2Informality rates by employment status in the countries under analysis. Source Authors' elaboration based on household surveysBolivia
Colombia
Ecuador
Peru
Venezuela
Employees
42.2
38.8
44.3
49.3
58.6
Self-employed
89.3
88.7
84.7
81.6
90.2
Table 3
3Data sources and microsimulation models. Source Authors' elaboration based on SOUTHMOD, LATINMOD, COLMOD, and PERUMOD documentationCountry
Data source
Year of data collection
Number of
individuals
Number of
households
Microsimulation model
Bolivia
Encuesta Nacional de Hogares (EH)
2015
37,364
10,171
LATINMOD-Bolivia
Colombia
Encuesta Nacional de Calidad de Vida (ENCV)
2014
67,332
20,141
COLMOD
Ecuador
Encuesta Nacional de Ingreso y Gastos de Hogares Urbanos y
Rurales (ENIGHUR)
2011-2012
153,341
39,617
ECUAMOD
Peru
Encuesta Nacional de Hogares (ENAHO)
2018
126,673
37,462
PERUMOD
Venezuela
IV Encuesta Nacional de Presupuestos Familiares (ENPF)
2009
158,840
37,122
Table 4
4Mean FTRs by population subgroups, 2015. Source Authors' calculations based on microsimulation models Income quintiles are based on per capita household disposable incomeBolivia
Colombia
Ecuador
Peru
Venezuela
All
23.1
42.0
18.3
23.9
8.5
Male
21.4
37.5
13.9
20.2
8.0
Female
26.2
49.7
25.8
29.0
9.3
Age (< 30)
19.5
39.0
16.0
20.8
7.6
Age (30-50)
23.1
41.3
18.7
23.4
8.7
Age (50 +)
28.6
49.1
21.5
29.2
9.7
Low-skilled
27.5
52.6
17.8
34.3
8.6
Medium-skilled
20.4
37.6
19.4
22.2
8.1
High-skilled
19.4
26.5
16.5
12.3
10.0
Employee
14.3
12.9
11.5
16.8
7.2
Self-employed
27.1
58.0
26.1
29.9
12.1
Rural
30.4
60.4
14.0
34.4
8.8
Urban
19.9
36.0
20.3
21.4
8.5
Agriculture and fishing
34.4
55.6
9.6
32.6
8.5
Mining, manufacturing, and utilities
21.0
39.5
21.4
22.6
8.9
Construction
15.5
29.8
12.4
15.0
8.8
Retail, wholesale, hotels, and restau-
rants
22.9
41.4
25.0
25.3
8.1
Transport and communication
18.4
39.2
17.3
16.1
9.3
Financial intermediation, real estate,
and business activities
12.7
39.2
19.8
18.8
8.5
Other industry sectors
19.6
35.8
22.0
20.0
7.2
Income quintile 1
43.6
84.5
20.5
45.7
10.1
Income quintile 2
25.0
47.3
19.0
25.4
8.2
Income quintile 3
21.2
37.3
18.9
20.6
7.7
Income quintile 4
18.9
29.7
17.7
17.5
7.3
Income quintile 5
16.9
20.3
15.5
14.5
9.4
Number of observations
7165
14,498
28,830
28,274
30,273
Table 5
5OLS regression estimates of FTRs. Source Authors' calculations based on microsimulation models Standard errors in parenthesis; significance level: *p < 0.1, **p < 0.05, ***p < 0.01Bolivia
Colombia
Ecuador
Peru
Venezuela
Female
− 0.577***
− 0.135
− 1.509***
− 0.936*** 0.625***
(0.237)
(0.629)
(0.249)
(0.191)
(0.0485)
Age
0.189***
0.190
0.668***
− 0.0963** 0.0222
(0.0572)
(0.151)
(0.0605)
(0.0459)
(0.0137)
Age 2
− 0.00201*** − 0.00194 − 0.00805*** 0.00232*** − 1.00e-05
(0.000729)
(0.00192)
(0.000778)
(0.000582) (0.000176)
Middle-skilled
− 0.106
1.311***
0.0369
− 0.0337
0.117**
(0.217)
(0.595)
(0.228)
(0.222)
(0.0470)
High-skilled
− 0.00784
11.54***
− 1.942***
4.192***
1.822***
(0.346)
(1.075)
(0.350)
(1.413)
(0.0821)
Rural
0.442*
3.289***
− 2.590***
0.138
0.255***
(0.293)
(0.627)
(0.262)
(0.196)
(0.0881)
Self-employed
7.605***
41.66***
10.40***
6.210***
3.407***
(0.224)
(0.559)
(0.233)
(0.171)
(0.0525)
Mining, manufacturing,
and utilities
− 1.171***
3.768***
13.17***
2.862***
0.223**
(0.399)
(1.024)
(0.386)
(0.327)
(0.103)
Construction
0.0141
− 0.905
11.04***
3.376***
− 0.0179
(0.404)
(1.087)
(0.383)
(0.335)
(0.0850)
Retail, wholesale, hotels,
and restaurants
− 0.156
0.550
13.48***
1.164***
− 0.201*
(0.379)
(0.823)
(0.343)
(0.249)
(0.0993)
Transport and communication − 0.542
− 1.254
11.99***
− 1.429*** 0.346***
(0.427)
(1.061)
(0.430)
(0.313)
(0.0995)
Financial intermediation,
real estate, and business activi-
ties
0.320
4.593***
13.72***
1.433***
0.0920
(1.995)
(1.301)
(0.622)
(0.489)
(0.141)
Other industry sectors
− 1.386***
− 0.429
13.19***
2.033***
− 0.906***
(0.437)
(1.014)
(0.396)
(0.302)
(0.0939)
Low earner (below 0.5*mini-
mum wage)
21.47***
6.118***
11.49***
24.02***
22.99***
(0.348)
(0.684)
(0.361)
(0.249)
(0.142)
Log earnings
− 7.716***
− 43.32*** − 10.53***
− 14.50*** 0.996***
(0.153)
(0.449)
(0.193)
(0.128)
(0.0389)
Constant
69.63***
570.5***
50.29***
112.0***
− 3.938***
(1.154)
(3.294)
(1.239)
(0.952)
(0.266)
Number of observations
7165
14,498
28,830
28,274
30,273
R 2
0.772
0.761
0.440
0.783
0.553
Table 6
6Probit estimates of the probability of being in formal employment. Source Authors' calculations based on microsimulation models Standard errors in parenthesis; significance level: *p < 0.1, **p < 0.05, ***p < 0.01Bolivia
Colombia
Ecuador
Peru
Venezuela
Male
0.160***
0.0912***
− 0.155*** − 0.177***
− 0.0932***
(0.0404)
(0.0294)
(0.0170)
(0.0186)
(0.0142)
Age
0.0389***
0.0428***
0.0192***
0.0658***
0.00821*
(0.0111)
(0.00811)
(0.00467)
(0.00538)
(0.00458)
Age 2
− 0.000365** − 0.000456*** − 0.000104* − 0.000631*** 2.73e-05
(0.000143)
(0.000104)
(6.02e-05)
(6.79e-05)
(5.89e-05)
Middle-skilled
0.378***
0.397***
0.193***
0.501***
0.216***
(0.0428)
(0.0335)
(0.0169)
(0.0315)
(0.0158)
High-skilled
0.825***
0.666***
0.379***
0.968***
0.191***
(0.0586)
(0.0453)
(0.0249)
(0.0781)
(0.0247)
Married
0.205***
0.0666**
0.242***
0.388***
0.135***
(0.0357)
(0.0289)
(0.0148)
(0.0185)
(0.0146)
Number of children
− 0.0588*** − 0.0385***
− 0.0417*** − 0.0389***
− 0.0246***
(0.0121)
(0.0103)
(0.00483)
(0.00695)
(0.00467)
Rural
0.0112
0.0766**
0.266***
− 0.362***
− 0.202***
(0.0523)
(0.0319)
(0.0183)
(0.0234)
(0.0309)
Self-employed
− 0.982***
− 1.307***
− 1.143*** − 0.695***
− 0.770***
(0.0389)
(0.0275)
(0.0187)
(0.0181)
(0.0209)
Log(earnings)
0.410***
0.787***
0.531***
0.416***
0.228***
(0.0249)
(0.0208)
(0.0106)
(0.0112)
(0.0111)
Constant
− 4.546***
0.335***
− 0.0349*
− 0.250***
0.102***
(0.262)
(0.0480)
(0.0197)
(0.0458)
(0.0247)
Industry dummies
Yes
Yes
Yes
Yes
Yes
Occupation dummies
Yes
Yes
Yes
Yes
Yes
Region dummies
Yes
Yes
Yes
Yes
Yes
Number of observations 11,368
23,449
51,639
46,840
47,829
Pseudo R 2
0.421
0.466
0.330
0.365
0.153
Table 7
7Tax Revenue under the baseline and counterfactual scenarios (in percent). Source Authors' elaboration based on own simulations and official sources a Baseline results for Venezuela are based on our own simulations due to lack of official information on tax revenue. b Authors' calculations based on microsimulation models. The counterfactual scenario corresponds to one with full formalization of informal workersBolivia
Colombia
Ecuador
Peru
Venezuela
Baseline tax revenue (% GDP) a
Personal income tax
0.22
0.80
1.03
1.45
0.80
Social insurance contributions
4.32
9.28
2.51
2.07
0.48
Additional tax revenue (% GDP) b
Personal income tax
0.55
0.02
0.16
0.10
1.30
Social insurance contributions
4.69
2.12
1.98
2.01
0.85
Table 8 Additional
8number
of taxpayers (in % of active
population). Source Authors'
calculations based on
microsimulation models
Bolivia Colombia Ecuador Peru Venezuela
PIT
23.20
0.34
0.89
2.47 14.50
Social insur-
ance contri-
butions
39.92
39.75
36.50
42.85 40.58
The following cash transfers have been simulated in the models: Bono Juancito Pinto, Bono Juana Azurduy and Renta Dignidad in Bolivia; Familias en acción and Colombia Mayor in Colombia; Bono de Desarrollo Humano and Bono Joaquín Gallegos Lara in Ecuador; Juntos in Peru; Misiones educativas: Robinson (I y II), Ribas y Sucre and Gran Misión en Amor Mayor1 3
Table 9
9Characteristics of employee social insurance contributions in the countries under analysis (2015). Source Authors' elaboration based on the 2015 legislation of employee social insurance contributions in each country CountryRate
Floor
Ceiling
Tax deductible
Bolivia
12.71% plus up to 10% on income above
21 minimum wages
12.71% of the minimum wage
-
Yes
Colombia
8% or 10%
8% of minimum wage
12% of 25 minimum wages
Yes
Ecuador
9.45% or 11.45%
9.45% of minimum wage
-
Yes
Peru
13%
13% of minimum wage
-
No
Venezuela
6%
6% of minimum wage
6% of 5 minimum wages
No
Table 10
10Characteristics of self-employed social insurance contributions in the countries under analysis(2015). Source Authors' elaboration based on the 2015 legislation of self-employed social insurance contributions in each countryCountry
Rate
Floor
Ceiling
Tax deductible
Bolivia
14.42% plus up to
10% on income
above 21 minimum
wages
14.42% of the minimum
wage
-
Yes
Colombia 28.5% or 30.5%
28.5% of minimum wage 30.5% of 25 minimum
wages
Yes
Ecuador
20.5%
20.5% of minimum wage -
Yes
Peru
Fixed amounts
between 0.18 and
0.29 times the
minimum wage
0.18 times the minimum
wage
0.29 times the minimum
wage
No
Venezuela 13%
13% of the minimum
wage
-
No
Table
Table 12
12Descriptive statistics of the selected sample of workers in informal employment. Source Authors' calculations based on microsimulation models The table reports the characteristics of the sample of informal workers in each country under analysis (e.g., % of female, % of self-employed, etc.)Table 13 Income thresholds for socio-economic categories, 2015 (in local currency units at current prices). Source Authors' elaboration based on IDBBolivia
Colombia
Ecuador
Peru
Venezuela
Population (unweighted)
7165
14,498
28,830
28,274
30,273
Population (weighted, in thousands)
2026
9608
2835
7069
5745
% female
34.1
37.2
37.2
42.2
37.9
% age (< 30)
27.3
28.0
29.5
26.8
29.5
% age (30-50)
55.9
55.4
54.7
54.5
56.7
% age (50 +)
16.8
16.6
15.8
18.7
13.8
% low-skilled
39.8
37.0
46.9
13.8
40.4
% medium-skilled
50.8
52.8
41.3
85.9
49.7
% high-skilled
9.4
10.2
11.8
0.3
9.9
% self-employed
69.6
64.6
40.2
55.6
25.9
% rural
31.1
24.9
31.6
18.9
6.2
% earning Q1
17.8
19.8
17.9
14.5
24.4
% earning Q2
25.2
33.3
29.2
28.3
24.3
% earning Q3
22.2
23.5
22.8
25.9
18.1
% earning Q4
20.0
13.6
18.4
20.7
15.6
% earning Q5
14.8
9.8
11.7
10.6
17.7
Table 14Descriptive statistics of 10 percent of workers in informal employment with the highest probability of being formal. Source Authors' calculations based on microsimulation models Financial disincentives to formal employment and tax-benefit… See Figs. 11, 12 and 13.Poor
Vulnerable middle class
Consolidated middle class
Rich
Bolivia
< 533.3
533.3-1333.2
1333.2-6665.9
> 6,665.9
Colombia
< 205,329.7 205,329.7-513,324.2
513,324.2-2,566,620.8
> 2,566,620.8
Ecuador
< 96.0
96.0-240.0
240.0-1200.0
> 1200.0
Peru
< 269.6
269.6-674.0
674.0-3370.1
> 3370.1
Venezuela
< 2546.2
2546.2-6365.6
6365.6-31,828.0
> 31,828.0
Bolivia
Colombia
Ecuador
Peru
Venezuela
Population (unweighted)
743
1044
2857
2020
2840
Population (weighted, in thousands)
203
961
283
707
575
% female
34.8
42.8
40.1
49.6
56.1
% age (< 30)
33.5
30.6
29.9
27.2
18.9
% age (30-50)
56.3
58.3
55.1
56.1
63.3
% age (50 +)
10.2
11.1
15.0
16.7
17.8
% low-skilled
10.4
2.3
15.0
0.2
2.6
% medium-skilled
51.8
61.3
43.5
97.6
54.8
% high-skilled
37.8
36.5
41.6
2.2
42.6
% self-employed
11.7
11.4
8.5
28.1
0.1
% rural
14.9
5.7
27.2
0.7
0.6
% earning Q1
2.8
0.0
1.1
0.3
1.3
% earning Q2
23.0
3.9
14.2
10.3
6.3
% earning Q3
31.2
24.8
25.0
27.5
12.0
% earning Q4
23.4
34.5
26.8
31.1
22.7
% earning Q5
19.6
36.8
32.9
30.8
57.8
1 3
According to the IDB's definition of labor informality, informal workers are those who do not contribute to social security (e.g., old-age pensions and health insurance). Specifically, informality is defined as the percentage of employed workers not contributing to old-age pensions. For a more detailed discussion, seeAlaimo et al. (2016) andBosch et al. (2013).
The model for Ecuador, ECUAMOD, has been developed and is maintained as part of the SOUTHMOD project. For more information see and https:// www. wider. unu. edu/ proje ct/ south mod-simul ating-tax-and-benefi t-polic ies-devel opment. The model for Colombia, COL-MOD, is developed and maintained by the Faculty of Economics at Universidad Externado de Colombia. For more information see Rodriguez (2019) and https:// www. uexte rnado. edu. co/ econo mia/ colmodel-primer-modelo-de-micro simul acion-en-colom bia/. The models for Bolivia and Venezuela have been developed as part of the LATINMOD project. LATINMOD is a regional tax-benefit microsimulation model for six Latin American countries (Argentina, Bolivia, Mexico, Paraguay, Uruguay and Venezuela). For more information, seeArancibia et al. (2019) andOliva (2018). The model for Peru, PERUMOD, has been developed as part of the project "Simulating Tax Policy Reforms and Fiscal Gains in the Andean Region," which was funded by the Inter-American Development Bank.
Market income is defined as the sum of employment and self-employment income, bonuses, in-kind income, own consumption from self-employment activities, capital and property income, inter-household payments, private transfers, minus alimony payments. Imputed rent is not included as part of market income.12 The following cash transfers are simulated in our models:Bono Juancito Pinto, Bono Juana Azurduy and Renta Dignidad in Bolivia; Familias en acción and Colombia Mayor in Colombia; Bono de Desarrollo Humano and Bono Joaquín Gallegos Lara in Ecuador; Juntos in Peru; Misiones educativas: Robinson (I y II), Ribas y Sucre and Gran Misión en Amor Mayor Venezuela in Venezuela.10 Data adjustments for the use in the microsimulation models are kept to a minimum. In particular, a minimum number of observations for domestic workers living in their employer's household have been dropped, as it is not possible to link them with information about their own households. An important shortcoming of the survey in Venezuela is that information about the household members' relationship to the head of the household is not released. Therefore, we have imputed information on mother and father identifiers for children that are less than 18 years old based on information about age, gender, and education level of adult household members.
Note that this assumption follows the original approach proposed byKoettl and Weber (2012), where earnings in formality are implicitly assumed to remain the same upon entry to formal employment. Jara and Rodriguez (2019) propose a different approach, whereby the earnings informal workers would face upon entry to formality are estimated based on the distribution of earnings of formal workers in the data. Potential changes in earnings upon entry to formal employment could be considered for future extensions of our work.13 In Venezuela, the social security law establishes the voluntary contribution of self-employed workers with a tax rate of 13 percent of declared income. In this case, the declared income is self-defined by workers but cannot be less than the minimum wage. Although no public information is available, assuming all affiliated self-employed contribute on the basis of the minimum wage seems a realistic assumption given the low social insurance contributions revenue in Venezuela.
In Ecuador, eligibility to social benefits (i.e., Bono de Desarrollo Humano) for the elderly and the disabled depends on non-affiliation to social security. However, these groups are not included in our sample of analysis.15 The monthly gross labor income of informal workers in the poor segments in Colombia is estimated at COL 300,000, equivalent to US$95. On the other hand, monthly labor income in Peru, Bolivia, and Ecuador amounts toUS$110, US$150 and US$212, respectively. 16 This information is based on legislation in force as of 2015. Minimum self-employed social insurance contribution rates equal 14 percent in Bolivia, 20.5 percent in Ecuador, 13 percent in Venezuela and fixed health insurance contribution payments depending on age in Peru.
Bolivia: 48.4 percent, Colombia: 56.4 percent, Ecuador: 46.4 percent, Peru: 48.2 percent, and Venezuela: 47.0 percent.
Household subscripts are omitted for simplicity.
Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
AcknowledgementsThe results presented herein are based on the following three projects: (i) LATIN-MOD, a project sponsored by the Centro Estratégico Latinoamericano de Geopolítica (CELAG), funded by The Venezuelan Economic and Social Development Bank (BANDES) and with the collaboration of EUROMOD; (ii) ECUAMOD v1.4. ECUAMOD is developed, maintained, and managed by UNU-WIDER in collaboration with the EUROMOD team at the Institute for Social and Economic Research (ISER) the Southern African Social Policy Research Institute (SASPRI), and local partners in selected developing countries (Ethiopia, Ghana, Mozambique, Tanzania, Zambia, Ecuador and Viet Nam) in the scope of the SOUTHMOD project. The local partner for ECUAMOD is the Instituto de Altos Estudios Nacionales (IAEN); and (iii) COLMOD v1.2, a project developed and managed by the Faculty of Economics at Universidad Externado de Colombia. The authors are indebted to the many people who have contributed to the development of LATINMOD, SOUTHMOD, ECUAMOD, and COLMOD, as well as to David Rodriguez for his helpful assistance and comments. The results and their interpretation presented in this publication are solely the authors' responsibility.DeclarationsConflict of interest The authors declare that they have no conflict of interest.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article
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| As the title says, I live in a country without tax.
Question is: Let's say I want to buy Alphabet stock and in one year I can (hopefully) sell my stocks in the company with a profit.
Do I only pay local tax (0% in my case), or do I have to pay tax in the country of origin of the stock/company. On this case the US.
TIA | Taxation in Georgia (country) royalty is 5%.There are few allowances deductible. Value-added tax (VAT) is collected at a flat rate of 18%.There is few exceptions granted, nearly all goods and services are subject to VAT. Medical care, exports and education are exempt from VAT. Regardless of turnover a taxpayer have to register for VAT if it produces or imports goods. Turnovers of less than 100,00 GEL is exempt. Corporate taxes are levied at a flat rate of 15%, which was enacted in 2008. From 2017 onward, non-distributed profits are exempt from taxation. Very few deductions are accessible. This system was set up to attract | Taxation in Georgia (country) royalty is 5%.There are few allowances deductible. Value-added tax (VAT) is collected at a flat rate of 18%.There is few exceptions granted, nearly all goods and services are subject to VAT. Medical care, exports and education are exempt from VAT. Regardless of turnover a taxpayer have to register for VAT if it produces or imports goods. Turnovers of less than 100,00 GEL is exempt. Corporate taxes are levied at a flat rate of 15%, which was enacted in 2008. From 2017 onward, non-distributed profits are exempt from taxation. Very few deductions are accessible. This system was set up to attract | If People and Corporations were taxed based on their net worth rather than on income, what percentage would be necessary for a basic income program that can actually not force the citizens of a country into the work place for survival? Would a tax at this percent be sustainable for the foreseeable future? Would this tax be too burdensome to allow businesses to flourish? Would this system be more beneficial to the average citizen than the progressive or flat income tax systems are today?
Edit: If this question is too broad and would need to be answered on a country to country bases then the United States is the main country I'm interested in. | Ajman Free Zone 100% foreign ownership and no taxation. Its services include the provision of visas for expatriate workers, land for development or pre-existing warehousing and business premises supporting a range of business types, including 'smart offices' - co-working spaces. Business licenses at AFZA are annually renewable and costs start from UAE Dhs 11,900. The UAE levies a 5% Value Added Tax, from which companies at AFZA are exempt when trading between free zones or internally. However, the tax is levied on goods and services traded onshore into the UAE. Indian-owned businesses comprise 40% of the companies operating from AFZA, which is growing | Ireland as a tax haven this, Ireland has a special lower salary tax rate scheme, and other tax bonuses, for employees of foreign multinationals earning over €75,000 ("SARP"). The OECD's "Hierarchy of Taxes" pyramid (from the Department of Finance Tax Strategy Group's 2011 tax policy document) summarises Ireland's tax strategy. EU and U.S. studies that attempted to find a consensus on the definition of a tax haven, have concluded that there is no consensus (see tax haven definitions). The Irish State, and its advisors, have refuted the tax haven label by invoking the 1998 OCED definition of a "tax haven" as the consensus definition: Most | Malaysia practises primarily a territorial system of taxation. Tax is levied on profits arising in or derived from carrying on a trade, business or profession in Malaysia. Since the territorial principle does not distinguish between residents and non-residents, a non-resident company or individual that derives income from a Malaysian source may be liable to pay tax in Malaysia. When a Malaysian-resident company or individual makes specific types of payments to a non-resident entity, a certain portion of the payment must be withheld and paid to the Inland Revenue Board of Malaysia on behalf of the non-resident. ::: The aim of this book is provide a working understanding of the Malaysian withholding tax system. It covers Malaysia’s withholding tax rules including the transactions and parties subject to withholding tax, payments that are subject to withholding tax, withholding tax rates and the consequences of non-compliance with withholding tax provisions. | 459 | It seems to be an issue with the new patch. Quite a few formerly colonizable provinces have there base tax set to 0, making them impossible to colonize. How can I change this in the game files? | Vassalisation Base Tax Bug | Your vassals might not be paying as much taxes as you might think | Spain: Interpretation of tax treaties | The Use of Neutralities in International Tax Policy | when was the last time foreign earned income was excluded | Brazil imposes a 2% tax on foreign capital inflows toward equities and fixed-income investments. | Unused Foreign Tax Credits | what is the capital of province no 1 |
460 | how long is the dorsal sepal of epidendrum anceps | result of the expansion of the trapezoid on the side of the palm. A small, lens-shaped radial sesamoid embedded into the APL tendon is a primitive state found in all known "Carnivora" genera except in the red and giant pandas and the extinct "Simocyon" where it is hypertrophied (enlarged) into a sixth digit or a so called "false thumb", a derived trait that first appeared in ursids. The APL sesamoid is present in all non-human primates, but only in about half of gorillas, and normally absent in humans. Abductor pollicis longus muscle In human anatomy, the abductor pollicis longus (APL) | How many lumber vertebrae doee a human have? | Epitrochleoanconeus muscle known as Osborne's band or Osborne's ligament, which is considered a vestigial form of the epitrochleoanconeus. There are also cases where there is no structure at all bridging the space occupied by the epitrochleoanconeus. In humans the m. epitrochleoanconeus has no known function beyond its putative role as a retaining element for the ulnar nerve. As a muscle it is considered a vestigial form with its retaining function being performed less problematically by the less bulky Osborne's ligament. The prevalence of the epitrochleoanconeus in humans has been estimated to be between 1-30% from various cadaver elbow dissection studies, conversely Osborne's | How long is a cricket pitch in feet? | had seven or eight digits. Pes (anatomy) The pes (Latin for "foot") is the zoological term for the distal portion of the hind limb of tetrapod animals. It is the part of the pentadactyl limb that includes the metatarsals and digits (phalanges). During evolution, it has taken many forms and served a variety of functions. It can be represented by the foot of primates, the lower hind limb of hoofed animals, the lower portion of the leg of birds and dinosaurs or the rear paw. It is also represented in the rear 'paddle' of extinct marine reptiles, such as plesiosaurs. | Introduction ::: An experimental study of experimental burst fractures in bovine spinal specimens was conducted to analyze the effects of transpedicular short-segment posterior fixation followed by reduction on indirect spinal canal decompression. | Determining the length of nail needed for intramedullary nailing can sometimes be difficult. This article describes a simple procedure for taking this important measurement. | Spinal nerve in the multifidus muscle. The laterals supply the erector spinae muscles. The upper three give off cutaneous nerves which pierce the aponeurosis of the latissimus dorsi at the lateral border of the erector spinae muscles, and descend across the posterior part of the iliac crest to the skin of the buttock, some of their twigs running as far as the level of the greater trochanter. Anterior divisions: The anterior divisions of the lumbar nerves (rami anteriores) increase in size from above downward. They are joined, near their origins, by gray rami communicantes from the lumbar ganglia of the sympathetic trunk. | 460 | Epidendrum anceps end of a long peduncle covered from its base by close, imbricating sheathes; sometimes additional racemes will arise from the nodes of the peduncle. The flowers typically contain significant amounts of chlorophyll and yellow pigment—these are often accompanied by enough purple pigment to give the flower a dingy, brown color. The oblong-ovate dorsal sepal can grow as long as 10 mm; the lateral sepals are often wider than the dorsal. The petals are linear. The adnate lip is heart- or kidney-shaped where it diverges from the column, is sufficiently three-lobed to be placed in the section "E." sect. "Schistochila", and | how long are the leaves of epidendrum anceps | what is the name of the narrow portion of a long bone between the epiphysis | what shape is epidendrum cristatum | how are the leaves of echinacea arranged | is apogee the same as apoapsis? | Epidendrum subg. Aulizeum | what kind of anatomy is the epidermis of leaves | Perennation in Epilobium (Onagraceae) and Its Relation to Classification and Ecology |
461 | when did big sur by jack johnson come out | Jack Johnson announces fall tour | Jack Johnson (musician) Johnson names Jimi Hendrix as his all-time favorite guitarist. Jack Johnson's big break was writing and contributing vocals for the song "Rodeo Clowns" which was featured on G. Love's 1999 album "Philadelphonic". The song would later become the most famous single of the album. In addition to his later success as a musician, Johnson is also an accomplished filmmaker. Johnson directed the surf films "Thicker Than Water" (2000) and "The September Sessions" (2002), in which he also starred. Both movie soundtracks were also products of Johnson. Johnson also starred in the 2004 surf film "A Brokedown Melody". Suela released a | Big Sur (film) Big Sur is a 2013 adventure drama film written and directed by Michael Polish. It is an adaptation of the 1962 novel of the same name by Jack Kerouac. The story is based on the time Kerouac spent in Big Sur, California, and his three brief sojourns to his friend Lawrence Ferlinghetti's cabin in Bixby Canyon. These trips were taken by Kerouac in an attempt to recuperate from his mental and physical deterioration due to his sudden success. The film debuted on January 23, 2013, at the 2013 Sundance Film Festival, where it received generally positive reviews. | was produced by the hip-hop duo Handsome Boy Modeling School (Nakamura & Huston). It is a more upbeat version of the original song, and was featured on their 2004 album, "White People". Breakdown (Jack Johnson song) "Breakdown" is a song written by Jack Johnson, Dan Nakamura & Paul Huston and sung by Jack Johnson. It is the eleventh track on the album "In Between Dreams" which was released in February 2005. It was released as a single in September 2005. The video features Jack Johnson surfing in Pichilemu, Chile. The single peaked at #73 in the United Kingdom. The song | Jack Jones (singer) Best Pop Male Performance), "The Race Is On", "Lollipops and Roses" (1962, Grammy Award, Best Pop Male Performance), "The Impossible Dream", "Call Me Irresponsible", "Lady", and "The Love Boat Theme". He was also the voice of Greg's frog in the 2014 animated television miniseries "Over the Garden Wall". John Allan Jones, the son of actors Allan Jones and Irene Hervey, was born in Los Angeles on the night his father recorded his signature song "The Donkey Serenade", causing him to say that he was "practically born in a trunk". The young Jones attended University High School in West Los Angeles | Jack Kerouac and musical performances focused on Kerouac's work and that of the Beat Generation. In the 2010s, there has been a surge in films based on the Beat Generation. Kerouac has been depicted in the films "Howl" and "Kill Your Darlings". A feature film version of "On the Road" was released internationally in 2012, and was directed by Walter Salles and produced by Francis Ford Coppola. Independent filmmaker Michael Polish directed "Big Sur", based on the novel, with Jean-Marc Barr cast as Kerouac. The film was released in 2013. A species of Indian platygastrid wasp that is phoretic (hitch-hiking) on grasshoppers | I Got You (Jack Johnson song) "I Got You" is a song by American musician Jack Johnson and is the lead single from his 2013 album "From Here to Now to You". The song was released on June 10, 2013. The song was released on June 10, 2013 as a CD single, 7' vinyl, and digital download along with the pre-order of the album. The song is mainly about his affection to his wife Kim, and about his love to his kids. On June 11, 2013, a colorful lyric video featuring a blue skied background was released. On June 28, | Better Together (Jack Johnson song) Better Together is a song written and performed by Jack Johnson. As well as being the first track on Johnson's album "In Between Dreams", which was released in February 2005, "Better Together" was released as a single in January 2006. It charted at #24 in the United Kingdom. In the United States, a live version from the "En Concert" live album was released as a single in 2009, peaking at #23 on the Mediabase Triple A chart. It was also included in a Cuban remix of the song which was included in Rhythms del Mundo. | 461 | Big Sur (Jack Johnson song) "Big Sur" is the fourth single by American musician Jack Johnson, and is featured on his 2017 studio album All the Light Above It Too. It was released as a single on September 18, 2017. The single was announced with its release on a live stream with Zane Lowe on Beats 1 on September 7, 2017. On the podcast, it was also announced that a CD Single of the song would be released which would feature a remix of the song by ex Beastie Boy Mike D. The song's remix premiered on episode 31 of | what's the name of big sean's last song | who released the album tribute to jack johnson | who wrote i do it big sean | Big Hits 2012 | who sings i'm a mighty o | who produced the 1976 sparks album big beat | what album is the song giants by lights on | what album is i do it on big sean |
462 | Recent findings have identified methylation occurring at the cytosine of mRNA as a new epitranscriptomic mark beyond the methylation of the adenine[1]. These marks open new biological horizons and define the existence of other possible mechanisms that do not correlate with alteration of the genetic sequence, epigenetic modifications or even post-translational modifications. The epitranscriptome identifies methylation occurring at the transcript level, which were once invisible[2], and that could be responsible for the maturation and translational processing of mRNAs. Once more, epi-marks claim their role in cellular processes and further confirm their involvement as key players in cell fate decisions.Epitranscriptomic modifications have been identified in cancer cells. In particular, methylation at the N6 adenosine of METTLR3 and IKHB5 mRNAs occur in bladder cancer, inhibit ITGA6 expression, and correlate with poor prognosis[3]. Recent advances have highlighted epitranscriptomic processes in pancreatic cancer too. In a study recently published in EBioMedicine, methylation occurring in total cellular mRNA at cytosine 5 decreased in patients affected by pancreatic cancer. The methylation status correlated with clinicopathological parameters such as T stage, proliferation index, tumour recurrence and survival. Furthermore, the authors identified that m5C level correlated with decreased expression of the methyltransferase NOP2/Sun RNA Methyltransferase 6 (NSUN6)[4]. The importance of its activity was recently determined by observing augmented translation of those mRNAs, that were methylated by NSUN6[5,6]. The emerging role of NSUN6, and m5C methylation mediated by its activity, has been also found in gastrointestinal and head and neck cancers[7,8]. The study of Yang and colleagues showed for the first time the involvement of NSUN6 and its relation to m5C methylation in pancreatic cancer.The peculiar mechanism of translational processing stands at the crossroads between epigenetic modification (occurring at the genome level), and the interaction between mRNAs and their counterparts with inhibitory function (small-non-coding RNAs). Interestingly, methylation occurring at the adenine and the cytosine of mRNA act differently than in the corresponding DNA. Epigenetic methylation of DNA is responsible for silencing the genetic sequence, to stop transcription. Instead, methylation occurring on mRNA bases promotes nucleic acid translation.Methylation could offer new insights in terms of interaction between mRNAs, the RNA-induced silencing complex (RISC), and miRNAs, highlighting new regulatory processes of RNA maturation and the translation machinery. This epitranscriptomic mark might not only affect mRNAs and tRNAs, but also small-non-coding RNA and the long-non-coding RNAs, by modulating affinity for their targets, thus amplifying the role exerted by the methylation of RNA. Of note, putative RNA-only methyltransferases have not yet been identified. It has been shown that DNA methyltransferases (DNMTs) and NSUNs are responsible for RNA methylation, but that they are capable of also methylating DNA[4]. Up to now, their dual function of inhibiting transcription and promoting translation is not clearly understood. Further studies are needed to clarify which regulatory processes modulate the functioning of those methyltransferases at the nuclear and cytosolic level.In the future, the epitranscriptome could be fully integrated in diagnostic tools for the identification of clinicopathological parameters and offer potential therapeutic insight for the treatment of different tumour types.Declaration of Competing InterestThe author reports no conflicts of interest. |
Background
Epitranscriptomics, also known as "RNA epigenetics", is a chemical modification for RNA regulation [1]. According to its function, RNA can be divided into two broad categories, including encoding protein mRNA and noncoding RNA. With the deep research of epitranscriptomics, the researchers found methylation modification on mRNA, which is involved in the regulation of eukaryotic gene expression [2][3][4].
The mRNA is a type of RNA with genetic information synthesized by DNA transcription, which acts as a template in protein synthesis and determines the amino acid sequence of the peptide chain [5]. It is an important RNA in the human body. The methylation is the process of catalytically transferring a methyl group from an active methyl compound such as S-adenosylmethionine (SAM) to another compound, which can chemically modify certain proteins or nucleic acids to form a methylated product [6]. In biological systems, methylation influences heavy metal modification, regulation of gene expression, regulation of protein function, RNA processing, etc. [7]. At the early 1970s, scientists discovered the presence of the methylation modification in mRNA [8,9]. The mRNA methylation modification mainly located in the nitrogen atom of the base group to form m 6 A, which is enriched in long exons and overrepresented in transcripts with alternative splicing variants [10]. The mRNA methylation modifications also include 5-methylcytosine (m 5 C), N1-methyladenosine (m 1 A), 5-hydroxymethylcytosine (5hmC), N6, 2′-O-dimethyladenosine (m 6 Am), 7-methylguanine (m 7 G) (Fig. 1). These modifications can affect regulation of various biological processes, such as RNA stability and mRNA translation, and abnormal mRNA methylation is linked to many diseases [11]. the presence of m 6 A was also detected in a variety of eukaryotes and viral mRNA [14].
In mammals, m 6 A is widely distributed in multiple tissues. Studies by Meyer showed that m 6 A expression was higher in liver, kidney and brain than in other tissues. It has also been found that the content of m 6 A is very different in various cancer cell lines [15]. With the help of high-throughput sequencing technology, a rough m 6 A modification map has been obtained. Meyer studied the m 6 A modification in mouse brain and found that it was mainly distributed inside the gene (94.8%), where the proportions in the protein coding region (CDS), untranslated regions (UTRs) and introns are 50.9%, 41.9%, and 2.0% respectively [16]. The m 6 A in the UTRs region tends to be enriched in the 3′UTR, while in the CDS region it is mainly enriched near the stop codon [17]. The m 6 A modification occurs mainly on the adenine in the RRACH sequence, where R is guanine or adenine, and H is uracil, adenine or cytosine [18] (Fig. 2).
mRNA m 6 A methylation modification enzyme
The methylation modification of m 6 A has been proved to be reversible, as it involves both methyltransferase and demethylase. The main role of methyltransferases is to catalyze the m 6 A modification of mRNA, while demethylases act on demethylation of bases that have had m 6 A modification [19,20].
m 6 A methyltransferase
The m 6 A methyltransferase, also known as "Writers", is an important kind of catalytic enzymes [21].
Methyltransferase like 3/14 (METTL3/14), Wilms' tumour 1-associating protein (WTAP), KIAA1429 and RNA binding motifs protein 15/15B (RBM15/15B) are core components of the m 6 A methyltransferase, which form complexes that work together to perform catalytic functions. Besides, E3 ubiquitin-protein ligase Hakai (HAKAI) and zinc finger CCCH-type containing 13 (ZC3H13) are also the part of the mRNA methyltransferase complex.
The METTL3 is identified as a SAM-binding component of the complex and has its own catalytic ability, which is highly conserved in eukaryotes [22]. METTL14 is closely homologous to METTL3. It does not bind to the SAM domain and does not with independently m 6 A methyltransferase function. Biochemical characterization has shown that METTL3 and METTL14 proteins form a stable complex with a stoichiometric ratio of 1:1, and the methylation activity of the complex is higher than that of METTL3 alone. Among them, METTL3 is a catalytically active subunit, and METTL14 plays a key role in substrate identification [23,24].
The WTAP is a regulatory subunit of the m 6 A methyltransferase complex, which can interact with METTL3 and METTL14. Knocking out WTAP can significantly reduce the m 6 A peak in cellular mRNA, even more effective than knocking down METTL3 or METTL14. The WTAP-bound gene has a change in alternative splicing patterns [25].
The KIAA1429, also known as vir-like m 6 A methyltransferase associated (VIRMA), is a homologous protein of the Virilizer protein in Drosophila, which is closely related to the methyltransferase complex. The N-terminus of KIAA1429 has the ability to gather methyltransferase-catalyzed core METTL3/METTL14/WTAP that can achieve the regulation of fixed-point m 6 A levels on mRNA [26].
It was identified by co-immunoprecipitation that the binding of RBM15/15B at the RRACH sequence site is three to fourfold o higher than that at the non-methylation site. Knocking down the RBM15 or RBM15B alone can reduce the m 6 A levels in cellular mRNA, and knocking down both RBM15 and RBM15B can result in a significant decrease of the m 6 A levels in mRNA [27]. The HAKAI, also known as CBL proto-oncogene like 1(CBLL1), is an E3 ubiquitin ligase. Down-regulation of the HAKAI in Arabidopsis can result in a decrease in m 6 A level [28]. ZC3H13 is also an important component of the methyltransferase complex and is key to anchor the complex in the nucleus [29]. Methyltransferase like 16 (METTL16) is a m 6 A methyltransferase of the mRNA precursor that maintains SAM homeostasis by regulating alternative splicing of methionine adenosyltransferase II alpha (MAT2a) [30][31][32].
m 6 A demethylase
The m 6 A demethylase, also known as the "Erasers". In eukaryotes, m 6 A demethylases are fat mass and obesity-associated protein (FTO) and alkB homolog 5 (alkB homolog 5, ALKHB5). In Arabidopsis, the alkB homolog 10B (ALKHB10B) has also been found as a m 6 A demethylase of mRNA.
The FTO also known as alkB homolog 9 (ALKBH9), which is a member of the Alkb protein family and associated with obesity. FTO is the first-discovered RNA demethylase. The long stem loop domain at the C-terminus of FTO enables the FTO proteins demethylate [33,34].
The ALKBH5 is another protein of the AlkB family and plays an important regulatory role in biological processes, such as mRNA processing. The ALKBH5 is similar to FTO and is also a Fe 2+ and α-Ketoglutaric acid dependent non-heme oxygenase. The ALKBH5 has an alaninerich region at the N-terminus and a unique coiled-coil structure. It only demethylates the m 6 A modification on single-stranded RNA/DNA, and the catalytic reaction removes methyl groups directly from m 6 A-methylated adenosine instead of oxidative demethylation [35,36].
The ALKBH10B is an m 6 A demethylase of mRNA in Arabidopsis, which regulates mRNA stability and affects the transformation of Arabidopsis from vegetative growth to reproductive growth [37].
mRNA m 6 A methylation binding protein
The m 6 A-modified mRNA that performs a specific biological function requires a specific RNA-binding protein-readers. Binding assays of RNA protein in vitro have identified a variety of binding proteins, including YTH domain containing RNA binding protein (YTP), heterogeneous nuclear ribonucleoprotein (hnRNP), eukaryotic initiation factor 3 (eIF3), Insulin-like growth factor 2 mRNA-binding protein (IGF2BP) and Proline rich coiled-coil 2A (Prrc2a). The functions of these binding proteins mainly include specific binding to the m 6 A methylation region, weakening the homologous binding to RNA reading proteins, and altering the secondary structure of RNA to alter protein-RNA interaction [38,39].
YTH domain containing RNA binding protein include YTH domain-containing family protein 1/2/3 (YTHDF1/2/3) and YTH domain-containing protein 1/2 (YTHDC1/2). YTHDF1/2/3 and YTHDC2 specifically recognize the m 6 A-modified mRNA in the cytoplasm, while the recognizing sites of YTHDC1 are mainly in the nucleus. These proteins all have a YTH domain at the C-terminus. They are capable of overlapping with the m 6 A RRACH fragment to mediate RNAspecific binding, while its proline/glutamine/asparagine enrichment (P/Q/N-rich) domain is related to subcellular localization [40,41].
YTHDF1 is combined with translation initiation factors and ribosomes, improving the translation efficiency. YTHDF2 is the first-discovered binding protein.
Specifically, it recognizes and binds m 6 A-containing RNAs, and regulates mRNA stability [42,43]. YTHDF3 promotes the translation of mRNA and regulates the mRNA stability. YTHDF3 and YTHDF1 coordinately control during translation [44,45]. YTHDC1 regulates the mRNA cleavage by recruiting splicing factors [46][47][48]. YTHDC2 accelerates the degradation of the modified mRNA and enhances the translation of the corresponding protein by recognizing m 6 A [49].
The hnRNP is a group of RNA-binding proteins that contain nearly 30 nucleic acid-binding proteins with molecular weights ranging from 30 to 120 kDa, which can interact with each other to form the complex, where A1, A2, B1, B2, C1 and C2 are the main core components. Heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) is capable of specifically recognizing the m 6 A modifications on transcripts, activating downstream variable shear events of partial genes [50,51]. Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is responsible for recognizing the m 6 A modifying group and mediating the processing of the mRNA precursor in the nucleus, affecting the abundance and alternative splicing of target transcripts. The m 6 A can increase the accessibility of its surrounding RNA sequences binding to heterogeneous nuclear ribonucleoprotein G (HNRNPG). In the transcriptome, the m 6 A site regulates RNA-HNRNPG interactions to alter target mRNA expression and alternative splicing patterns [52].
In mammalian cells, eIF3 is the largest eukaryotic initiation factor and plays a key role in the initiation of eukaryotic translation. It is able to directly bind to the 5′UTR m 6 A of mRNA, thereby facilitating translation of mRNA. IGF2BP protein is a unique m 6 A reader. The family mainly includes IGF2BP1/2/3. IGF2BP can make the target gene and corresponding translation more stable [53]. Prrc2a is a new m 6 A reader that stabilizes mRNA expression by binding to a consensus GGACU motif in the CDS region in an m 6 A-dependent manner [54] (Fig. 3).
Neurobiological function of mRNA m 6 A methylation
Sequencing results have showed that the level of RNA m 6 A modification increase significantly during embryogenesis [55][56][57][58]. Compared to other organs or tissues, the overall level of m 6 A in the head is significantly higher. This suggests that the mRNA m 6 A modification has potential neurobiological functions in the nervous system and is worthy of further study [59][60][61][62][63].
Effect of m 6 A on neural stem cells
Neural stem cells maintain cell populations through self-renewal, and can differentiate into various nerve cells such as neurons, astrocytes, and oligodendrocytes [64][65][66][67]. A number of studies have shown that the mRNA m 6 A modification can affect the self-renewal and differentiation of neural stem cells [68][69][70]. These new findings will promote stem cell therapy and gene-targeted therapy for neurological diseases.
Inactivation of Mettl3 in mouse and human embryonic stem cells leads to a decrease in m 6 A, and severely impairs the transition of neurons from self-renewal to differentiation. The knockout of Mettl3 can cause early embryonic lethality and impair the formation of mature neurons in the embryoid body [58,71]. Wang et al. found that when knocking out Mettl14 in embryonic neural stem cells in a mouse model, the proliferation of neural stem cells was significantly reduced and differentiated prematurely. It indicates that m 6 A can promote the proliferation of neural stem cells and prevent premature differentiation of cells, thus ensuring the reserve of neural stem cell bank [72,73]. Mettl14 and Mettl3 can participate in neurogenesis by regulating the cell cycle progression of cortical neural stem cells, which acts in a m 6 A-dependent way [74]. The SMAD2/3 protein binds to the METTL3-METTL14-WTAP complex and promotes the differentiation of embryonic stem cells into neuroendodermal cells [75].
The Ythdf2-mediated m 6 A mRNA clearance has a regulatory effect on neurodevelopment in mice. Proliferation and differentiation of neural stem cells are seriously affected by the deletion of embryonic Ythdf2 [76].
Effect of m 6 A on learning and memory
In the emerging field of epitranscriptomic mechanisms, mRNA m 6 A modification has potential role in learning and memory [77]. It regulates physiological and stressinduced behavior in the adult mammalian brain, and augments the strength of weak memories [78][79][80]. As a newly identified element in the region-specific gene regulatory network in the mouse brain, mRNA m 6 A modification plays a vital role in the death of dopaminergic neuron [81,82].
Mettl3-mediated RNA m 6 A modification has the direct effect on regulating hippocampal-dependent long-term memory formation. The decrease of Mettl3 in the mice hippocampus may reduce its memory consolidation, and adequate training or restoration would restore the ability of learn and memory. The abundance of Mettl3 in the hippocampus of wild-type mice is positively correlated with learning efficiency, and the overexpression of Mettl3 can significantly enhance the long-term memory consolidation [83]. METTL14 is critical for striatum function and transcriptional regulation of learning epitopes. In cell experiments, the deletion of METTL14 reduces striatum m 6 A levels without altering cell number or morphology, increases neuronal excitability and severely impaired striatal-mediated behavior [84].
Fto can regulate the activity of dopaminergic midbrain circuits. Inactivation of the Fto gene weaken neuronal activity and behavioral responses that are dependent on dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively D2-like receptors) [85]. FTO also regulates dopaminergic neurotransmission deficits caused by arsenite [86]. Walters [87] has found that Fto plays an important role in the formation of mouse hippocampal-dependent memory. The decrease in Fto protein observed shortly after the situational fear reflex indicates that Fto typically limits memory formation. The m 6 A is regulated in the activity-dependent way in the adult brain, and may finetune mRNA turnover during memory-related processes [88]. When knocking out the Fto gene in the prefrontal cortex of mice, the intensity of m 6 A on several fearrelated genes in neurons increases significantly, and the knockdown of Fto further enhances the consolidation of fear memory [89]. FTO plays important roles in learning and memory. The loss of FTO led to the altered expression of several key components of the brain derived neurotrophic factor pathway that were marked by m 6 A [90].
In the adult mouse hippocampus, the m 6 A binding protein Ythdf1 can promote neuronal stimulation of protein translation of target transcripts, thereby facilitating learning and memory. Mice with a genetic deletion of Ythdf1 have showed the deficits of learning and memory, impaired hippocampal synaptic transmission and longterm potentiation [91]. Prrc2a controls the specification and myelination of oligodendrocyte, and Prrc2a knockout induces cognitive defects in a mouse model [54].
Effect of m 6 A on brain development
Widespread and dynamic m 6 A methylation were identified in the developing mouse cerebellum. RNA m 6 A methylation is controlled in a precise spatiotemporal manner and participates in the regulation of postnatal development of the mouse cerebellum [92,93].
Specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3-mediated m 6 A participates in cerebellar development by controlling mRNA stability of genes involved in cerebellar development and apoptosis [94].
Under the low pressure and hypoxia, the level of RNA m 6 A methylation in the cerebellar of Alkbh5-deficient mouse is imbalanced, which leads to an increase in the efficiency of extranuclear RNA excretion and a significant change in cerebellar phenotype, including neuronal structural disorder, abnormal cell proliferation and differentiation, and other phenotypes [92].
Effect of m 6 A on synaptic growth
The m 6 A modification plays a key role in synaptic regeneration of mature mouse neurons. Increased m 6 A in somatic neurons alters the transcriptome response to synaptic plasticity [77,89]. The m 6 A methylation of neurological function-related genes in the hippocampus of human immunodeficiency virus transgenic rats is significantly different, suggesting synaptic damage and neurodegeneration [95]. The m 6 A methylation of synaptic mRNAs critically contribute to synaptic function in healthy adult mouse forebrains [96]. Deletion of Mettl14 reduces functional axonal regeneration in the peripheral nervous system of the body. After knockdown of Mettl14, the axonal regeneration of retinal ganglion neurons in the central nervous system is also diminished [97].
The m 6 A modification can affect axon growth by regulating local translation of mRNA in neuronal axons. FTO is highly expressed in axons of neurons. Local translation in axons plays an important role in neurodevelopment, including axon guidance, axon growth, and neuronal specifications [90,98].
The mRNA m 6 A modification of synaptic plays a key role in synaptic function. After knocking out the dendritic positioning readers Ythdf1 and Ythdf3 in cultured hippocampal neurons, m 6 A-reader-deficient neurons have abnormal spine morphology and the spines are reduced. Knocking out the Ythdf1 gene of mouse, in the peripheral and peripheral nervous system, functional axon regeneration is reduced [97,99]. The neurons of YTHDF2 −/− could not produce normal synapses [76].
Effect of m 6 A on glioblastoma
Several studies have revealed the role of m 6 A witers and erasers in glioblastoma. Changes of the m 6 A level in glioblastoma stem cell-like cells (GSC) severely affect the growth, self-renewal and development of tumor. The mRNA m 6 A methylation is expected to be a new target for the treatment of glioblastoma [100].
Decreasing the m 6 A levels by knocking down METTL3 and/or METTL14 enhance growth and self-renewal of GSCs in vitro, and promote the ability of GSCs to form brain tumors in vivo. The Mettl3-mediated m 6 A modification plays a key role in neurosphere maintenance and glioma cell dedifferentiation [101][102][103]. Ethyl form of methylbenzoic acid (MA2) is a selective inhibitor of FTO, which can significantly inhibit tumor progression and prolong the lifespan of GSC mice. Therefore, The Fto may play a key carcinogenic role in GSC self-renewal and is required for the development of glioblastoma [101]. ALKBH5 is able to maintain stem cell in malignant glioma cells, and ALKBH5-mediated m 6 A modification on forkhead box M1 (FOXM1) mRNA is involved in the maintenance of tumor stem cell. High expression of ALKBH5 predicts poor prognosis in glioblastoma patients [104,105] (Table 1).
Conclusion
In summary, the mRNA methylation is an important epitranscriptomic modification and the m 6 A is highly expressed in the brain. The mRNA m 6 A methylation has a wide range of effects on the nervous system, and plays an important part in self-renewal of neural stem cells, learning memory, brain development, synaptic growth and proliferation of glioma cells. This new regulatory system will promote targeted therapy for neurological diseases.
However, mRNA m 6 A methylation is a relatively new field and many problems remain unknown. Up till now, all of the demethylases found belong to the AlkB family, and whether other proteins in the AlkB family are also involved in mRNA demethylation is worthy for further study. HNRNPA1, HNRNPG and HNRNPM play a key role in the methylation of protein arginine. These proteins are similar to HNRNPA2B1 and HNRNPC, and belong to the hnRNP binding protein family. It is worth exploring its role in mRNA m 6 A methylation.
Variations in the FTO gene can not only regulate D2Rdependent reward learning [106][107][108], but also affect nerve adjust food visual, produce more frequent rewards [109][110][111], affect the control of mood and impulse [112][113][114], and affect obesity by regulating brain signaling pathways [115,116]. The homozygous mutation of FTO gene can reduce the brain capacity of healthy elderly people, increase the susceptibility to brain atrophy during aging, and even affect the brain volume of adolescents [117,118]. The genetic polymorphism of FTO is related to attention-deficit/hyperactivity disorder (ADHD), Alzheimer's disease and depression [119][120][121][122][123][124]. Whether it is as demethylase that affects these diseases, is worthy of further study.
The genetic polymorphism of ZC3H13 is associated with schizophrenia. Nito, another member of the m 6 A methyltransferase complex in Drosophila, called RBM15 in human, controls the axonal growth and differentiation and regulates the synapse formation through neuronal activity. Whether human ZC3H13 and RBM15 genes have the effect on synaptic growth, is worthy of further study.
To study the methylation mechanism of mRNA m 6 A and find potential targets for treatment, it is hopeful to develop inhibitors or agonists of related proteins for clinical treatment in the future.
Fig. 1
1Different types of mRNA methylation modification
Fig. 2
2RRACH
Fig. 3
3mRNA m 6 A methylation-associated protein
5hmC: 5-hydroxymethylcytosine; ADHD: attention-deficit/hyperactivity disorder; ALKHB5/9/10B: alkB homolog 5/9/10B; CBLL1: CBL proto-oncogene like 1; CDS: coding region; D2/3R: dopamine receptor type 2/3; DNA: deoxyribonucleic acid; eIF3: eukaryotic initiation factor 3; FOXM1: forkhead box M1; FTO: fat mass and obesity-associated protein; GSC: glioblastoma stem cell-like cells; HAKAI: E3 ubiquitin-protein ligase Hakai; hnRNP: heterogeneous nuclear ribonucleoprotein; HNRNPA2B1/C/G: heterogeneous nuclear ribonucleoprotein A2B1/C/G; IGF2BP: insulin-like growth factor 2 mRNA-binding protein; m 1 A: N1-methyladenosine; m 5 C: 5-methylcytosine; m 6 A: N6-methyladenosine; m 6 Am: N6, 2′-O-dimethyladenosine; m 7 G: 7-methylguanine; MA2: methylbenzoic acid; MAT2a: methionine adenosyltransferase II Alpha; METTL3/14/16: methyltransferase like 3/14/16; mRNA: messenger RNA; P/Q/N-rich: proline/ glutamine/asparagine enrichment; PolyA: polyadenosinic acid; Prrc2a: proline rich coiled-coil 2A; RBM15/15B: RNA binding motifs protein 15/15B; RNA: ribonucleic acid; SAM: S-adenosylmethionine; UTRs: untranslated regions; VIRMA: vir-like m 6 A methyltransferase associated; WTAP: wilms' tumour 1-associating protein; YTHDC1/2: YTH domain-containing protein 1/2; YTHDF1/2/3: YTH domain-containing family protein 1/2/3; YTP: YTH domain containing RNA binding protein; ZC3H13: zinc finger CCCH-type containing 13.
Table 1
1Neurobiological functions of mRNA m 6 A methylationNeurological disease
Related enzymes and proteins
Neural stem cell
METTL3, METTL14 and YTHDF2
Learning memory
METTL3, METTL14, FTO, YTHDF1 and Prrc2a
Brain development
METTL3 and ALKBH5
Synaptic growth
METTL14, FTO, YTHDF1, YTHDF2 and YTHDF3
Glioblastoma
METTL3, METTL14, FTO and ALKBH5
AcknowledgementsWe would like to acknowledge Qixue Li, Fengdi Wu, Minghui Li, Xianchao Du, Bingchen Liu and Haiying Wang for their advice.Authors' contributionsEach author substantially contributed to the review. JL: conception and design, drafting the review; XY, ZQ, YS, YL, BX, WL and ZX: revising the manuscript; YD: conception and design, revising it critically for important intellectual content, and final approval of the version to be published. All authors read and approved the final manuscript.
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FTO affects food cravings and interacts with age to influence age-related decline in food cravings. L C Dang, G R Samanez-Larkin, C T Smith, J J Castrellon, S F Perkins, R L Cowan, 10.1016/j.physbeh.2017.12.013Physiol Behav. 192Dang LC, Samanez-Larkin GR, Smith CT, Castrellon JJ, Perkins SF, Cowan RL, et al. FTO affects food cravings and interacts with age to influence age-related decline in food cravings. Physiol Behav. 2018;192:188-93. https ://doi.org/10.1016/j.physb eh.2017.12.013.
FTO genotype impacts food intake and corticolimbic activation. S J Melhorn, M K Askren, W K Chung, M Kratz, T A Bosch, V Tyagi, 10.1093/ajcn/nqx029Am J Clin Nutr. 107Melhorn SJ, Askren MK, Chung WK, Kratz M, Bosch TA, Tyagi V, et al. FTO genotype impacts food intake and corticolimbic activation. Am J Clin Nutr. 2018;107:145-54. https ://doi.org/10.1093/ajcn/nqx02 9.
An obesity-associated risk allele within the FTO gene affects human brain activity for areas important for emotion, impulse control and reward in response to food images. L Wiemerslage, E K Nilsson, Solstrand Dahlberg, L Ence-Eriksson, F Castillo, S Larsen, A L , 10.1111/ejn.13177Eur J Neurosci. 43Wiemerslage L, Nilsson EK, Solstrand Dahlberg L, Ence-Eriksson F, Castillo S, Larsen AL, et al. An obesity-associated risk allele within the FTO gene affects human brain activity for areas important for emotion, impulse control and reward in response to food images. Eur J Neurosci. 2016;43:1173-80. https ://doi.org/10.1111/ejn.13177 .
Fat mass and obesity-associated gene (FTO) is associated to eating disorders susceptibility and moderates the expression of psychopathological traits. G Castellini, M Franzago, S Bagnoli, L Lelli, M Balsamo, M Mancini, 10.1371/journal.pone.0173560PLoS ONE. 12173560Castellini G, Franzago M, Bagnoli S, Lelli L, Balsamo M, Mancini M, et al. Fat mass and obesity-associated gene (FTO) is associated to eating disorders susceptibility and moderates the expression of psychopatho- logical traits. PLoS ONE. 2017;12:e0173560. https ://doi.org/10.1371/ journ al.pone.01735 60.
FTO genotype and aging: pleiotropic longitudinal effects on adiposity, brain function, impulsivity and diet. Y F Chuang, T Tanaka, L L Beason-Held, An Y Terracciano, A Sutin, A R , 10.1038/mp.2014.49Mol Psychiatry. 20Chuang YF, Tanaka T, Beason-Held LL, An Y, Terracciano A, Sutin AR, et al. FTO genotype and aging: pleiotropic longitudinal effects on adiposity, brain function, impulsivity and diet. Mol Psychiatry. 2015;20:133-9. https ://doi.org/10.1038/mp.2014.49.
Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway. L Lin, C M Hales, K Garber, Jin P , 10.1093/hmg/ddu043Hum Mol Genet. 23Lin L, Hales CM, Garber K, Jin P. Fat mass and obesity-associated (FTO) protein interacts with CaMKII and modulates the activity of CREB signaling pathway. Hum Mol Genet. 2014;23:3299-306. https ://doi. org/10.1093/hmg/ddu04 3.
The growth hormone-IGF-I axis as a mediator for the association between FTO variants and body mass index: results of the Study of Health in Pomerania. D Rosskopf, C Schwahn, F Neumann, A Bornhorst, C Rimmbach, M Mischke, 10.1038/ijo.2010.158Int J Obes. 35Rosskopf D, Schwahn C, Neumann F, Bornhorst A, Rimmbach C, Mischke M, et al. The growth hormone-IGF-I axis as a mediator for the association between FTO variants and body mass index: results of the Study of Health in Pomerania. Int J Obes. 2011;35:364-72. https ://doi. org/10.1038/ijo.2010.158.
research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC. A J Ho, J L Stein, X Hua, S Lee, D P Hibar, A D Leow, 10.1073/pnas.0910878107Proc Natl Acad Sci USA. 107A commonly carried allele of the obesity-related FTO gene is associated with reduced brain volume in the healthy elderly. research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit fromHo AJ, Stein JL, Hua X, Lee S, Hibar DP, Leow AD, et al. A commonly carried allele of the obesity-related FTO gene is associated with reduced brain volume in the healthy elderly. Proc Natl Acad Sci USA. 2010;107:8404-9. https ://doi.org/10.1073/pnas.09108 78107 . research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research ? Choose BMC and benefit from:
FTO, obesity and the adolescent brain. M G Melka, J Gillis, M Bernard, M Abrahamowicz, M M Chakravarty, G T Leonard, 10.1093/hmg/dds504Hum Mol Genet. 22Melka MG, Gillis J, Bernard M, Abrahamowicz M, Chakravarty MM, Leon- ard GT, et al. FTO, obesity and the adolescent brain. Hum Mol Genet. 2010;22:1050-8. https ://doi.org/10.1093/hmg/dds50 4.
Association between obesity-related gene FTO and ADHD. Z Choudhry, S M Sengupta, N Grizenko, G A Thakur, M E Fortier, N Schmitz, 10.1002/oby.20444Obesity. 21Choudhry Z, Sengupta SM, Grizenko N, Thakur GA, Fortier ME, Schmitz N, et al. Association between obesity-related gene FTO and ADHD. Obesity. 2013;21:e738-44. https ://doi.org/10.1002/oby.20444 .
The obesity related gene, FTO, interacts with APOE, and is associated with Alzheimer's disease risk: a prospective cohort study. L Keller, W Xu, H X Wang, B Winblad, L Fratiglioni, C Graff, 10.3233/JAD-2010-101068J Alzheimers Dis. 23Keller L, Xu W, Wang HX, Winblad B, Fratiglioni L, Graff C. The obesity related gene, FTO, interacts with APOE, and is associated with Alzheimer's disease risk: a prospective cohort study. J Alzheimers Dis. 2011;23:461-9. https ://doi.org/10.3233/JAD-2010-10106 8.
Genetic variants in the fat and obesity associated (FTO) gene and risk of Alzheimer's disease. C Reitz, G Tosto, R Mayeux, J A Luchsinger, 10.1371/journal.pone.0050354PLoS ONE. 7Reitz C, Tosto G, Mayeux R, Luchsinger JA, et al. Genetic variants in the fat and obesity associated (FTO) gene and risk of Alzheimer's disease. PLoS ONE. 2012;7:e50354. https ://doi.org/10.1371/journ al.pone.00503 54.
FTO is involved in Alzheimer's disease by targeting TSC1-mTOR-Tau signaling. H Li, Y Ren, K Mao, F Hua, Y Yang, N Wei, 10.1016/j.bbrc.2018.02.201Biochem Biophys Res Commun. 498Li H, Ren Y, Mao K, Hua F, Yang Y, Wei N, et al. FTO is involved in Alzheimer's disease by targeting TSC1-mTOR-Tau signaling. Biochem Biophys Res Commun. 2018;498:234-9. https ://doi.org/10.1016/j. bbrc.2018.02.201.
The protective effect of the obesity-associated rs9939609 A variant in fat mass-and obesity-associated gene on depression. Z Samaan, S S Anand, X Zhang, D Desai, M Rivera, G Pare, 10.1038/mp.2012.160Mol Psychiatry. 18Samaan Z, Anand SS, Zhang X, Desai D, Rivera M, Pare G, et al. The protective effect of the obesity-associated rs9939609 A variant in fat mass-and obesity-associated gene on depression. Mol Psychiatry. 2013;18:1281-6. https ://doi.org/10.1038/mp.2012.160.
Interaction between the FTO gene, body mass index and depression: meta-analysis of 13701 individuals. M Rivera, A E Locke, T Corre, D Czamara, C Wolf, A Ching-Lopez, 10.1192/bjp.bp.116.183475Br J Psychiatry. 211Rivera M, Locke AE, Corre T, Czamara D, Wolf C, Ching-Lopez A, et al. Interaction between the FTO gene, body mass index and depression: meta-analysis of 13701 individuals. Br J Psychiatry. 2017;211:70-6. https ://doi.org/10.1192/bjp.bp.116.18347 5.
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Introduction
The emerging "epitranscriptomics" field studies the impact of RNA chemical modifications on gene expression regulation. Among hundreds of RNA chemical modifications, the methylation in position N 6 of adenine, which characterized both N 6 -methyladenosine (m 6 A m ) and N 6 ,2 -O-dimethyladenosine (m 6 A m ), is better characterized in mRNA molecules ( Figure 1) [1].
Introduction
The emerging "epitranscriptomics" field studies the impact of RNA chemical modifications on gene expression regulation. Among hundreds of RNA chemical modifications, the methylation in position N 6 of adenine, which characterized both N 6 -methyladenosine (m 6 Am) and N 6 ,2′-O-dimethyladenosine (m 6 Am), is better characterized in mRNA molecules ( Figure 1) [1]. m 6 A is the most abundant internal chemical modification in mRNA. Several studies reported its relevant role in regulating different steps of the mRNA expression, includ-ing splicing, nuclear export, stability, and translation (reviewed in [1]). On the other hand, m 6 A m is found in the first position adjacent to the 5 -end cap structure, which is constituted by a N 7 -methylguanosine (m 7 G) connected via a 5 to 5 triphosphate bond (m7GpppN; also referred to as cap 0), in many mammalian mRNAs [2]. m7GpppN is added cotranscriptionally by three sequentially enzymatic activities and is followed by one or two 2 -O-methylated nucleotides to produce cap1 (m7GpppN m ) and cap2 structures (m7GpppN m N m ), respectively [2]. When the first transcribed nucleotide is an adenine, this can be further modified cotranscriptionally at the N 6 position to produce m 6 A m [3]. Quantification studies revealed that the percentage of m 6 A m in mRNA species varies according to different organisms and cell types, spanning from 10% to almost 50% [4]. Moreover, m 6 A m profiling performed in human and mouse tissues showed that m 6 A m levels vary greatly between different tissues [5]. In view of its vicinity with the mRNA cap-structure, it has been hypothesized that there is a role for m 6 A m in regulating decapping, and eventually stability and translation. However, its role in gene expression is still controversial. In addition, m 6 A m can be found as an internal modified nucleotide within the small nuclear RNAs (snRNAs) U2, where it has been shown to regulate pre-mRNA splicing.
In this review, we describe the methodologies to profile m 6 A m , the enzymes responsible for installing and removing m 6 A m from RNA, and the impact of this RNA modification in gene expression regulation. Furthermore, we discuss the emerging roles of m 6 A m regulators in tumorigenesis.
Methodologies for m 6 A m Detection
As for other RNA modifications, global quantification of m 6 A m levels can be obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS) or thin-layer chromatography (TLC) on single-nucleotide digested RNAs [6,7]. Analysis can be restricted to mRNA species by affinity purification using poly-dT oligonucleotides followed by in vitro decapping. Nevertheless, one of the major challenges in RNA modification studies is the identification and mapping of the modified nucleotide within specific transcripts. Highthroughput sequencing methods coupled with immunoprecipitation of fragmented RNAs with m 6 A-specific antibodies have been developed to identify m 6 A m containing RNAs. Even if the anti-m 6 A antibodies cannot distinguish between m 6 A m and m 6 A, m 6 A m is limited to the first transcribed position in mRNAs, while m 6 A is enriched in internal regions of the RNA molecules within a specific consensus motif. Thus, sequencing methods utilized for m 6 A identification, such as MeRIP-seq (m6A-seq) and miCLIP, can be applied to m 6 A m mapping with specific bioinformatics analysis [8,9] (Table 1). However, m 6 A sequencing methods do not efficiently capture m 6 A m -containing transcripts. Therein, specific protocols for the identification of m 6 A m methylomes have been developed, each with its advantages and limitations (Table 1).
Method
Principle Advantages Limitations
MeRIP-seq (m6A-seq) [8] Utilizes anti-m6A antibody to enrich m 6 A-and m 6 A m -containing fragments Easy to perform, kit available from different suppliers Low resolution, required dedicated bioinformatic analysis for m 6 A m identification, cannot distinguish between cap-m 6 A m and m 6 A in 5 -RNA fragments, antibody cross-reactivity miCLIP [9] Utilizes CLIP with anti-m6A antibody to identify m 6 A-and m 6 A m in RNA fragments
Single-nucleotide resolution
Required dedicated bioinformatic analysis for m 6 A m identification, antibody cross-reactivity Cap-m 6 A m are enzymatically propargylated by PCIF1, using synthetic AdoMet analog, selectively biotinylated and enriched with magnetic streptavidin-beads
Antibody-independent method
Never applied to transcriptome studies m6Am-Exo-Seq [10] utilizes a 5 -> 3 exonuclease to degrade uncapped RNAs after fragmentation, leaving only capped 5 -end fragments. These are decapped in vitro, to favor the m 6 A m recognition by the m6A antibody, immunoprecipitated with an anti-m6A antibody and sequenced (Figure 2a). A similar method called m6A-crosslinking-exonucleasesequencing (m6ACE-seq) allows the detection of both m 6 A m and m 6 A modifications [11]. In the m6ACE-seq the m6A antibody, which recognizes both modifications, is crosslinked to RNA after fragmentation, thus protecting the fragments from digestion with 5 -> 3 exoribonuclease (Figure 2b). Sequencing of protected RNA, followed by a dedicated bioinformatics analysis, allows the mapping of 5 -end (m 6 A m ) and internal (m 6 A) modifications. An additional methodology developed for specific m 6 A m identification is the m6Amseq [12] (Figure 2c), in which capped RNAs are immunoprecipitated with an m7G antibody after fragmentation and treated with recombinant FTO RNA demethylases, which removes the methyl group from the N 6 position. Control and demethylated RNAs are then immunoprecipitated by an anti-m6A antibody. Comparison of the two samples, FTO-treated and FTO-untreated, allows the specific identification of m 6 A m sites ( Figure 2c). Recently, an antibody-free approach has been developed for the enrichment of m 6 A m containing RNAs, CAPturAM [13]. However, it has not yet been applied to transcriptome-wide studies.
Figure 2.
Sequencing methods for m 6 Am mapping. All methods work on fragmented RNA. (a) In the m6a-exo-seq [10], RNAs are treated with a 5′-3′ exonuclease to enrich for m7G-capped RNAs. After decapping, fragments are immunoprecipitated with an anti-m6A antibody and sequenced. (b) In the m6ACE-seq [11], the anti-m6A antibody is covalently bound to m6A containing RNA fragments with UV crosslinking. Fragments are then treated with a 5′-3′ exonuclease, which is blocked by the bound antibody, to enrich modified RNAs before sequencing. (c) In the m6Am-seq method [12], m7G-capped fragments are immunoprecipitated with an anti m7G-antibody and then treated with FTO demethylases, which in vitro has better activity towards m6Am modification, before sequencing. FTO-untreated RNAs are utilized as control.
Regulators of m 6 Am Levels
Levels of m 6 Am are regulated by specific proteins that install and remove m 6 Am modification from specific transcripts ( Figure 3). Specific writer proteins modify mRNA and snRNA molecules, while the removal of m 6 Am depends on a single eraser protein. [10], RNAs are treated with a 5 -3 exonuclease to enrich for m7G-capped RNAs. After decapping, fragments are immunoprecipitated with an anti-m6A antibody and sequenced. (b) In the m6ACE-seq [11], the anti-m6A antibody is covalently bound to m6A containing RNA fragments with UV crosslinking. Fragments are then treated with a 5 -3 exonuclease, which is blocked by the bound antibody, to enrich modified RNAs before sequencing. (c) In the m6Am-seq method [12], m7G-capped fragments are immunoprecipitated with an anti m7G-antibody and then treated with FTO demethylases, which in vitro has better activity towards m6Am modification, before sequencing. FTO-untreated RNAs are utilized as control.
Regulators of m 6 A m Levels
Levels of m 6 A m are regulated by specific proteins that install and remove m 6 A m modification from specific transcripts ( Figure 3). Specific writer proteins modify mRNA and snRNA molecules, while the removal of m 6 A m depends on a single eraser protein. It was reported that m 6 Am can increase mRNA translation, decrease mRNA translation, increase mRNA stability, and not affect mRNA stability (see main text for details). (b) Different snRNAs, including U1 and U2, contain m 6 Am on their caps and this methylation is established by PCIF1 and removed by FTO. In addition, the U2 snRNA contains an internal m 6 Am site that is installed by METTL4 and whose deletion causes altered alternative splicing.
m 6 Am Writers: PCIF1 and METTL4
• PCIF1 (CAPAM)
The enzyme responsible for the methylation in the N 6 position of the 2′-O-methyladenosine residue next to the m 7 G-cap of mRNAs and some small nuclear RNAs (snR-NAs) was discovered from fractionated HeLa extract in the early 1970s [14]. By using in vitro modified mRNAs and purified germ or reticulocyte ribosomes, the authors also show that in their experimental conditions the presence of m7Gpppm 6 Am had a small positive effect on ribosome binding [14]. Nevertheless, the gene coding for the modifying enzyme, known as "phosphorylated CTD-interacting factor 1" (PCIF1) or "cap-specific adenosine methyltransferase" (CAPAM), was only identified in 2019 by four independent groups [10,[15][16][17]. PCIF1 contains an N-terminal WW domain that mediates interactions with the Ser5-phosphorylated carboxy-terminal domain (CTD) of the major RNA polymerase II (Pol II) subunit, which is present at transcription initiation, and a core region that contains the "helical" and "methyltransferase" (MTase) domains [14]. Like capping enzymes, which interact with Ser5-phosphorylated CTD, the WW domain provides coupling of the m 6 Am modification with transcription initiation. The helical domain is specific for the PCIF1 protein and forms a positively charged groove that is thought to function as (b) Different snRNAs, including U1 and U2, contain m 6 A m on their caps and this methylation is established by PCIF1 and removed by FTO. In addition, the U2 snRNA contains an internal m 6 A m site that is installed by METTL4 and whose deletion causes altered alternative splicing.
m 6 A m Writers: PCIF1 and METTL4
• PCIF1 (CAPAM)
The enzyme responsible for the methylation in the N 6 position of the 2 -O-methyladenosine residue next to the m 7 G-cap of mRNAs and some small nuclear RNAs (snRNAs) was discovered from fractionated HeLa extract in the early 1970s [14]. By using in vitro modified mRNAs and purified germ or reticulocyte ribosomes, the authors also show that in their experimental conditions the presence of m7Gpppm 6 A m had a small positive effect on ribosome binding [14]. Nevertheless, the gene coding for the modifying enzyme, known as "phosphorylated CTD-interacting factor 1" (PCIF1) or "cap-specific adenosine methyltransferase" (CAPAM), was only identified in 2019 by four independent groups [10,[15][16][17]. PCIF1 contains an N-terminal WW domain that mediates interactions with the Ser5-phosphorylated carboxy-terminal domain (CTD) of the major RNA polymerase II (Pol II) subunit, which is present at transcription initiation, and a core region that contains the "helical" and "methyltransferase" (MTase) domains [14]. Like capping enzymes, which interact with Ser5-phosphorylated CTD, the WW domain provides coupling of the m 6 A m modification with transcription initiation. The helical domain is specific for the PCIF1 protein and forms a positively charged groove that is thought to function as the RNA-binding surface. The MTase domain is highly homologous to that of class I MTases, which includes, among many, DNA and RNA SAM-dependent m 6 A methyltransferases. It is characterized by a Rossman fold catalytic domain containing a conserved sequence motif (NPPF). The specific recognition of the m7G cap occurs through a specific site (m7Gsite) located between the helical and MTase domains [15]. Recombinant PCIF1 protein alone is sufficient to methylate in vitro m7G capped mRNA [10,[15][16][17]. It is the only methyltransferase responsible for N 6 -methylation of the 2 -O-methyladenosine nucleotide next to the m 7 G-cap of mRNA. Therein, its depletion produces the complete loss of m 6 A m in mRNA. Surprisingly, PCIF1 deletion in mice has no effects on viability and fertility but produces reduced body weight [18]. Similarly, in the human HEK293T cell line, PCIF1 knockout did not produced any growth defect but altered cell proliferation under oxidative stress conditions [15]. PCIF1 methyltransferase is conserved only in vertebrates, and it is absent in yeasts, insects, and worms. However, in there is present in Drosophila a catalytically dead PCIF1 that is unable to methylate RNA, but it is still able to bind Ser5-phosporilated CTD [17]. Interestingly, in human cells it has been shown that PCIF1 expression inhibits transcriptional activation of reporter genes [19]. Thus, these results suggest a methyltransferase-independent role in the regulation of RNA Pol II activity by PCIF1 that has been conserved in fly.
• METTL4
The spliceosomal small nuclear RNA (snRNA) U2 contains an internal 2 -O methylated adenine, at position 30 in human U2 snRNA, that is specifically N 6methylated by the METTL4 protein [20,21]. METTL4 belongs to MT-A70-like protein family containing a C-terminal MTase domain with the catalytic DPPW motif followed by a middle domain (MID) and a N-terminal domain (NTD) [22]. Interestingly, the catalytic site of METTL4 is very similar to the methyltransferase METTL3, which is responsible for the formation of m 6 A within mRNA molecules [1]. However, while METTL3 requires the METTL14 protein partner to form a positively charged groove for RNA binding, METTL4 works as a monomer and the function of METTL14 is played by its NTD domain [21]. Contrary to PCIF1, METTL4 is conserved during evolution and U2 snRNA appears to be its only RNA target in vivo [20][21][22]. In in vitro methylation assays, recombinant METTL4 has a preference for A m over A for N 6 -methylation, within the CAAGUG sequence (A is the methylation site) found in U2 snRNA [19,20]. However, when overexpressed in cells, METTL4 can also modify A instead of A m in mRNAs containing the consensus HMAGKD (H = A/C/U, M = A/C, K = G/U, D = A/G/U), which also includes the U2 snRNA target sequence [20]. Interestingly, in human cell lines METTL4 was also found to localize in mitochondria where it acts as an mtDNA m 6 A methyltransferase [23]. The depletion of METTL4 in the HEK293T cell line completely abolished m 6 A m from U2 snRNA but did not alter cell viability [19,20]. However, METTL4 knockdown in mouse 3T3-L1 cells resulted in altered adipocyte differentiation [24], and the loss of METTL4 in Drosophila cells greatly impaired the proliferation rate [25]. These disparate results indicate cell-specific phenotypes.
m 6 A m Eraser: FTO
• FTO FTO (fat mass and obesity-associated protein) belongs to the Fe(II)/α-ketoglutarate acid (α-KG)-dependent AlkB family of dioxygenases. Various in vivo studies reported that FTO can demethylate both cap m 6 A m and internal m 6 A sites in mRNAs and snR-NAs [26,27]. Notably, different cell types exhibit distinctive FTO cellular localization that greatly influence substrate demethylation by FTO [26]. Nuclear FTO preferentially demethylates cap-adjacent m 6 A m in RNA Pol II-transcribed snRNAs, and internal m 6 A m and m 6 A in the snRNAs U2 and U6, respectively. On the other hand, cytoplasmic FTO acts on cap-m 6 A m and internal m 6 A acts on mRNAs [26]. The localization of FTO has been shown to be dependent on the cell cycle phase and to be regulated by casein kinase II-mediated phosphorylation [27,28]. Still, different cell types exhibit different FTO nuclear/cytoplasmic ratios [26]. FTO exhibits the same demethylation activity in vitro toward internal m 6 A and m 6 A m positioned in the same RNA but is influenced by the sequence and tertiary structure of the RNA molecule [26,27]. Structural studies confirmed that the catalytic activity of FTO is mediated by the recognition of the methyl group in N 6 position of adenine rather than the 2 -O methyl group of the ribose [27]. Therefore, the substrate specificity of FTO depends on its localization more than on the type of modification. Since cellular m 6 A levels are considerably higher than those of m 6 A m , many of the effects of FTO have been ascribed to the m 6 A demethylation activity [26].
Function of m 6 A m in mRNA Expression
The development of transcriptome-wide methodologies for m 6 A m mapping and the possibility to alter m 6 A m levels by modulating m 6 A m regulators expression allowed for the study of the specific contribution of m 6 A m in gene expression regulation. However, the results have been controversial so far ( Table 2). Reduced stability of low expressed mRNAs/no effect on translation HEK293T PCIF1/KO [18] Reduced stability of pseudogenes in testis/no effect on translation
The Role of m 6 A m in mRNA Stability
The m 6 A m modification was initially shown to promote the mRNA stability of highly expressed genes [29]. The knockdown of FTO in the HEK293T cell line and the mapping of m 6 A m sites by miCLIP [9] indicated that m 6 A m methylated mRNAs exhibited increased expression levels [29]. Direct transfection of synthetic mRNAs in HEK293T showed that m 6 A m modified transcripts were more stable than their nonmodified counterparts, and the presence of m 6 A m next to the cap decreased in vitro decapping by Dcp2 [29]. Furthermore, the enforced expression of a cytoplasmic FTO in HEK293T caused a significant decrease of m 6 A m containing mRNA but did not affect m 6 A containing mRNA [29]. However, in a later publication the same group demonstrated that cytoplasmic FTO had a significant impact on m 6 A levels within mRNAs and their stability when localized in the cytoplasm [26]. Furthermore, a major problem in utilizing FTO modulation to assess m 6 A m function is that the dual activity of FTO demethylase makes it difficult to discriminate between m 6 A-and m 6 A m -mediated effects. Later, the influence of cap-m 6 A m on mRNA stability was analyzed in additional cell lines: 3T3-L1 (mouse embryonic cells), HeLa (cervical cancer) and JAWS II (mouse immortalized immature dendritic cells) by direct transfection of m 6 A m -modified mRNAs [30]. Interestingly, the initial observation in HEK293 of the positive contribution of m 6 A m in mRNA stability was confirmed only in JAWS II. Thus, these results indicate that the effect of m 6 A m on mRNA stability is strongly dependent on the cell type. Furthermore, it was also shown that mRNAs bearing a 2 -O methylated adenine (cap1), even if not methylated in N 6 , had increased stability. However, it was also shown that neither the 2 -O-methylation nor N 6 -methylation of adenosine influenced in vitro sensitivity to decapping by hDcp2, which, on the other hand, was strongly affected by the type of starting nucleotide [30]. It should be mentioned that Dcp2 is not the only decapping enzyme in human cells [33]. Thus, it is possible that the presence of m 6 A m close to the cap might affect other decapping activities. Moreover, decapping activity in vivo is strongly influenced by RNA structure, RNA uridylation, and RNA binding proteins [34].
As PCIF1 is the only enzyme capable of modifying the adenine next to the cap, its deletion should have given clear results on the role of m 6 A m in mRNA stability. However, even in this case, the results from various studies were not in agreement [10,15,16]. Analyses performed in mouse and human cell lines reported that PCIF1 knockout affected the stability of a subset of m 6 A m -modified mRNAs [16,18]. Particularly, PCIF1 knockout in three different mice tissues (brain, spleen, and testis), which resulted in complete loss of m 6 A m , produced deregulation of different transcripts, with a major impact on pseudogene expression in the testis [18]. However, while in testis transcripts beginning with an A were prevalently downregulated, in the other tissues the presence of an A close to cap did not discriminate between positive and negative variation of RNA levels [18]. An additional study performed in mice analyzed the dynamic regulation of m 6 A m in the fat liver from animals on a high-fat diet [31]. The authors utilized m6A-seq methodology to map m 6 A m sites in liver by subtracting m 6 A sites identified in mESC knockout for the mRNA m 6 A methyltransferase METTL3. Interestingly, they found that fat mice presented significant changes in m 6 A m levels in mRNAs involved in metabolic and obesity-related processes [31]. Furthermore, by using RNA-seq to measure levels of modified mRNAs, they found a positive correlation between m 6 A m levels and mRNA stability. These results suggest that the effect of m 6 A m on mRNA stability might depend on tissue-specific factors. PCIF1 knockout in the HEK293T cell line confirmed that the deletion of m 6 A m correlates with a decrease in mRNA expression [16]. However, by using SLAM-seq (Thiol SH-Linked Alkylation for the Metabolic sequencing of RNA) [35], a method that enables the detection of RNA synthesis and degradation kinetics, and miCLIP [9], which allows m 6 A mapping, they found an effect only on the stability of low-expressed m 6 A m -marked mRNAs [16]. To sum up, the deletion of PCIF1 in different model systems indicated that the m 6 A m modification has a positive effect on mRNA stability. However, this did not always apply to all modified mRNAs. Moreover, the results depended on the experimental conditions and the cell type utilized for the analysis.
In contrast to the data showing a positive role for m 6 A m in cap 1 on mRNA stability, other studies reported opposite results [10,15]. By combining m6Am-Exo-Seq, a method developed for m 6 A m mapping, RNA-seq, to measure steady-state levels of mRNAs, and PRO-Seq (Precision nuclear run-on sequencing) [36], to assess nascent RNA levels, in human MEL624 melanoma and the HEK293T cell line deleted for PCIF1, they did not detect a direct effect of m 6 A m on mRNA stability [10]. On the contrary, changes in RNA abundance upon PCIF1 deletion were due to variations in transcription [10]. An additional recent study that developed m6Am-seq, a specific sequencing method for m 6 A m mapping (Figure 2), confirmed that PCIF1 was not required for the stability of m 6 A m -modified mRNAs [17]. These discrepancies might result from the different methods utilized in these studies for m 6 A m mapping and measurement of transcription dynamics. In particular, the studies that found a positive role for PCIF1 in mRNA stability have utilized m 6 A mapping protocols instead of specific m 6 A m mapping methodologies. Moreover, as mentioned before, the activity of decapping depends on several factors (e.g., 5 -end sequence, uridylation, RNA binding proteins), including the 2 -O-methylation status of the second transcribed nucleotide (cap2), which has been recently shown to inhibit mRNA decapping independently from hDCP2 [32].
Finally, m 6 A m close to the cap in mRNAs was also shown to inhibit microRNAmediated gene silencing, which also involved decapping [29]. However, the initial observation was not followed by mechanistic studies.
The Role of m 6 A m in mRNA Translation
Considering that the cap structure in mRNAs plays a critical role in translation, various studies aimed at understanding the contribution of m 6 A m in cap1 (m7Gpppm 6 A m ) on protein production. However, as for the role of m 6 A m in mRNA stability, these studies gave controversial results. The translation initiation factor eIF4E recognizes the mRNA cap structure to stimulate translation initiation. However, the eIF4E binding to cap is achieved by the specific contacts with the m 7 G and the three phosphate groups but not of the first transcribed nucleoside [37]. Analysis of eIF4E-cap binding affinity by EMSA (electrophoretic mobility shift assay) and competitive assays, which analyzed the displacement of the synthetic cap from eIF4E by capped transcripts, showed that the presence of m 6 A m has a modest effect on eIF4E-cap interaction [15,32]. Different studies analyzed the effect of caps containing m 6 A m modification by ribosome profiling and reporter assays. Ribosome profiling performed in PCIF1-deleted HEK293T cells did not produce significant changes in mRNA translational efficiency while, in the same study, it was reported to affect the stability of a subset of modified mRNAs [16]. Conversely, a different study, which utilized the transfection of reporter GFP-encoding mRNAs, found that m 7 G-m 6 A m -containing mRNAs were translated less efficiently [10]. This result was also confirmed in in vitro translation assay. Moreover, by combining quantitative proteomic with m6Am-Exo-Seq, it was reported that, in PCIF1-deleted cells, m 6 A m -modified mRNA that are not altered at the transcript level presented increased protein levels [10]. This was also confirmed across the human tissues where m 6 A m modifications identified by m6A-seq were found to be negatively correlated with the protein level obtained from the Human Proteome Map database [5]. Thus, these results suggest a negative impact of m 6 A m on protein synthesis. On the other hand, a different study, which combined ribosome profiling and RNA-seq in PCIF1 knockout HEK293T cells, reported a positive effect of m 6 A m on mRNA translation but no effect on mRNA stability [15]. Finally, ribosome profiling analysis performed on PCIF1 knockout mice found no correlation between changes in translation rates and the presence of m 6 A m in the cap1 of mRNAs [18].
These discrepancies might depend again on the different methods utilized for identifying m 6 A m -modified mRNAs or from additional mRNA features that might impact protein production, such as the 2 -O-methylation of the cap structure, which has been recently shown to influence translation in a cell-specific manner [32]. In a recent study, which utilized the transfection of in vitro transcribed luciferase reporter RNAs carrying different adenine modifications in cap 0, cap 1, and cap 2, it was observed that the effect of m 6 A modification in the cap structure was cell-specific [32]. In human lung carcinoma A549 cells, the presence of m 6 A in cap 0 (m7Gpppm 6 A) did not impact translation efficiency. However, the presence of conventional cap1 (m7Gpppm 6 A m ) and cap 2 (m7Gpppm 6 A m N m ) resulted in an increase in protein synthesis. Interestingly, protein production was also stimulated by single 2 -O-methylation in cap2 (m7Gpppm 6 AN m ). In contrast, in mouse dendritic JAWS II cells the presence of m 6 A in cap 0 decreased translation efficiency compared to transcripts with cap 0 without m6A. Conversely, the presence of m 6 A m resulted in an increase in protein production. However, the introduction of a single methyl group at the second transcribed nucleotide in transcripts starting with adenine (m7GppAN m and m7Gpppm 6 AN m ) produced a decrease of translation efficiency compared to canonical cap1 [32]. The contribution of m 6 A m to the translation efficiency of transfected in vitro transcribed mRNAs was also analyzed in human THP1 leukemia cell lines and mouse 3T3-L1 embryonic fibroblasts [32]. In this case, the presence of m 6 A m , cap 1, decreased the translation in 3T3-L1 compared to the presence of only cap 0, while it had no effect on THP1 cells. Thus, the effect of m 6 A m observed in A549 and JAWS II cells is lost in THP1 cells while it was the opposite in the 3T3-L1 cell line. The presence of cap2 (m7Gpppm 6 A m N m ) gave, in both 3T3-L1 and 3T3-L1 cells, results consistent with JAWS II cells, with a negative effect on translation, while it was positive in A549.
These data indicate that the presence of m 6 A m modification in the mRNA cap impacts translation in a cell-specific manner, and that its effects also depend on that of 2 -Omethylation modification in the second nucleotide of the cap-structure. However, there are no current methodologies that allow high-throughput mapping of 2 -O-methylation in the cap structure of mRNAs.
The Role of m 6 A m in Splicing Regulation
Different RNA-pol II-transcribed snRNAs, such as U1 and U2, contain cap-adjacent m 6 A m installed by PCIF1 [38], and can be removed by FTO in the nuclear compartment [38]. FTO knockout HEK293T cells exhibited increased exon inclusion but, in view of their different demethylating activity, it is still not clear if this can be ascribed to altered m 6 A m levels in snRNAs. Moreover, splicing defects were never reported in PCIF1 knockout cells. The spliceosomal snRNA U2 also contains an internal m 6 A m modification. The depletion of METTL4 did not alter U2 snRNA expression levels but rather, both in human and fly cells, led to altered splicing regulation [20,21,25]. Bioinformatics analysis performed in HEK293T cells showed that the lack of METTL4-mediated U2 snRNA modification resulted in an increase in splicing of retained introns and inclusion of exons [20]. Thus, the internal m 6 A m modification appears to have a negative effect on the U2 snRNA function in splicing. Mechanistically, it was hypothesized that the lack of m 6 A m in U2 snRNA might affect the recruitment of spliceosome components, such as U2AF, or splicing regulators. Indeed, it was also found that most of the affected introns are enriched for the GGGAGGG motif that is recognized by the splicing regulatory protein hnRNP H2 [20].
The Role of m 6 A m in Cancer
The first indication of the role of m 6 A m in cancer came from a functional RNAi screening performed in human bladder cancer cells and xenograft mice that identified PCIF1 as a novel tumor suppressor [39]. However, this was not followed by further functional studies. Instead, a later study performed in colorectal cancer (CRC) indicated an opposite role for this modification [40]. Knockdown of the RNA demethylases' FTOs in CRC cell lines promoted staminality and resistance to chemotherapy drugs. This was related to a global increase of m 6 A m levels but not of m 6 A in mRNAs, measured by LC-MS/MS [40]. Moreover, mapping of m 6 A peaks by m6A-seq upon FTO silencing did not reveal any changes in m 6 A sites within specific mRNAs. However, it should be considered that FTO downregulation could produce slight variation in m6A levels across genes, which cannot be precisely quantified by standard m6A-seq methodology. Nevertheless, the knockdown of PCIF1 partially rescued the phenotype of FTO downregulation, therein indicating that high m 6 A m levels play an oncogenic role in CRC. Notably, in primary tumors, FTO protein was found predominantly in the nucleus in healthy adjacent tissue and in the initial precursor lesion of CRC while it translocated to the cytoplasm during infiltration in submucosa [39]. Thus, these data indicate that FTO might acquire specific cytoplasmic demethylation functions versus m 6 A m in CRC. PCIF1 protein and m 6 A m levels were also found to be upregulated in gastric cancer cases (GC) [41]. Furthermore, PCIF expression increased with increasing disease aggressiveness and correlated with a poor survival rate [41]. The knockdown of PCIF1 in GC cell lines produced a strong decrease in proliferation and invasion potential. More importantly, the oncogenic effect was also confirmed in GC patient-derived xenografts, where PCIF1 silencing decreased tumor volume and inhibited lung metastases [41]. Analysis of differential m 6 A m methylated transcript upon PCIF1 silencing by m6A-seq identified TM9SF1 (Transmembrane Protein 9 Superfamily Member 1), a regulator of autophagy [42], as a relevant PCIF1 target mRNA. Interestingly, by using polysome profiling upon PCIF1 modulation, this study reported that PCIF1 specifically repressed TM9SF1 mRNA translation without affecting its stability and global mRNA translation. However, the same study, by using transfection of reporter GFP mRNAs containing m7G-A m or m7G-m 6 A m cap1, found that the presence of m 6 A m modification had a negative effect on reporter mRNA translation [41]. Thus, in this latter case a general effect of m 6 A m on mRNA translation was observed. The oncogenic role of PCIF1 was also confirmed by Pan-cancer analysis and PCIF1 RNA was found to be upregulated in most tumors compared to normal tissues [43]. However, recently, in gliomas a tumor suppressor role was again demonstrated for PCIF1 [44]. In this case, PCIF1 knockdown promoted the proliferation of primary glioma cells, glioma cell lines, and glioma xenograft mice. Furthermore, increased PCIF1 levels in glioma cell lines impaired proliferation and promoted apoptosis [44], while overexpression of PCIF1 in glioma cells injected into the brains of mice reduced the growth of the tumors and extended the survival rates of the animals [44]. However, the study did not analyze changes in m 6 A m levels or identification of differentially m 6 A m -methylated mRNAs upon PCIF1 modulation.
Conclusions
Cap structure plays a crucial role in gene expression regulation by controlling mRNA stability and translation. The discovery of the methyltransferase responsible for the m6Am modification in the cap structure opened interesting perspectives on the possible role of this modification in regulating mRNA levels and protein production. However, a major problem in the epitranscriptomics field is the mapping and quantification of specific modification within the transcriptome. Most of the studies performed on m 6 A m have utilized methodologies developed for internal m 6 A mapping followed by specific bioinformatics pipelines. Only a few studies have utilized specific protocols for m 6 A m identification. These caused the lack of reproducibility and produced controversial results on the effect of m 6 A m on gene expression. Hopefully, more sensitive methods for m 6 A m detection will be developed to resolve the apparently contentious results. Furthermore, the modulation of m 6 A m modification can result in cell-specific effects and, in transfection experiments using in vitro transcribed mRNAs, these effects are influenced by the 2 -O-methylation statuses of cap1 and cap2. In conclusion, the role of m 6 A m in gene expression regulation needs to be clarified by further and more detailed investigation.
Interestingly, the deletion of m 6 A m regulator proteins is well-tolerated in vivo but greatly affects the survival of different types of cancer. Thus, these data indicate that inhibitors against these proteins might have future applications in clinics.
Conflicts of Interest:
The authors declare no conflict of interest.
Figure 1 .
1Chemical structures of adenosine (A), N 6 -methyladenosine (m 6 A), and N 6 ,2′-O-dimethyladenosine (m 6 Am). Methyl groups are indicated in red.
Figure 1 .
1Chemical structures of adenosine (A), N 6 -methyladenosine (m 6 A), and N 6 ,2 -Odimethyladenosine (m 6 A m ). Methyl groups are indicated in red.
Figure 2 .
2Sequencing methods for m 6 A m mapping. All methods work on fragmented RNA. (a) In the m6a-exo-seq
Figure 3 .
3Regulators of m 6 Am modification. (a) PCIF1 methylates the first transcribed 2′-O-methyladenosines, when present, producing m 6 Am during primary mRNA transcription. This modification can be removed in the cytoplasm by FTO demethylases. The consequences of cap m 6 Am deletion are still under debate.
Figure 3 .
3Regulators of m 6 A m modification. (a) PCIF1 methylates the first transcribed 2 -Omethyladenosines, when present, producing m 6 A m during primary mRNA transcription. This modification can be removed in the cytoplasm by FTO demethylases. The consequences of cap m 6 A m deletion are still under debate. It was reported that m 6 A m can increase mRNA translation, decrease mRNA translation, increase mRNA stability, and not affect mRNA stability (see main text for details).
Author
Contributions: Writing-review and editing, B.C. and M.T.; writing-original draft, A.F. All authors have read and agreed to the published version of the manuscript.
Funding:
A.F is funded by NextGenerationEU-PNRR M4C2-Investment 1.4-CN00000041, and "Progetti Ateneo Sapienza", RP1201729D714976.Data Availability Statement: Not applicable.
Table 1 .
1Methods utilized for m 6 A m mapping.
Table 1 .
1Cont.Method
Principle
Advantages
Limitations
m6am-exo-seq [10]
Utilizes 5 -> 3 digestion to
degrade uncapped RNAs after
fragmentation followed by
m6A IP
Allows sequencing of
cap-m6Am fragments
Cannot distinguish between
cap-m 6 A m and m 6 A in 5 -RNA
fragments, antibody
cross-reactivity
m6ACE-seq [11]
Utilizes crosslinking of
anti-m6A antibody, followed
by 5 -> 3 digestion and
m6A-IP to identify m 6 A-and
m 6 A m in RNA fragments
Single-nucleotide resolution,
allows mapping of both m 6 A
and cap-m 6 A m
Required dedicated
bioinformatic analysis, antibody
cross-reactivity
m6Am-seq [12]
Utilizes anti-m7G-antibody to
purify 5 -RNA fragments,
followed by digestion with
recombinant FTO and m6A IP
Allows mapping of both m 6 A
and cap-m 6 A m
Cannot distinguish between
cap-m 6 A m and m 6 A in 5 -RNA
fragments, requires recombinant
FTO protein, FTO activity is not
specific for m 6 A m and is
influenced by sequence and
structure, antibody
cross-reactivity
CAPturAM [13]
Table 2 .
2Effects of m 6 A m in mRNA stability and translation.Enzyme/
Experimental Procedure
Molecular Effect of m 6 A m
Model System
FTO/KO [29]
Enhanced mRNA stability
HEK293T
FTO/OE [29]
Reduced mRNA stability
HEK293T
PCIF/KO [10,15,17]
No effect on mRNA stability
[10,15,17]/increased
translation [10]/reduced
translation [15]
HEK293T, MEL624
PCIF1/KO [16]
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Structural insights into FTO s catalytic mechanism for the demethylation of multiple RNA substrates. X Zhang, L.-H Wei, Y Wang, Y Xiao, J Liu, W Zhang, N Yan, G Amu, X Tang, L Zhang, 10.1073/pnas.1820574116Proc. Natl. Acad. Sci. Natl. Acad. Sci116Zhang, X.; Wei, L.-H.; Wang, Y.; Xiao, Y.; Liu, J.; Zhang, W.; Yan, N.; Amu, G.; Tang, X.; Zhang, L.; et al. Structural insights into FTO s catalytic mechanism for the demethylation of multiple RNA substrates. Proc. Natl. Acad. Sci. 2019, 116, 2919-2924. [CrossRef]
FTO Demethylates Cyclin D1 mRNA and Controls Cell-Cycle Progression. M Hirayama, F.-Y Wei, T Chujo, S Oki, M Yakita, D Kobayashi, N Araki, N Takahashi, R Yoshida, H Nakayama, 10.1016/j.celrep.2020.03.028Cell Rep. 2020, 31, 107464. [CrossRefHirayama, M.; Wei, F.-Y.; Chujo, T.; Oki, S.; Yakita, M.; Kobayashi, D.; Araki, N.; Takahashi, N.; Yoshida, R.; Nakayama, H.; et al. FTO Demethylates Cyclin D1 mRNA and Controls Cell-Cycle Progression. Cell Rep. 2020, 31, 107464. [CrossRef]
Reversible methylation of m 6 A m in the 5 cap controls mRNA stability. J Mauer, X Luo, A Blanjoie, X Jiao, A V Grozhik, D P Patil, B Linder, B F Pickering, J J Vasseur, Q Chen, 10.1038/nature21022Nature. 541Mauer, J.; Luo, X.; Blanjoie, A.; Jiao, X.; Grozhik, A.V.; Patil, D.P.; Linder, B.; Pickering, B.F.; Vasseur, J.J.; Chen, Q.; et al. Reversible methylation of m 6 A m in the 5 cap controls mRNA stability. Nature 2017, 541, 371-375. [CrossRef]
The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5 cap modulates protein expression in living cells. P J Sikorski, M Warminski, D Kubacka, T Ratajczak, D Nowis, J Kowalska, J Jemielity, 10.1093/nar/gkaa032Nucleic Acids Res. 48Sikorski, P.J.; Warminski, M.; Kubacka, D.; Ratajczak, T.; Nowis, D.; Kowalska, J.; Jemielity, J. The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5 cap modulates protein expression in living cells. Nucleic Acids Res. 2020, 48, 1607-1626. [CrossRef]
Dynamic regulation of N 6 ,2 -O-dimethyladenosine (m 6 Am) in obesity. M S Ben-Haim, Y Pinto, S Moshitch-Moshkovitz, V Hershkovitz, N Kol, T Diamant-Levi, M S Beeri, N Amariglio, H Y Cohen, G Rechavi, 10.1038/s41467-021-27421-2Nat. Commun. 2021. 12PubMedBen-Haim, M.S.; Pinto, Y.; Moshitch-Moshkovitz, S.; Hershkovitz, V.; Kol, N.; Diamant-Levi, T.; Beeri, M.S.; Amariglio, N.; Cohen, H.Y.; Rechavi, G. Dynamic regulation of N 6 ,2 -O-dimethyladenosine (m 6 Am) in obesity. Nat. Commun. 2021, 12, 7185. [CrossRef] [PubMed]
2 -O -Methylation of the second transcribed nucleotide within the mRNA 5 cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion. K Drazkowska, R Tomecki, M Warminski, N Baran, D Cysewski, A Depaix, R Kasprzyk, J Kowalska, J Jemielity, P J Sikorski, 10.1093/nar/gkac722Nucleic Acids Res. 50PubMedDrazkowska, K.; Tomecki, R.; Warminski, M.; Baran, N.; Cysewski, D.; Depaix, A.; Kasprzyk, R.; Kowalska, J.; Jemielity, J.; Sikorski, P.J. 2 -O -Methylation of the second transcribed nucleotide within the mRNA 5 cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion. Nucleic Acids Res. 2022, 50, 9051-9071. [CrossRef] [PubMed]
New insights into decapping enzymes and selective mRNA decay. E Grudzien-Nogalska, M Kiledjian, 10.1002/wrna.1379Wiley Interdisciplinary Reviews. RNA. 8PubMedGrudzien-Nogalska, E.; Kiledjian, M. New insights into decapping enzymes and selective mRNA decay. Wiley Interdisciplinary Reviews. RNA 2017, 8, e1379. [CrossRef] [PubMed]
Eukaryotic mRNA Decapping Activation. E Vidya, T F Duchaine, 10.3389/fgene.2022.83254713Front. Genet. 2022Vidya, E.; Duchaine, T.F. Eukaryotic mRNA Decapping Activation. Front. Genet. 2022, 13, 832547. [CrossRef]
Thiol-linked alkylation of RNA to assess expression dynamics. V A Herzog, B Reichholf, T Neumann, P Rescheneder, P Bhat, T R Burkard, W Wlotzka, A Von Haeseler, J Zuber, S L Ameres, 10.1038/nmeth.4435Nat. Methods. 14Herzog, V.A.; Reichholf, B.; Neumann, T.; Rescheneder, P.; Bhat, P.; Burkard, T.R.; Wlotzka, W.; von Haeseler, A.; Zuber, J.; Ameres, S.L. Thiol-linked alkylation of RNA to assess expression dynamics. Nat. Methods 2017, 14, 1198-1204. [CrossRef]
Base-pairresolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). D B Mahat, H Kwak, G T Booth, I H Jonkers, C G Danko, R K Patel, C T Waters, K Munson, L J Core, J T Lis, 10.1038/nprot.2016.086Nat. Protoc. 11Mahat, D.B.; Kwak, H.; Booth, G.T.; Jonkers, I.H.; Danko, C.G.; Patel, R.K.; Waters, C.T.; Munson, K.; Core, L.J.; Lis, J.T. Base-pair- resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat. Protoc. 2016, 11, 1455-1476. [CrossRef]
Biophysical studies of eIF4E cap-binding protein: Recognition of mRNA 5 cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. A Niedzwiecka, J Marcotrigiano, J Stepinski, M Jankowska-Anyszka, A Wyslouch-Cieszynska, M Dadlez, A Gingras, P Mak, E Darzynkiewicz, N Sonenberg, 10.1016/S0022-2836(02)00328-5J. Mol. Biol. 319PubMedNiedzwiecka, A.; Marcotrigiano, J.; Stepinski, J.; Jankowska-Anyszka, M.; Wyslouch-Cieszynska, A.; Dadlez, M.; Gingras, A.; Mak, P.; Darzynkiewicz, E.; Sonenberg, N.; et al. Biophysical studies of eIF4E cap-binding protein: Recognition of mRNA 5 cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins. J. Mol. Biol. 2002, 319, 615-635. [CrossRef] [PubMed]
FTO controls reversible m 6 A m RNA methylation during snRNA biogenesis. J Mauer, M Sindelar, V Despic, T Guez, B R Hawley, J J Vasseur, A Rentmeister, S S Gross, L Pellizzoni, F Debart, 10.1038/s41589-019-0231-8Nat. Che. Biol. 15Mauer, J.; Sindelar, M.; Despic, V.; Guez, T.; Hawley, B.R.; Vasseur, J.J.; Rentmeister, A.; Gross, S.S.; Pellizzoni, L.; Debart, F.; et al. FTO controls reversible m 6 A m RNA methylation during snRNA biogenesis. Nat. Che. Biol. 2019, 15, 340-347. [CrossRef]
Patient Mutation Directed shRNA Screen Uncovers Novel Bladder Tumor Growth Suppressors. J Hensel, J E Duex, C Owens, G M Dancik, M G Edwards, H F Frierson, D Theodorescu, 10.1158/1541-7786.MCR-15-0130Mol. Cancer Res. 13Hensel, J.; Duex, J.E.; Owens, C.; Dancik, G.M.; Edwards, M.G.; Frierson, H.F.; Theodorescu, D. Patient Mutation Directed shRNA Screen Uncovers Novel Bladder Tumor Growth Suppressors. Mol. Cancer Res. 2015, 13, 1306-1315. [CrossRef]
FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell. S Relier, J Ripoll, H Guillorit, A Amalric, C Achour, F Boissière, J Vialaret, A Attina, F Debart, A Choquet, 10.1038/s41467-021-21758-4Nat. Commun. 2021. 12Relier, S.; Ripoll, J.; Guillorit, H.; Amalric, A.; Achour, C.; Boissière, F.; Vialaret, J.; Attina, A.; Debart, F.; Choquet, A.; et al. FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell. Nat. Commun. 2021, 12, 1716. [CrossRef]
m6Am methyltransferase PCIF1 is essential for aggressiveness of gastric cancer cells by inhibiting TM9SF1 mRNA translation. W Zhuo, M Sun, K Wang, L Zhang, K Li, D Yi, M Li, Q Sun, X Ma, W Liu, 10.1038/s41421-022-00395-1Cell Discov. 2022, 8, 48. [CrossRef. PubMedZhuo, W.; Sun, M.; Wang, K.; Zhang, L.; Li, K.; Yi, D.; Li, M.; Sun, Q.; Ma, X.; Liu, W.; et al. m6Am methyltransferase PCIF1 is essential for aggressiveness of gastric cancer cells by inhibiting TM9SF1 mRNA translation. Cell Discov. 2022, 8, 48. [CrossRef] [PubMed]
High-throughput functional screening for autophagyrelated genes and identification of TM9SF1 as an autophagosome-inducing gene. P He, Z Peng, Y Luo, L Wang, P Yu, W Deng, Y An, T Shi, D Ma, 10.4161/auto.5.1.7247Autophagy. 5PubMedHe, P.; Peng, Z.; Luo, Y.; Wang, L.; Yu, P.; Deng, W.; An, Y.; Shi, T.; Ma, D. High-throughput functional screening for autophagy- related genes and identification of TM9SF1 as an autophagosome-inducing gene. Autophagy 2009, 5, 52-60. [CrossRef] [PubMed]
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Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas. S Gao, J Zhou, Z Hu, S Zhang, Y Wu, P P Musunuru, T Zhang, L Yang, X Luo, J Bai, 10.1016/j.bbadis.2022.166498Biochim. Biophys. Acta Mol. Basis Dis. 2022, 1868, 166498. [CrossRef. PubMedGao, S.; Zhou, J.; Hu, Z.; Zhang, S.; Wu, Y.; Musunuru, P.P.; Zhang, T.; Yang, L.; Luo, X.; Bai, J.; et al. Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas. Biochim. Biophys. Acta Mol. Basis Dis. 2022, 1868, 166498. [CrossRef] [PubMed]
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Introduction
N6-methyladenosine (m 6 A) modifications in RNA were first identified in the 1870s [1]. The enzyme that catalyzes the formation of m 6 A is known as m 6 A "writer", methyl transferase-like 3 (METTL3), which is the only catalytic subunit of the methyltransferase complex and can synthesize almost all m 6 A modifications in mRNAs [2]. As research progressed, it was gradually realized that m 6 A modifications are an essential regulatory modality in biological development, affecting cell differentiation and other physiological processes. Since the rise of m 6 A high-throughput sequencing methods in 2012 [3,4], more and more studies have found that the epitranscriptome plays a key role in regulating the fate and function of mRNAs in cells. m 6 A is a selective modification enriched in specific mRNAs [5]. Some mRNAs contain only a single m 6 A site, but some contain 20 or even more m 6 A sites [4]. Overall, about 50-80% of mammalian mRNAs may m 6 A sites be absent [4,[6][7][8]. Under physiological conditions, m 6 A is enriched in the 3' untranslated region (3 UTR) and near the stop codons of the transcripts [3]. Analysis of mRNAs enriched in m 6 A modifications showed enrichment of developmental regulation and cell fate-related genes [9]. In contrast, transcripts of some highly stable "housekeeping" genes, including ribosomal proteins, showed a de-enrichment of m 6 A [9]. However, in pathological situations, some m 6 A sites may be regulated in a disease-specific manner. In various cellular stresses, the investigators also observed changes in m 6 A levels in the 5 UTR as well [10]. Thus, the role of m 6 A modifications in different diseases and the role they play remains to be elucidated.
In recent years, the role of m 6 A modifications in cancer has received increasing attention as epigenomics and oncology studies continue to progress. It has been found that m 6 A modifications in tumors can regulate the stability, splicing, nuclear translocation, and Biomolecules 2023, 13, 243 2 of 14 translation efficiency of various mRNAs [11,12], which in turn leads to a complex series of molecular events. m 6 A is added to RNA by the m 6 A writer-complex which includes METTL3, METTL14, WTAP, VIRMA, RBM15/15B, ZC3H13, and CBLL1 [13,14]. m 6 A readers include YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, HNRNPC/G/A2B1 [13]. They act as RNA binding proteins to exert their effect on the RNA life cycle subsequently. m 6 A erasers, ALKBH5 and FTO, are demethylases that can remove m 6 A from RNA. As the key catalytic subunit forming m 6 A modification, there is increasing evidence in recent years that the m 6 A writer METTL3 is significantly aberrantly expressed in tumors and can play a key role as an oncogene in most cases, leading to different phenotypic changes in tumors, resulting in proliferation, invasion, metastasis, and drug resistance. For example, in bladder cancer, METTL3 is significantly overexpressed and is associated with proliferation, invasion, and tumorigenic capacity in in vivo, and METTL3 promotes tumor progression through m 6 A modification on AFF4 and NF-κB mRNA, which in turn activates MYC transcription [15]. In hepatoblastoma, abnormally high expression of METTL3 leads to a significant increase in m 6 A levels in the tumor, and m 6 A is enriched not only near the mRNA stop codon but also in the coding sequence (CDS) region. The elevated stability of CTNNB1 due to its m 6 A modification leads to significant activation of the Wnt/β-catenin signaling pathway, which in turn promotes malignant proliferation of tumors [16]. In esophageal cancer, METTL3 is also significantly overexpressed and can lead to mRNA degradation by upregulating the m 6 A level of APC mRNA and recruiting the m 6 A "reader" protein YTHDF2. The reduced expression of APC leads to abnormal activation of the Wnt/β-catenin signaling pathway, thus promoting the glycolytic process and malignant cell proliferation in tumors [17]. Interestingly, sometimes METTL3 also acts as a tumor suppressor. Cui et al. [18] reported that knocking down METTL3 altered m 6 A enrichment on ADAM19 and promoted the malignancy of glioblastoma stem cells. Wu et al. revealed that METTL3 mediated m 6 A modification of FBXW7 and suppressed the development of lung adenocarcinoma subsequently [19]. Thus, METTL3 regulates the fate of these RNAs through m 6 A modification at key transcripts, which in turn affects the development of many cancers, including hematologic malignancies and solid tumors.
Aberrant Translation in Cancer
In the life cycle of mRNA, translation, the process of protein synthesis, is the most energy-consuming step in the entire cell [20], and this step plays a key role in the regulation of gene expression. With the rapid development of high-throughput sequencing technologies in recent years, mathematical modeling and multi-omics analysis have revealed that the magnitude of translational regulation in cells exceeds the sum of transcription, mRNA degradation, and protein degradation [21]. Components of the translational machinery integrate almost all oncogenic signals [22], and dysregulation of the translational process is considered one of the hallmarks of tumors and is associated with abnormal proliferation, angiogenesis, differentiation, and immune response [23,24]. Aberrant mRNA translation is a common feature of tumors, in which the process cannot be separated from the involvement of RNA-binding proteins (RBPs) in canonical translation machinery, including eukaryotic initiation factor (eIF) and elongation factor (eEF) (Figure 1). Their signals are aberrantly amplified in tumors [25,26].
Figure 1.
Translation factors participate in the mRNA translation process. The left panel shows the translation initiation process: eIF2 subunits combine with initiator methionyl tRNA and GTP to form the ternary complex (TC). TC associates with the 40S ribosomal subunit complex which consists of eIF3, eIF1, eIF1A, and eIF5 to form the 43S pre-initiation complex (43S PIC). Then 43S PIC is recruited to the mRNA template by combining to eIF4F complex and they form the 48S pre-initiation complex (48S PIC). The phosphorylation of eIF6 allows the 60S ribosomal subunit to join the 40S subunit, which leads to the formation of the translation-competent 80S ribosome and marks the end of translation initiation. The right panel shows the translation elongation cycle: eEF1A-GTP helps to deliver the aa-tRNA to the Aminoacyl site in ribosome and eEF1B recycles the released eEF1A-GDP subsequently. eEF2-GTP mediates the translocation of the elongating peptide to the Peptidyl site of the ribosome.
Translation Factors in Eukaryotic Translation
Translation of mRNA in eukaryotic cells includes cap-dependent and cap-independent translation, in which the eIF4F complex plays an important role. eIF4F contains three components (eIF4E, eIF4G, eIF4A), of which eIF4E is the cap-binding subunit of the eIF4F complex and is required for cap-dependent translation of all nuclear-encoded mRNAs [27]. In addition, eIF4E can also stimulate the RNA unwinding enzyme activity of eIF4A independently of its cap-binding function and thus promotes translation [28]. eIF4E interacts with eIF4G and binds the m7G cap structure of mRNA, which in turn promotes translation. eIF3 plays a central role in the translation initiation of classical cap-dependent translation and cap-independent translation [29][30][31]. Different subunits of eIF3 confer different functions to the eIF3 core complex. Besides, other eukaryotic initiation factors also play important role in the translation process. Translation initiation is generally regulated by the 43S pre-initiation complex (43S PIC), which consists of eIF1, eIF1A, eIF3, eIF5, and the ternary complex (TC) [22]. The TC is formed by eIF2 (containing α, β, γ subunits), tRNA, and GTP. When eIF2α is phosphorylated under stress, the TC formation is inhibited and the global translation is downregulated subsequently [32,33]. eIF6 was first reported to participate in the biogenesis of the 60S ribosomal subunit in the nucleus as an anti-association factor [34,35]. However, Gandin et al. [36] found that eIF6 is rate-limiting Figure 1. Translation factors participate in the mRNA translation process. The left panel shows the translation initiation process: eIF2 subunits combine with initiator methionyl tRNA and GTP to form the ternary complex (TC). TC associates with the 40S ribosomal subunit complex which consists of eIF3, eIF1, eIF1A, and eIF5 to form the 43S pre-initiation complex (43S PIC). Then 43S PIC is recruited to the mRNA template by combining to eIF4F complex and they form the 48S pre-initiation complex (48S PIC). The phosphorylation of eIF6 allows the 60S ribosomal subunit to join the 40S subunit, which leads to the formation of the translation-competent 80S ribosome and marks the end of translation initiation. The right panel shows the translation elongation cycle: eEF1A-GTP helps to deliver the aa-tRNA to the Aminoacyl site in ribosome and eEF1B recycles the released eEF1A-GDP subsequently. eEF2-GTP mediates the translocation of the elongating peptide to the Peptidyl site of the ribosome.
Translation Factors in Eukaryotic Translation
Translation of mRNA in eukaryotic cells includes cap-dependent and cap-independent translation, in which the eIF4F complex plays an important role. eIF4F contains three components (eIF4E, eIF4G, eIF4A), of which eIF4E is the cap-binding subunit of the eIF4F complex and is required for cap-dependent translation of all nuclear-encoded mRNAs [27]. In addition, eIF4E can also stimulate the RNA unwinding enzyme activity of eIF4A independently of its cap-binding function and thus promotes translation [28]. eIF4E interacts with eIF4G and binds the m7G cap structure of mRNA, which in turn promotes translation. eIF3 plays a central role in the translation initiation of classical cap-dependent translation and cap-independent translation [29][30][31]. Different subunits of eIF3 confer different functions to the eIF3 core complex. Besides, other eukaryotic initiation factors also play important role in the translation process. Translation initiation is generally regulated by the 43S pre-initiation complex (43S PIC), which consists of eIF1, eIF1A, eIF3, eIF5, and the ternary complex (TC) [22]. The TC is formed by eIF2 (containing α, β, γ subunits), tRNA, and GTP. When eIF2α is phosphorylated under stress, the TC formation is inhibited and the global translation is downregulated subsequently [32,33]. eIF6 was first reported to participate in the biogenesis of the 60S ribosomal subunit in the nucleus as an anti-association factor [34,35]. However, Gandin et al. [36] found that eIF6 is rate-limiting for efficient translation initiation. In the cytoplasm of mammalian cells, the phosphorylation of eIF6 on Ser235 leads to its release from the 60S, which promotes the formation of a translationcompetent 80S ribosome [37]. The translation elongation process is carried out by the ribosome with the assistance of eEFs. Among them, eEF1A is an important component of the translational apparatus as it interacts with tRNA [26]. eEF2 possesses an RNA binding site that interacts with tRNAs and promotes conformational changes, thus allowing the latter to interact with the coding region of mRNAs, mediating the translocation of peptide chains in extension to the P-site of the ribosome [38].
Dysregulation of Translation Factors in Cancer
Most of the eIF4E-sensitive mRNAs have a long and highly structured 5 UTR region [39], and these mRNAs encode many proteins associated with cell proliferation and tumor progression, including MYC, VEGF, cyclin, and others [40]. Many studies have reported that overexpression of eIF4E is associated with poor prognosis in cancer patients and can lead to tumor vascularization and invasion [41]. In recent years, various subunits of eIF3 have been found to have altered expression in malignant tumors, affecting translation of oncogenic mRNAs. eIF3a expression level were first found to be elevated in breast cancer tissues compared to paired normal breast tissues by Bachmann et al. [42], and eIF3a might play an important role in regulating translation of specific mRNAs encoding α-microtubulin, RRM2, and proteins associated with the cell cycle [43]. In virus-induced murine mammary tumors, the eIF3e gene was identified as a common insertion site and suggested that production of truncated eIF3e could lead to malignant transformation of mammary epithelial cells [44]. In prostate cancer, elevation of eIF3h positively correlates with tumor stages. The expression level of eIF3h is higher in metastatic prostate cancer than in primary prostate cancer, and eIF3h may play an important regulatory role in the translation of specific mRNAs [45]. Therefore, aberrant overexpression of eIF3h may contribute to tumor development by upregulating the translation of important mRNAs associated with cell proliferation [46]. Other translation factors are also dysregulated in cancer. eIF1 expression is downregulated in pancreatic ductal adenocarcinoma [47]. eIF1A is essential for cell proliferation and the cell cycle in cancer [48]. Interestingly, although eIF2α phosphorylation leads to reduced global translation, the translation of a restricted subset of mRNAs is enhanced, which facilitates glycolysis and cell invasion in cancer [49,50]. eIF5 is overexpressed in colorectal cancer and hepatocellular carcinoma and predicts poor prognosis [22,51]. eIF6 is reported to be markedly upregulated in hepatocellular carcinoma, colorectal cancer, and gallbladder cancer, which lead to tumor progression via mTOR and AKT-related signaling pathways [52][53][54][55]. Targeting eIF6-mediated translation blunts lipid accumulation and oncogenic transformation in the liver [56]. eEF1A plays an important and well-defined role in cancer development and progression [26,57], and eEF1A is aberrantly highly expressed in a variety of tumors and suggests a poor prognosis [58,59]. eEF2 also plays an important role in promoting the progression of tumors such as breast cancer [38,60,61]. Taken together, these translation factors affect the translation initiation and elongation process of multiple mRNAs in different types of cancer.
As a key enzyme regulating mRNA fate, the regulation of METTL3 on the translation process in tumors cannot be ignored. In recent years, many studies have reported that METTL3 could lead to changes in the expression of target genes through the regulation of mRNA translation process, which in turn caused tumor progression. The importance of RBPs in canonical translation machinery in the aberrant translation of m 6 A-modified mRNAs was also mentioned in many studies [7,62]. Therefore, the following is intended to introduce the various mechanisms involved in the translational regulation of mRNA by METTL3 in cancer and the interconnection between various m 6 A reading proteins and canonical translation machinery in these processes, so as to provide new ideas and possibilities targeting METTL3-mediated translation regulation for cancer treatment.
METTL3 Functions as a Translation Regulator in Cancer
The life cycle of m 6 A-modified mRNA begins with the transcriptional process, and m 6 A modifications are mainly mediated by METTL3 in the nucleus. In general, when mRNA is transported to the cytoplasm, specific m 6 A reader proteins bind to m 6 A, which in turn affects the translation of mRNA [63]. Many studies in recent years have found that METTL3 could be directly or indirectly involved in the translational regulation of mRNAs through a variety of mechanisms, as summarized in Table 1. 6 A reader YTHDF1 selectively recognized the m 6 A sites. Eukaryotic translation initiation factors were recruited by YTHDF1 subsequently, which in turn promoted ribosome loading and assembling on target mRNA, and advanced cap-dependent or cap-independent translation initiation [7,64].
Subsequently, a large number of studies have emerged to support and enrich this theory. Song et al. discovered that in colorectal cancer, METTL3 catalyzed m 6 A modification in 3 UTR of HSF1 mRNA and protein expression of HSF1 was significantly downregulated after knockdown of YTHDF1, demonstrating that METTL3-mediated m 6 A could promote translation through the m 6 A reader YTHDF1 [65]. In endometrial cancer, reduced METTL3 expression leads to a decrease in m 6 A modification on PHLPP2 mRNA, resulting in attenuated YTHDF1-mediated translation of PHLPP2, which in turn caused de-repression of the AKT pathway and promotes tumor progression [66]. In melanoma, knockdown of METTL3 in bone marrow cells results in the lack of m 6 A modification on SPRED2, which in turn disrupts YTHDF1-mediated mRNA translation, leading to enhanced activation of NF-κB and STAT3 via the ERK pathway, contributing to tumor progression [67]. In gastric cancer, METTL3-mediated SPHK2 m 6 A modification followed by YTHDF1 facilitates translation initiation through interacting with eIF3a, which in turn upregulates the translation efficiency and promotes tumor progression [68]. In lung adenocarcinoma, m 6 A modification promotes YTHDF1-mediated translation of ENO1 and SLC7A11, thereby enhancing tumor glycolysis and ferroptosis [69,70]. In ocular melanoma, down-regulated METTL3-mediated m 6 A modification leads to attenuated YTHDF1-mediated translation of tumor suppressor HINT2, which promotes tumor progression [71]. In hepatocellular carcinoma, researchers discovered that METTL3 could catalyze m 6 A modification in CDS and 3'UTR of SNAI1 mRNA, and YTHDF1 tended to bind to m 6 A in CDS of SNAI1 to mediate translation. Researchers then treated cells with rapamycin and found that YTHDF1 mediated the cap-independent translation of SNAI1 and that YTHDF1 could synergize with eEF2 to promote translation extension of SNAI1 mRNA [72]. In gastrointestinal stromal tumors, METTL3 recruits YTHDF1 through m 6 A modification of MRP1 mRNA in 5 UTR and promotes translation extension by eEF1, which in turn leads to intracellular translocation of MRP1 to imatinib and promotes drug resistance [73]. In cervical and liver cancer, METTL3 mediates m 6 A modification of PDK4 in 5 UTR, and subsequently YTHDF1 synergizes with eEF2 to promote the translation of PDK4, which in turn enhances the glycolytic process in tumors [74]. In breast cancer, METTL3 mediates the m 6 A modification of KRT7 in CDS, followed by enhanced translation elongation with the involvement of YTHDF1/eEF1 [75].
In addition to YTHDF1, METTL3 can also regulate translation of mRNAs through YTHDF3. Researchers identified a certain overlap between YTHDF3 and YTHDF1-bound proteins in the cytoplasm, and it was found that YTHDF3 and YTHDF1 could simultaneously interact with eIF4A, which in turn accelerated the translation process. It was suggested that the m 6 A reader YTHDF3 could enhance YTHDF1-mediated translation after METTL3-mediated m 6 A modification in some mRNAs [64]. This mechanism was validated in the subsequent studies: METTL3 could promote translation initiation complex formation through m 6 A modification of YAP mRNA in lung cancer by recruiting YTHDF1/3 as well as eIF3b, which in turn improved the translation efficiency and stability of YAP mRNA [76], resulting in tumor metastasis. In bladder cancer, after m 6 A modification of ITGA6, YTHDF1 cooperated with YTHDF3 to promote the translation of ITGA6 and mediated tumor progression [77].
METTL3 also enhances mRNA translation through other m 6 A readers. Liu et al. found that METTL3 mediated m 6 A modification in 3 UTR of BMI1 in oral cancer [78]. Overexpression of METTL3 increased the binding of BMI1 mRNA to polysomes without altering the stability of BMI1 mRNA and the rate of protein degradation, suggesting an enhancement of the translation process. Subsequently, to investigate through which m 6 A reader protein METTL3 promotes translation, authors knocked down IGF2BP1, IGF2BP2, IGF2BP3, and YTHDF1 and found that BMI1 mRNA expression was not altered, while knockdown of IGF2BP1 downregulated BMI1 protein level. The above experiments illustrated that METTL3 could also promote the translation of mRNAs such as BMI1 through m 6 A readers other than YTHDF1/3, such as IGF2BP1. In esophageal cancer, METTL3 mediates m 6 A modification in 3 UTR of TNFR1 mRNA [79]. ATXN2 acts as a novel RNA binding protein and enhances the translation of m 6 A-modified TNFR1 mRNA.
METTL3 Enters the Cytoplasm to Facilitate the Translation Process
In oncology studies, researchers found that after m 6 A modification in the 3 UTR of a large subset of mRNAs at sites close to the stop codon, METTL3 itself could tether to the mRNA as an m 6 A reader in the cytoplasm. Subsequently, METTL3 formed as a "bridge" between the 3 UTR and the 5 cap-binding proteins of mRNA, which supported an mRNA looping mechanism for ribosome recycling and translational control. In addition, the researchers observed the close proximity of METTL3 and individual polyribosomes with cap-binding proteins such as eIF4E by electron microscopy, and found that METTL3 and eIF3h had direct physical and functional interactions, thus promoting the translation of a large number of oncogenic mRNAs including BRD4 [62,80].
Several studies subsequently reported evidence for the direct involvement of METTL3 in translation regulation in the cytoplasm. Song et al. found that METTL3 deletion in colorectal cancer significantly reduced the level of HSF1 mRNA in the polyribosome fractions and increased its level in the non-translating ribosome fractions. m 6 A-modified HSF1 resulted in direct tethering of METTL3 to HSF1 mRNA in the cytoplasm to facilitate the translation process [65]. In addition, interestingly, miR455-3p could also inhibit the translation of HSF1 mRNA by interacting with the m 6 A site of HSF1 located in 3 UTR, preventing METTL3-mediated m 6 A modification as well as the direct binding. In cervical cancer, researchers found that METTL3 was mainly localized in the cytoplasm. Knockdown of YTHDF1 did not affect protein expression of AXL, but protein expression of AXL was significantly upregulated after overexpression of both wild-type or catalytic mutant METTL3, suggesting that METTL3 was directly involved in the translation process of AXL in the cytoplasm [81]. In chronic myeloid leukemia (CML), investigators found the presence of METTL3 in the cytoplasm, then they verified that METTL3 knockdown led to a reduction in global translation efficiency in CML cells and showed a critical role for METTL3 in maintaining ribosome levels and translational potential [82]. Subsequent knockdown of METTL3 resulted in a significant decrease in m 6 A levels of genes involved in ribosome biogenesis and translation such PES1. After overexpression of wild-type and mutant METTL3 in cells, it was found that the protein levels of PES1 were both significantly increased, while the mRNA levels were unchanged, and both wild-type and catalytic mutant METTL3 were found to bind to 3 UTR of PES1 mRNA in the cytoplasm. Thus, this study revealed that METTL3 could promote the production of PES1 protein by directly binding to m 6 A-modified PES1 in the cytoplasm, which in turn upregulated the translation efficiency of its mRNA, ultimately allowing for enhanced ribosome synthesis and translation processes of other oncogenic mRNAs. In bladder cancer, investigators found that m 6 A levels in the 3 UTR of CDCP1 mRNA were increased during malignant transformation [83,84]. Mechanistically, after METTL3 mediated the m 6 A modification of CDCP1, METTL3 cooperated with YTHDF1 to bind to the 3 UTR m 6 A site of CPCP1 and thus promoted translation. Overexpression of METTL3 had no effect on the expression level and stability of CPCP1 mRNA, but upregulated the protein level of CDCP1 without changing the protein degradation rate, and significantly upregulated the polysome-bound CPCP1 mRNA. The catalytic mutant METTL3 can also promote the translation of m 6 A-modified CDCP1 mRNA, although with weaker activity compared with the wild type METTL3.
In 2022, Wei et al. [85] revealed a novel m 6 A-independent mechanism for METTL3 to regulate translation in gastric cancer progression. Cytoplasm-anchored METTL3 can promote the looping of some non-m 6 A-modified oncogenic mRNAs by interacting with PABPC1 and eIF4F complex. This study assigned a new function to cytoplasmic distributed METTL3 and expanded the ways in which METTL3 facilitates translation. We look forward to further studies to provide more evidence for this important and interesting finding.
Promoter-Bound METTL3 Enhances Translation
In addition to METTL3's ability to upregulate translation efficiency through the binding of other reading proteins or binding to m 6 A-modified mRNAs directly, researchers have identified a mechanism by which METTL3 promotes m 6 A-dependent translation regulation through binding to the promoter of target genes. They found that in acute myeloid leukemia (AML), METTL3 could be localized to the transcription initiation site of target genes in chromatin independently of METTL14 which was essential for m 6 A modification [86]. Since the majority of target genes had the CAATT-box binding protein CEBPZ at the transcription initiation site, METTL3 could induce m 6 A modifications within the CDS region of related transcripts such as SP1 and SP2 mRNAs after their transcription by interacting with the CEBPZ protein and binding to these transcription initiation sites on chromosomes. Researchers found that the transcripts of METTL3-bound target genes were enriched in [GAG]n sequences that could cause ribosomal arrest during translation. When these sequences were modified by METTL3-mediated m 6 A methylation, ribosomal stalling was lifted, thereby contributing to enhanced translation. As transcription factors, SP1 and SP2 proteins played an important role in promoting AML progression. The transcriptional activity of SP1 and SP2 was unaffected by METTL3 deletion, but due to the lack of m 6 A modification on the transcripts, the transcripts were shifted to low molecular weight polyribosomes, resulting in reduced translational efficiency and less protein production, ultimately leading to reduced malignancy of AML cells.
Other Possible Pathways
Protein translation usually begins with the recruitment of the 43S pre-initiation complex to the 5' cap structure of the mRNA via the cap-binding complex. However, some transcripts can be translated in a cap-independent manner through certain mechanisms. A study found that a single 5 UTR m 6 A could directly bind eIF3, which in turn allowed it to recruit the 43S complex to initiate translation without the involvement of the capbinding protein eIF4E. The inhibition of adenosine methylation also selectively reduced the translation efficiency of the 5 UTR m 6 A-modified mRNA. This study revealed that cells under different stresses induced a redistribution of m 6 A modifications at the transcriptome level and could generate a translation pattern resulting from a 5 UTR m 6 A modification that bypassed the involvement of the eIF4F complex with eIF3 as a novel m 6 A reading protein [87]. Meanwhile, a similar mechanism was reported: in mammalian cells, the asymmetric distribution of m 6 A along mRNA resulted in relatively little methylation in the 5 UTR. However, in the heat shock stress response, certain adenosines in the 5 UTR of the newly transcribed mRNA were preferentially methylated, and increased m 6 A modification in this region promoted cap-independent translation initiation, revealing a novel mechanism of translation regulation under stress [88]. Therefore, it is worthwhile to further explore whether this mechanism of mRNA translation with eIF3 directly as an m 6 A reader involved in 5 UTR m 6 A modification exists in cancer.
In summary, METTL3 is involved in translation regulation in a variety of ways in cancer, which can be summarized as follows (see Figure 2):
(1) METTL3 modifies m 6 A in target mRNAs and then recruits canonical translation machinery through classical or novel m 6 A readers YTHDF1/YTHDF3/IGF2BP1/ATXN2 to promote translation. (2) After m 6 A modification of target mRNAs, METTL3 directly binds m 6 A sites in the cytoplasm and recruits canonical translation machinery to promote translation. (3) METTL3 interacts with CEBPZ to bind the promoter of target genes and enhances the translation of the associated mRNAs by relieving ribosomal arrest through m 6 A modification. chinery through classical or novel m 6 A readers YTHDF1/YTHDF3/IGF2BP1/ATXN2 to promote translation. (2) After m 6 A modification of target mRNAs, METTL3 directly binds m 6 A sites in the cytoplasm and recruits canonical translation machinery to promote translation. (3) METTL3 interacts with CEBPZ to bind the promoter of target genes and enhances the translation of the associated mRNAs by relieving ribosomal arrest through m 6 A modification. Figure 2. Molecular mechanisms underlying translation regulation of METTL3 on mRNAs in cancers. METTL3 methylates target mRNA transcripts in the nucleus and enhances its translation in the following ways: 1. METTL3 methylates target mRNA transcripts and recruits canonical translation machinery through classical reader proteins YTHDF1/YTHDF3/IGF2BP1 or novel reader protein ATXN2 to enhance translation. 2. METTL3 directly binds to 3′UTR of methylated mRNA in the cytoplasm and recruits canonical translation machinery to promote translation. 3. METTL3 methylates target mRNA transcripts and eIF3 binds to 5′UTR m 6 A to enhance translation in a cap-independent manner, but this mechanism has not been reported in cancer cells. 4. METTL3 interacts with CEBPZ to bind the promoter of target genes and relieves ribosome stalling through m 6 A modification to enhance translation.
Conclusions and Perspectives
In recent years, more and more studies have reported that METTL3-mediated m 6 A modification of mRNAs affected various processes in the mRNA life cycle through multiple intermolecular interactions, which in turn mediated various phenotypic changes in cancer. In this review, we focused on the involvement of METTL3 in the regulation of mRNA translation. First, we briefly introduced the aberrant translation in cancer and its relationship with canonical translation machinery. Then, we introduced in detail the different roles of METTL3 in the aberrant translation process of m 6 A-modified mRNA. With METTL3 methylates target mRNA transcripts in the nucleus and enhances its translation in the following ways: 1. METTL3 methylates target mRNA transcripts and recruits canonical translation machinery through classical reader proteins YTHDF1/YTHDF3/IGF2BP1 or novel reader protein ATXN2 to enhance translation. 2. METTL3 directly binds to 3 UTR of methylated mRNA in the cytoplasm and recruits canonical translation machinery to promote translation. 3. METTL3 methylates target mRNA transcripts and eIF3 binds to 5 UTR m 6 A to enhance translation in a cap-independent manner, but this mechanism has not been reported in cancer cells. 4. METTL3 interacts with CEBPZ to bind the promoter of target genes and relieves ribosome stalling through m 6 A modification to enhance translation.
Conclusions and Perspectives
In recent years, more and more studies have reported that METTL3-mediated m 6 A modification of mRNAs affected various processes in the mRNA life cycle through multiple intermolecular interactions, which in turn mediated various phenotypic changes in cancer. In this review, we focused on the involvement of METTL3 in the regulation of mRNA translation. First, we briefly introduced the aberrant translation in cancer and its relationship with canonical translation machinery. Then, we introduced in detail the different roles of METTL3 in the aberrant translation process of m 6 A-modified mRNA. With the gradual clarification of the mechanism of aberrant translation regulation in cancer, targeting the METTL3-mediated translation process has become a possibility. Since METTL3 interacts with multiple translation-related RBPs in the process of translation promotion, the use of small molecule inhibitors targeting canonical translation machinery for cancer treatment seems possible. Small molecule inhibitors targeting eIF4A and eIF4E have been introduced for a long time. For example, 4EGI-1, an inhibitor of eIF4E-eIF4G interaction [89], can decrease eIF4E-sensitive mRNA translation level and demonstrate a favorable antitumor effect. However, advancing to clinical application has been delayed [89,90]. The inhibitors against the binding of eIF6 to the 60S were also identified and might have dose-and cell-specific effects [91]. We cannot help but wonder whether targeting key molecules upstream of the translation machinery in cancer translation regulation could produce a broader spectrum and better efficacy of inhibitory effects on aberrant translation. Encouragingly, the first launch of STM2457, a small molecule inhibitor targeting METTL3, was published in 2021, and its promising antitumor effect was validated in AML [92]. Moreover, Du et al. [93] used virtual screening of 1042 natural products and identified quercetin as a qualified METTL3 small molecule inhibitor. Moroz et al. [94] developed a METTL3 inhibitor UZH1a using a structure-based drug discovery approach. Lee et al. [95] reported eltrombopag as an allosteric inhibitor of the METTL3-14 complex. Since the determinants of successful clinical application of small molecule inhibitors include confirmation of the compound-mechanism hypothesis, compound-target action and pharmacodynamic activity [96], if the likelihood of successful drug development is to be maximized, a high standard of screening for inhibitors targeting translational regulation is required in preclinical studies. Therefore, whether drugs such as STM2457 can target METTL3 and thereby inhibit aberrant translation in other types of cancer such as lung cancer remains to be explored and validated through numerous studies. Combining small molecule inhibitors of METTL3 and canonical translation machinery inhibitors to overcome intra-tumor heterogeneity and inhibit the oncogenic signal of aberrant translation integration in cancer cells is also promising. Since the initial success of therapeutic approaches targeting abnormal translation in cancer has been achieved, we believe that in the near future, these drugs will move from the laboratory to the clinic and achieve breakthroughs in anti-cancer therapy.
Figure 2 .
2Molecular mechanisms underlying translation regulation of METTL3 on mRNAs in cancers.
Author
Contributions: Conceptualization, W.M.; investigation, W.M. and H.X.; writing-original draft preparation, P.M.; writing-review and editing, J.C., Y.W. and R.Z.; visualization, J.C., Y.W. and R.Z.; supervision, Y.L., W.M. and H.X. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by the National Natural Science Foundation of China (No. 82072593), and Department of Science and Technology of Hubei Province (No. 2020BCB027).
Table 1 .
1Mechanisms of METTL3 involved in translation regulation through m 6 A modification in human cancers. Wang et al. reported in 2015 that after m 6 A modification of the 3 UTR and stop codon regions of mRNA by METTL3 in the nucleus, mRNA translocated to the cytoplasm, where the mPMID
Cancer Types
Reader Proteins *
Targets
Function
m 6 A Sites
RBPs of
Canonical
Translational
Machinery
31061416
Liver cancer
YTHDF1
SNAI1
Epithelial-
mesenchymal
transition
CDS
eEF2
35032557
Gastrointestinal
stromal tumor
YTHDF1
MRP1
Drug resistance
5 UTR
eEF1
32444598
Cervical and liver
cancer
YTHDF1
PDK4
Glycolysis
5 UTR
eEF2
33795252
Breast cancer
YTHDF1
KRT7
Metastasis
CDS
eEF1
33654093
Melanoma
YTHDF1
SPRED2
Tumor growth
and metastasis
CDS, 3 UTR
30154548
Endometrial
cancer
YTHDF1
PHLPP2
Proliferation and
tumorigenicity
33758320
Gastric cancer
YTHDF1
SPHK2
Progression
eIF3a
35078505
Lung
adenocarcinoma
YTHDF1
ENO1
Glycolysis and
tumorigenesis
eIF3e
31722709
Ocular melanoma
YTHDF1
HINT2
Progression
3 UTR
34996469
Lung
adenocarcinoma
YTHDF1
SLC7A11
Progression
33618740
NSCLC
YTHDF1, YTHDF3
YAP
Drug resistance
and metastasis
3 UTR
eIF3b
31409574
Bladder cancer
YTHDF1, YTHDF3
ITGA6
Progression
3 UTR
32621798
Oral squamous
cell carcinoma
YTHDF1,
IGF2BP1
BMI1
Tumorigenesis
and metastasis
3 UTR
32838807
Colorectal cancer
METTL3,
YTHDF1
HSF1
Progression
3 UTR
30796352
Bladder cancer
METTL3, YTHDF1
CDCP1
Chemical
carcinogenesis
3 UTR
27117702
Lung cancer
METTL3
EGFR, TAZ
Progression
3 UTR
eIF4E, eIF3
30232453
Lung cancer
METTL3
BRD4
Tumorigenesis
3 UTR
eIF3h, eIF4E
34561421
Chronic myeloid
leukemia
METTL3
PES1
Proliferation and
drug resistance
3 UTR
30249526
Ovarian
carcinoma
METTL3
AXL
Epithelial-
mesenchymal
transition
34995801
Esophageal
cancer
ATXN2
TNFR1
Progression and
tumorigenesis
3 UTR
29186125
Acute myeloid
leukemia
SP1,SP2
Proliferation
CDS
34631715
Kidney cancer
ABCD1
Progression
5 UTR
33676554
Lung
adenocarcinoma
FBXW7
Apoptosis and
proliferation
CDS
34530048
Melanoma
EGFR
Drug resistance
3 UTR
33217448
Colorectal cancer
GLUT1
Progression
3 UTR
28920958
Acute myeloid
leukemia
c-MYC, BCL-2,
PTEN
Proliferation
3 UTR
33267838
Bladder cancer
CDCP1
Progression
3 UTR
31454538
Breast cancer
BCL-2
Progression
* The reader proteins here refer to the classical or novel m 6 A readers as well as METTL3, which can also act as an
m 6 A reader directly in some cases.
Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement:The data that support the findings of this study are available from the corresponding author upon reasonable request.Conflicts of Interest:The authors declare that they have no conflicts of interest.
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N6-methyladenosine modification regulates imatinib resistance of gastrointestinal stromal tumor by enhancing the expression of multidrug transporter MRP1. K Xu, Q Zhang, M Chen, B Li, N Wang, C Li, Z Gao, D Zhang, L Yang, Z Xu, 10.1016/j.canlet.2022.01.008Cancer Lett. 2022PubMedXu, K.; Zhang, Q.; Chen, M.; Li, B.; Wang, N.; Li, C.; Gao, Z.; Zhang, D.; Yang, L.; Xu, Z.; et al. N6-methyladenosine modification regulates imatinib resistance of gastrointestinal stromal tumor by enhancing the expression of multidrug transporter MRP1. Cancer Lett. 2022, 530, 85-99. [CrossRef] [PubMed]
N6-methyladenosine regulates glycolysis of cancer cells through PDK4. Z Li, Y Peng, J Li, 10.1038/s41467-020-16306-5Nat. Commun. Li, Z.; Peng, Y.; Li, J. N6-methyladenosine regulates glycolysis of cancer cells through PDK4. Nat. Commun. 2020, 11, 2578. [CrossRef]
N6-methyladenosine regulates mRNA stability and translation efficiency of KRT7 to promote breast cancer lung metastasis. F Chen, Z Chen, T Guan, 10.1158/0008-5472.CAN-20-3779Cancer Res. 81Chen, F.; Chen, Z.; Guan, T. N6-methyladenosine regulates mRNA stability and translation efficiency of KRT7 to promote breast cancer lung metastasis. Cancer Res. 2021, 81, 2847-2860. [CrossRef]
)A mRNA methylation initiated by METTL3 directly promotes YAP translation and increases YAP activity by regulating the MALAT1-miR-1914-3p-YAP axis to induce NSCLC drug resistance and metastasis. D Jin, J Guo, Y Wu, J Du, L Yang, X Wang, W Di, B Hu, J An, L Kong, 10.1186/s13045-019-0830-6J. Hematol. Oncol. 126PubMedJin, D.; Guo, J.; Wu, Y.; Du, J.; Yang, L.; Wang, X.; Di, W.; Hu, B.; An, J.; Kong, L.; et al. m(6)A mRNA methylation initiated by METTL3 directly promotes YAP translation and increases YAP activity by regulating the MALAT1-miR-1914-3p-YAP axis to induce NSCLC drug resistance and metastasis. J. Hematol. Oncol. 2019, 12, 135. [CrossRef] [PubMed]
N6-methyladenosine modification of ITGA6mRNA promotes the development and progression of bladder cancer. H Jin, X Ying, B Que, 10.1016/j.ebiom.2019.07.06847Jin, H.; Ying, X.; Que, B. N6-methyladenosine modification of ITGA6mRNA promotes the development and progression of bladder cancer. EBioMedicine 2019, 47, 195-207. [CrossRef]
He, Q. METTL3 Promotes Tumorigenesis and Metastasis through BMI1 m6A Methylation in Oral Squamous Cell Carcinoma. L Liu, Y Wu, Q Li, J Liang, 10.1016/j.ymthe.2020.06.024Mol. Ther. 28Liu, L.; Wu, Y.; Li, Q.; Liang, J.; He, Q. METTL3 Promotes Tumorigenesis and Metastasis through BMI1 m6A Methylation in Oral Squamous Cell Carcinoma. Mol. Ther. 2020, 28, 2177-2190. [CrossRef]
ATXN2-mediated translation of TNFR1 promotes esophageal squamous cell carcinoma via m(6)A-dependent manner. R Li, L Zeng, H Zhao, J Deng, L Pan, S Zhang, G Wu, Y Ye, J Zhang, J Su, 10.1016/j.ymthe.2022.01.006Mol. Ther. 2022Li, R.; Zeng, L.; Zhao, H.; Deng, J.; Pan, L.; Zhang, S.; Wu, G.; Ye, Y.; Zhang, J.; Su, J.; et al. ATXN2-mediated translation of TNFR1 promotes esophageal squamous cell carcinoma via m(6)A-dependent manner. Mol. Ther. 2022, 30, 1089-1103. [CrossRef]
The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. S Lin, J Choe, P Du, R Triboulet, R I Gregory, 10.1016/j.molcel.2016.03.021Mol. Cell. 62Lin, S.; Choe, J.; Du, P.; Triboulet, R.; Gregory, R.I. The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol. Cell 2016, 62, 335-345. [CrossRef]
METTL3 promotes ovarian carcinoma growth and invasion through the regulation of AXL translation and epithelial to mesenchymal transition. W Hua, Y Zhao, X Jin, D Yu, J He, D Xie, P Duan, 10.1016/j.ygyno.2018.09.015Gynecol. Oncol. 151Hua, W.; Zhao, Y.; Jin, X.; Yu, D.; He, J.; Xie, D.; Duan, P. METTL3 promotes ovarian carcinoma growth and invasion through the regulation of AXL translation and epithelial to mesenchymal transition. Gynecol. Oncol. 2018, 151, 356-365. [CrossRef]
New insight into the catalytic -dependent and -independent roles of METTL3 in sustaining aberrant translation in chronic myeloid leukemia. Z Ianniello, M Sorci, L C Ginistrelli, A Iaiza, M Marchioni, C Tito, E Capuano, S Masciarelli, T Ottone, C Attrotto, 10.1038/s41419-021-04169-7Cell Death Dis. 12Ianniello, Z.; Sorci, M.; Ginistrelli, L.C.; Iaiza, A.; Marchioni, M.; Tito, C.; Capuano, E.; Masciarelli, S.; Ottone, T.; Attrotto, C.; et al. New insight into the catalytic -dependent and -independent roles of METTL3 in sustaining aberrant translation in chronic myeloid leukemia. Cell Death Dis. 2021, 12, 870. [CrossRef]
Dynamic m6A mRNA methylation reveals the role of METTL3-m6A-CDCP1 signaling axis in chemical carcinogenesis. F Yang, H Jin, B Que, Y Chao, H Zhang, X Ying, Z Zhou, Z Yuan, J Su, B Wu, 10.1038/s41388-019-0755-0Oncogene. 38PubMedYang, F.; Jin, H.; Que, B.; Chao, Y.; Zhang, H.; Ying, X.; Zhou, Z.; Yuan, Z.; Su, J.; Wu, B.; et al. Dynamic m6A mRNA methylation reveals the role of METTL3-m6A-CDCP1 signaling axis in chemical carcinogenesis. Oncogene 2019, 38, 4755-4772. [CrossRef] [PubMed]
Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development. X Ying, X Jiang, H Zhang, B Liu, 10.1186/s12943-020-01289-0Mol. Cancer. 19PubMedYing, X.; Jiang, X.; Zhang, H.; Liu, B. Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9- methyltransferase like 3 conjugates promotes bladder cancer development. Mol. Cancer 2020, 19, 169. [CrossRef] [PubMed]
METTL3 preferentially enhances non-m(6)A translation of epigenetic factors and promotes tumourigenesis. X Wei, Y Huo, J Pi, Y Gao, S Rao, M He, Q Wei, P Song, Y Chen, D Lu, 10.1038/s41556-022-00968-yNat. Cell Biol. 24Wei, X.; Huo, Y.; Pi, J.; Gao, Y.; Rao, S.; He, M.; Wei, Q.; Song, P.; Chen, Y.; Lu, D.; et al. METTL3 preferentially enhances non-m(6)A translation of epigenetic factors and promotes tumourigenesis. Nat. Cell Biol. 2022, 24, 1278-1290. [CrossRef]
Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control. I Barbieri, K Tzelepis, L Pandolfini, J Shi, G Millán-Zambrano, S C Robson, D Aspris, V Migliori, A J Bannister, N Han, 10.1038/nature24678Nature. 552PubMedBarbieri, I.; Tzelepis, K.; Pandolfini, L.; Shi, J.; Millán-Zambrano, G.; Robson, S.C.; Aspris, D.; Migliori, V.; Bannister, A.J.; Han, N.; et al. Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control. Nature 2017, 552, 126-131. [CrossRef] [PubMed]
5 UTR m(6)A Promotes Cap-Independent Translation. K D Meyer, D P Patil, J Zhou, A Zinoviev, M A Skabkin, O Elemento, T V Pestova, S.-B Qian, S R Jaffrey, 10.1016/j.cell.2015.10.012Cell. 163Meyer, K.D.; Patil, D.P.; Zhou, J.; Zinoviev, A.; Skabkin, M.A.; Elemento, O.; Pestova, T.V.; Qian, S.-B.; Jaffrey, S.R. 5 UTR m(6)A Promotes Cap-Independent Translation. Cell 2015, 163, 999-1010. [CrossRef]
Dynamic m(6)A mRNA methylation directs translational control of heat shock response. J Zhou, J Wan, X Gao, X Zhang, S R Jaffrey, S.-B Qian, 10.1038/nature15377Nature. 526Zhou, J.; Wan, J.; Gao, X.; Zhang, X.; Jaffrey, S.R.; Qian, S.-B. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 2015, 526, 591-594. [CrossRef]
Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. N J Moerke, H Aktas, H Chen, S Cantel, M Y Reibarkh, A Fahmy, J D Gross, A Degterev, J Yuan, M Chorev, 10.1016/j.cell.2006.11.046Cell. 128Moerke, N.J.; Aktas, H.; Chen, H.; Cantel, S.; Reibarkh, M.Y.; Fahmy, A.; Gross, J.D.; Degterev, A.; Yuan, J.; Chorev, M.; et al. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 2007, 128, 257-267. [CrossRef]
Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A. M.-E Bordeleau, A Mori, M Oberer, L Lindqvist, L S Chard, T Higa, G J Belsham, G Wagner, J Tanaka, J Pelletier, 10.1038/nchembio776Nat. Chem. Biol. 2Bordeleau, M.-E.; Mori, A.; Oberer, M.; Lindqvist, L.; Chard, L.S.; Higa, T.; Belsham, G.J.; Wagner, G.; Tanaka, J.; Pelletier, J. Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A. Nat. Chem. Biol. 2006, 2, 213-220. [CrossRef]
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits. E Pesce, A Miluzio, L Turcano, C Minici, D Cirino, P Calamita, N Manfrini, S Oliveto, S Ricciardi, R Grifantini, 10.3390/cells9010172Cells. 2020172PubMedPesce, E.; Miluzio, A.; Turcano, L.; Minici, C.; Cirino, D.; Calamita, P.; Manfrini, N.; Oliveto, S.; Ricciardi, S.; Grifantini, R.; et al. Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits. Cells 2020, 9, 172. [CrossRef] [PubMed]
Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. E Yankova, W Blackaby, M Albertella, 10.1038/s41586-021-03536-wNature. 593PubMedYankova, E.; Blackaby, W.; Albertella, M. Small molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature 2021, 593, 597-601. [CrossRef] [PubMed]
Discovery of METTL3 Small Molecule Inhibitors by Virtual Screening of Natural Products. Y Du, Y Yuan, L Xu, F Zhao, W Wang, Y Xu, X Tian, 10.3389/fphar.2022.878135Front. Pharmacol. 2022PubMedDu, Y.; Yuan, Y.; Xu, L.; Zhao, F.; Wang, W.; Xu, Y.; Tian, X. Discovery of METTL3 Small Molecule Inhibitors by Virtual Screening of Natural Products. Front. Pharmacol. 2022, 13, 878135. [CrossRef] [PubMed]
Caflisch, A. METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes. E V Moroz-Omori, D Huang, R K Bedi, S J Cheriyamkunnel, E Bochenkova, A Dolbois, M D Rzeczkowski, Y Li, L Wiedmer, 10.1002/cmdc.202100291ChemMedChem. 16PubMedMoroz-Omori, E.V.; Huang, D.; Bedi, R.K.; Cheriyamkunnel, S.J.; Bochenkova, E.; Dolbois, A.; Rzeczkowski, M.D.; Li, Y.; Wiedmer, L.; Caflisch, A. METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes. ChemMedChem 2021, 16, 3035-3043. [CrossRef] [PubMed]
Eltrombopag as an Allosteric Inhibitor of the METTL3-14 Complex Affecting the m(6)A Methylation of RNA in Acute Myeloid Leukemia Cells. J H Lee, N Choi, 10.3390/ph15040440Pharmaceuticals. 2022440Lee, J.H.; Choi, N. Eltrombopag as an Allosteric Inhibitor of the METTL3-14 Complex Affecting the m(6)A Methylation of RNA in Acute Myeloid Leukemia Cells. Pharmaceuticals 2022, 15, 440. [CrossRef]
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INTRODUCTION
Reversible DNA and histone modifications have a wellestablished role in the regulation of gene expression. RNA modifications, namely N6-methyladenosine (m 6 A), were recently recognized as another important layer of regulation ((1,2), reviewed in (3) and (4)). Mammalian cells possess dedicated cellular machinery to write, erase and read m 6 A and m 6 Am (found adjacent to the 5 cap) marks. Their substrate specificity, regulation and roles in RNA metabolism, development and disease undergo intensive investigations.
In mammals, METTL3 and METTL16 are two m 6 A methyltransferases (MTs) that can modify mRNAs. Although both catalyse the formation of m 6 A in RNA using S-adenosylmethionine (SAM) as a methyl donor, they display several rather distinct features. METTL16 appears to act as a monomer (5)(6)(7), whereas METTL3 forms a heterodimeric complex with METTL14 (METTL3/14) (8). The METTL3/14 core further assembles with auxiliary proteins that enhance its methylation activity and specificity in vivo; WTAP, HAKAI, VIRMA (also known as KIAA1429), RBM15 and ZC3H13 (8)(9)(10)(11)(12)(13)(14)(15)(16). METTL16 preferentially methylates structured RNAs carrying a nonameric consensus sequence (UACAGAGAA, the methylated adenosine is underlined) (5,6), whereas METTL3/14 prefers single-stranded RNAs with a short and degenerated motif (DRACH, D = A/G/U; R = A/G; H = A/C/U) (1,2,17).
In mRNAs, the first transcribed adenosines directly adjacent to the 7-methylguanosine (m 7 G) cap can be dimethylated (m 6 Am). The N6 position is methylated by the capspecific N6-adenosine RNA MT PCIF1 (also known as CA-PAM) (18). PCIF1 N6-adenosine methylation depends on prior 2 -O-methylation of adenosine, as m 6 A was not detected as the first cap-adjacent adenosine (19,20). PCIF1 is recruited to nascent transcripts via direct interaction with Ser5-phosphorylated (Ser5-P) C-terminal domain (CTD) of RNA polymerase II (RNAPII) (18,21).
The levels of m 6 A and m 6 Am in the cells are fine-tuned by the action of at least two demethylases (DMTs): FTO (22) and ALKBH5 (23). FTO was reported to bind the transcription co-activator TRIP4 in vitro (24), the protein kinase CaMKII (25) and the tRNA methyltransferase TRMT10A (26). ALKBH5 interacts with two members of the DEADbox (DDX) family of RNA helicases, DDX46 and DDX3 (27,28). However, the role of most of these interactions and the mechanisms of DMTs regulation remain largely unknown.
The activity of MTs and DMTs determines the N6methylation status of mRNAs and non-coding (ncRNAs) in the cell and, in turn, their metabolism and function. Several pieces of evidence support a model of co-transcriptional m 6 A and m 6 Am dynamics (18,(29)(30)(31)(32)(33)(34)(35). METTL3 and PCIF1 are recruited to active chromatin by RNAPII (18,21,33) and histone H3 trimethylation at lysine 36 guides m 6 A deposition (34). In turn, m 6 A modulates gene expression via the regulation of histone modifications (36). Therefore, a better understanding of the regulation of MTs and DMTs is key to establish their role in cellular metabolism. To date, the possibility of crosstalk between individual MTs and DMTs and their spatial contacts has not been explored.
To address this question, we employed a proteomic approach and mapped the interaction networks of the key enzymes of the m 6 A and m 6 Am pathways METTL3, METTL16, PCIF1, FTO and ALKBH5 in the human cell line HEK293 T-REx Flp-In (293T). We used the proximitydependent labelling approach BioID coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) (37,38). In this method, the bait protein is fused to a promiscuous biotin ligase derived from Escherichia coli BirA (R118G, BirA*) to label proteins in vivo in an approximate radius of 10 nm (39), detecting stable and transient protein-protein interactions. Among others, this method has been successfully used to identify protein interactors of other RNA modifying proteins (40).
MATERIALS AND METHODS
Preparation of vectors for mammalian expression
To prepare the common backbone for inducible expression of the modified version of BirA (R118G, BirA*), the BirA* sequence was amplified from pcDNA3.1 mycBioID vector (Addgene #35700) with primers flanked by NotI and XhoI restriction enzyme sites. The PCR product was digested and ligated into pcDNA5/FRT/TO™ vector (Invitrogen) containing an N-terminal 3xFlag tag. To create the NLS-BirA*vector, the SV40 nuclear localization signal (NLS) (PKKKRKV) was cloned upstream of the Nterminal 3xFlag tag via KpnI restriction enzyme site. The full-length coding sequences (CDS) of the bait genes were amplified with iProof high-fidelity DNA polymerase (Bio-Rad) from cDNA prepared from 293T cells. For cDNA preparation, total RNA was isolated with TriPure reagent (Roche) according to manufacturer's protocol, treated with Turbo DNase (Ambion) and 2 g of RNA was used for reverse transcription (RT) with oligo dT primers and Su-perScript III RT (Invitrogen). The cDNA was then treated with RNase H (Invitrogen) before being used as a template for PCR. METTL3, METTL16, FTO and ALKBH5 were subcloned by restriction endonucleases. The PCR products were digested and ligated into the pcDNA5/FRT/TO™ vector (Invitrogen) containing the 3xFlag tag-BirA* insert. Two N-and C-terminal BirA* fusion versions were prepared for METTL3. FTO and ALKBH5 have the BirA* tag at the N-terminus and METTL16 at the C-terminus. The PCIF1 construct was prepared by Gateway cloning (Invitrogen). MAC-tag (HA-Strep II-BirA*)-N terminal vector, a gift from Markku Varjosalo (Addgene plasmid # 108078; http://n2t.net/addgene:108078) (38) was used as a destination vector for inducible expression of the BirA* tagged fusion protein. The HA-Strep II-eGFP vector was prepared by Gateway cloning (Invitrogen). pTO-HA-StrepII vector (gift from Markku Varjosalo) was used as a destination vector for inducible expression of eGFP fusion protein for control Strep II tag pull-downs. The C-terminal tagged ALKBH5-Strep II-HA vector was prepared by recloning the CDS of ALKBH5 from the pcDNA5/FRT/TO-Flag-ALKBH5-BirA* to the pcDNA5/FRT/TO™-Strep II tag between the HindIII and KpnI restriction sites. All constructs were verified by Sanger sequencing. The sequences of cloning primers are in Supplementary Table S6.
Cell culture and stable cell lines preparation
Human 293 Flp-In™ T-REx™ (293T) (Invitrogen) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 • C in the presence of 5% CO 2 . To prepare stable cell lines with inducible expression of the fusion proteins, 293T cells were grown to 70% confluency in 6-well plates format and cotransfected with 300 ng of the corresponding expression vector (pcDNA5/FRT/TO™, Invitrogen, MAC-tag-N or HA-Strep II tag) and 2.7 g of pOG44 vector (Invitrogen) (ratio 1:9) using 5 l of TurboFect reagent (Invitrogen) following manufacture's protocol. One day after transfection, cells were transferred to a 150 mm dish and selected in 60 g/ml of hygromycin B until individual clones were formed. Doxycycline (dox)-inducible expression of the tagged proteins were confirmed by western blot with anti-Flag antibodies (Sigma, 1:5000) or anti-Strep tag II (Abcam, 1:2000) and individual clones were selected.
Immunofluorescence analysis
Cells were grown on polyethyleneimine-coated coverslips. The expression of BirA* tagged proteins was induced by the addition of 200 ng/ml of dox for 24 h at 37 • C. All the subsequent steps were performed at room temperature. Cells were fixed in 3.7% paraformaldehyde for 20 min. Fixed cells were washed with PBS, permeabilized by 0.2% Triton X-100 in PBS for 20 min and blocked for 1 h in 5% horse serum in PBS, then incubated with anti-Flag primary antibody (Sigma, 1:500) or anti-HA (Santa Cruz, 1:100) in 3% horse serum for 1 h. After three washes with PBS for 10 min, cells were incubated with a mix of Alexa 594 secondary antibodies (Invitrogen, 1:500) and DAPI (Sigma, 1:500) in PBS for 30 min in the dark and consequently washed with PBST, PBS and finally fixed on slides in mowiol (Mowiol ® , Nucleic Acids Research, 2021, Vol. 49, No. 19 10897 Sigma) with DABCO. Samples were imaged with upright microscope Zeiss AxioImager.Z2 combined with Hamamatsu ORCA Flash 4.0 camera. Images were processed using the open-source platform Fiji.
Biotin-mediated proximity labelling and streptavidin affinity purification (BioID)
Cells were checked for mycoplasma contamination by RT-PCR prior affinity purification (AP) by BioID. PCR primers sequences are in the Supplementary Table S6. For each bait, two 100 mm cell culture dishes were grown to 90% confluency. Twenty-four hours prior cell harvesting the protein expression was induced with 200 ng/ml of dox and 50 M biotin (Sigma) was added to the medium to allow in vivo biotinylation. Cells from the two dishes were combined, pelleted as one biological replicate, flash frozen and short-term stored at -80 • C until affinity purification was performed. At least three independent biological replicates were prepared per cell line.
AP was performed according to (41) with minor modifications. Cell pellets were thawed on ice, lysed in 1.2 ml of lysis buffer per frozen pellet and homogenized with a 2 ml syringe and a 21 G × 1-1/2" needle followed by three cycles of sonication on ice (5s on, 10s off, amplitude 35, microtip). The insoluble part was removed by centrifugation 16 500 g 4 • C 10 min. The supernatants (from two 100 mm cell culture dishes) were incubated with 150 l of magnetic streptavidin beads (Dynabeads™ MyOne™ Streptavidin C1, Invitrogen) O/N at 4 • C and washed according (41). After washing with wash 3 buffer, beads were washed thoroughly three times with 750 l of 50 mM Tris-HCl pH 8 to remove any detergents present in the sample that would interfere with mass spectrometry analysis. 1/20th of the beads resuspended in 50 mM Tris-HCl were saved for western blot analysis with HRP-conjugated streptavidin (Thermo Scientific, 1:10 000) prior to mass spectrometry analysis.
Strep II-tag pull-downs
293T HA-Strep II-eGFP, MAC-PCIF1 and ALKBH5-Strep II-HA cell lines were grown in 15 cm dishes under standard cell culture conditions. At a cell-confluency of 60%, transgene expression was induced with 200 ng/l doxycyclin. Cells were harvested 24 h after induction in icecold PBS and immediately pelleted by centrifugation for 5 min at 500 g, 4 • C. To remove residual DMEM and fetal calf serum, cell pellets were washed one additional round with ice cold PBS. For total cell lysates preparation, cell pellets from two 15 cm dishes were resuspended in 1.5 ml ice cold lysis buffer [20 mM HEPES-KOH pH 8; 150 mM KCl, 1 mM EDTA, 0.2% IGEPAL, 1 mM DTT, 1× complete Mini (Roche), 15% glycerol], briefly vortexed and placed on ice for 15 min. Cell homogenates were then sonicated on ice using QSonica sonicator Model Q700 [Sonication setting: microtip probe; amplitude: 35%; 10× (1 s on; 9 s off)]. To deplete cell debris and intact cells, crude cell lysates were centrifuged for 20 min at 16 000 g, 4 • C. Clarified total lysates were transferred into new clean tubes and placed on ice (20 l of each lysate were stored at -20 • C for western blot analysis, input). For Strep II-tag pull-down, 50 l StrepTact-inXT bead slurry (iba) were pelleted on a magnetic stand.
Beads were washed and equilibrated for 40 min at 4 • C in lysis buffer. Equilibrated beads were resuspended in 1 ml total lysate (protein concentration ∼2 mg/ml). For pull-downs of 'intact RNA' condition (-RNase A), 40 units of placental RNase Inhibitor complex (Biotechrabbit) were added to the lysates and for 'digested RNA' condition (+RNase A), lysates were supplemented with 2 g/ml RNAse A (Thermo). Pull-downs were performed for 4 h at 4 • C in rotation. After incubation, the bead-lysate homogenate was placed for 2 min on an ice-chilled magnetic stand and 20 l of supernatant were stored at −20 • C for western blot analysis (FT, unbound fraction). Beads were washed three times in 1.5 ml of ice chilled high-salt wash buffer [20 mM HEPES-KOH pH 8; 350 mM KCl, 1 mM EDTA, 0.2% IGEPAL, 0.1% Na-deoxycholate, 0.05 mg/ml heparin, 1 mM DTT, 1× complete Mini (Roche), 15% glycerol] for 15 min at 4 • C on rotation. After the last wash, beads were resuspended in lysis buffer and transferred to a new tube. Beads were washed three times with lysis buffer 10 min at 4 • C on rotation. To reduce the content of detergent prior LC-MS/MS analysis, beads were washed four times in 1.5 ml ice cold PBS 5 min at 4 • C on rotation. After the last wash, beads were resuspended in 20 l of PBS. Two l were kept in −20 • C for western blot analysis with Anti-Strep II tag (1:2000, Abcam) and the rest of the beads were frozen in liquid nitrogen and stored at −80 • C until LC-MS/MS analysis.
Protein mass spectrometry sample preparation and data acquisition
Proteins retrieved by streptavidin AP were digested on beads with trypsin and the resulting peptide mixtures were analyzed using RSLCnano system connected to either Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) or Orbitrap Elite hybrid spectrometer (Thermo Fisher Scientific). For details, see Supplementary methods.
Mass spectrometry data analysis
For Limma filtering pipeline, the MS RAW data files were analyzed using the MaxQuant software and further processed using the software container environment (https://github.com/OmicsWorkflows). For SAINT filtering pipeline, the MS RAW data files were searched with Proteome Discoverer 1.4 (Thermo Scientific) and further processed using Significance Analysis of INTeractome (SAINT) -express version 3.6.3 (42). For details, see Supplementary methods.
DNA damage assays
For siRNA knockdowns, 293T or U2OS cells were seeded in a six-well plate format and transfected with 20 nM FTO siRNAs (ON-TARGET plus FTO SMARTpool, Dharmacon) or non-target control siRNAs (ON-TARGET plus non-targeting pool, Dharmacon) with Lipofectamine RNAiMAX (ThermoFisher) following manufacture's protocol. 48 h after transfection (or 24 h after seeding for WT and FTO KO cells), 2 mM hydroxyurea (HU) or 1 M camptothecin (CPT) were added to the cell culture medium and cells were collected after the indicated hours. Western blotting was performed with the following antibodies: Anti-FTO (Abcam, ab126605), Anti-␥ -H2AX (Sigma, 05-636-I), Anti-tubulin (Sigma, T6074), Anti-histone 3 (Abcam, ab1791), Anti-pSer317 Chk1 (Merk, DR1025), Anti-pRPA(S4/S8) (Bethyl, A300-245A). Quantification of western blot signals were performed using the software ImageJ.
GO terms analysis
GO terms enrichment analysis was perform using Panther (http://www.pantherdb.org/) with the following parameters: analysis type PANTHER Overrepresentation Test (GO biological process complete, GO cellular component complete or GO molecular function complete), reference list Homo sapiens (all genes in the database), test type Fisher's with FDR corrected for multiple tests. Main figures include manually curated terms due to space constraints. Supplementary Table S5 contains the full list of significant (<0.05 FDR) GO terms. Plots were created using ggplot2 R package.
RESULTS AND DISCUSSION
Validation of the BioID approach for m 6 A and m 6 Am modifiers
To identify the stable and transient protein interactomes of the key mammalian m 6 A and m 6 Am RNA MTs and DMTs, we performed BioID pull-down assays coupled to LC-MS/MS analysis (37). For each bait, we prepared 293T stable cell lines with inducible expression of BirA* fusion proteins ( Figure 1A). All BirA*-fused proteins showed mostly nuclear localization (Supplementary Figure S1A) which is in agreement with previous reports for the endogenous proteins (8,23,30,(43)(44)(45). As a background control, we used BirA* alone, with mostly cytoplasmic localization, and BirA* fused to a nuclear localisation signal (NLS-BirA*) that targeted BirA* to the nucleoplasm ( Figure 1A, Supplementary Figure S1A). Protein lysates of 293T cells expressing the BirA*-fusion proteins were processed in parallel with BirA* and NLS-BirA* controls following the protocol established by Roux el at. (41) with the modifications described in the Methods section ( Figure 1B). The biological replicates of each bait displayed good reproducibility and distinct pattern as compared to the other baits (Supplementary Figure S1B). The western blot analysis of proteins precipitated with streptavidin beads indicated efficient enrichment of biotinylated proteins prior to mass spectrometry analysis and already anticipated differences between the individual baits (Supplementary Figure S1C).
BioID recapitulates the known METTL3 stable interactors and identifies new factors contacting METTL3 in vivo
METTL3 is an exception among our bait set, as it has been extensively studied and shown to form a stable complex with other proteins in vivo. Therefore, to test whether our BioID approach and analysis of MS/MS can recapitulate known interactors, we first performed LC-MS/MS analysis of the BioID samples from METTL3-BirA* cell lines together with the two background controls, BirA* and NLS-BirA*.
To obtain a high-confident list of interacting proteins (hits), the raw MS/MS data were analysed by two independent parallel filtering pipelines--Limma and SAINT (see Materials and Methods and Supplementary Methods). When using the Limma analytical pipeline, only proteins with an enrichment >4-fold and an adjusted P-value smaller than 0.01 relative to the control were considered as significant hits. For SAINT we established a SAINT score cut-off of >0. 74.
The results from N-and C-terminal BirA* fusions of METTL3 revealed the other essential counterpart METTL14 and all the known auxiliary components of the METTL3/14 complex--WTAP, VIRMA, HAKAI, RBM15 and ZC3H13 -among the top significant Limma hits when BirA* cell line was used as a reference ( Figure 1C, Supplementary Figure S2). Importantly, all the known auxiliary components of the METTL3/14 complex except RBM15 were among the shared hits between both filtering pipelines whereas highly probable contaminants were filtered out ( Figure 1C, Supplementary Table S1). RBM15 was identified with both N-and C-terminal BirA* fusions of METTL3 by using Limma analysis (Supplementary Table S2). The interaction between RBM15 and METTL3 is mediated by WTAP (9). This likely increases the distance between RBM15 and METTL3 which resulted in less efficient biotinylation of RMB15. Our approach did not detect some of the previously reported METTL3 inetractors, such as SETDB1 and its associated factor TRIM28 previously found in mouse stem cells (46) nor the translation initiation factor eIF3h (47) and TREX mRNA export complex components (48). Nevertheless, we used the combined filtering workflow as it greatly increased confidence while maintaining a high sensitivity of detection. It is also important to point out that due to the nature of the in vivo labelling technique used in BioID, we cannot exclude that some of the hits could be biotinylated due to the colocalization and close proximity within the same cellular compartment as the bait proteins, and may not have direct functional consequences.
Apart from the known auxiliary components of the METTL3 complex, our results revealed STAT5B among the top hits in N-and C-terminal METTL3 BioID (Figure 1D, Supplementary Table S1). STAT5B belongs to the STAT (Signal Transducer and Activator of Transcription) family of transcription factors (TFs) which get activated upon binding of cytokines and growth factors to cell surface receptors and then translocate to the nucleus to bind the promoters of their target genes and activate transcription (49). We hypothesize that STAT5B could promote the binding and subsequent methylation of METTL3/14 of certain transcripts, similarly to the reported role of SMAD2/3 in stem cells (50).
METLL16 BioID detects proteins involved in the biogenesis of RNAs transcribed by the three different RNA polymerases
We confirmed that our approach can recapitulate known interactors of METTL3 and allows us to obtain highly confident datasets. Therefore, we proceeded to perform the analysis of the other baits. BioID-MS/MS of METTL16, the second human m 6 A MT, identified 78 significant protein hits (Supplementary Table S1). The gene ontology (GO) analysis revealed an enrichment in biological processes (BP) GO terms for mRNA and ncRNA processing and ribosome biogenesis (Figure 2A, Supplementary Table S5). METTL16 targets a wide spectrum of RNAs transcribed by all three RNA polymerases (44), which is reflected in the diversity of METTL16 protein network. Specifically, we identified U6 snRNA biogenesis factors, constituents of 7SK and 7SL particles, tRNA and other ncRNAs modifiers and precursor rRNA processing factors ( Figure 2B). METTL16 methylates U6 snRNA at position A43 (5,6,51) and interacts with the early U6 biogenesis factors La, LARP7 and MePCE in an RNA-dependent manner (44), all of which were significant interactors in our dataset ( Figure 2B, Supplementary Tables S1, S2). However, LARP7 and MePCE are also stable core components of the 7SK small nuclear ribonucleoprotein (snRNP) particle (52)(53)(54)(55) and La protein transiently interacts with 7SK RNA during its biogenesis (54). METTL16 targets 7SK RNA in vivo (44). Our BioID experiments revealed additional 7SK snRNP components HEXIM1 and Cyclin-T2 as significant protein hits (Supplementary Tables S2 and S4). The 5 -terminal hairpin of 7SK recognized by HEXIM1 contains triple-based interactions (56) and METTL16 has an affinity to triplestranded RNA structures (57). Altogether, we hypothesize that METTL16 binding to 7SK could regulate 7SK snRNP assembly and function in vivo. We also observed interaction with a component of another RNP particle, the signal recognition particle (SRP) SRP14 ( Figure 2B). Notably, 7SL RNA, an RNA component of the SRP, was also identified as a METTL16 substrate (44).
In addition to U6 and 7SK snRNPs, we observed other factors linked to RNAPIII transcripts, the RNA modifying enzymes ADAT1, NSUN2 and TRUB1 ( Figure 2B, C Supplementary Tables S1, S4). METTL16 and NSUN2 both target similar types of ncRNAs, such as vault RNAs, Y RNAs, U6 snRNA and lncRNAs (44,58,59). HITS-CLIP analysis of TRUB1 revealed binding to several classes of ncRNAs, but the extent of overlap with METTL16 RNA targets remained unknown as the analysis focused mainly on pri-miRNAs and tRNAs (60). Future studies will address whether the potential interactions between these different modifiers detected by BioID result from their activity on the same RNA molecule or whether they localize to specific nuclear foci that are formed to facilitate RNA processing.
In coding transcripts, METTL16 preferentially binds intronic regions (44). However, so far, only splicing of MAT2A transcript was experimentally shown to be regulated by METTL16 activity (51). METTL16 BioID revealed several spliceosome components and splicing factors among the enriched hits ( Figure 2C). Among those, SNRNP27 and PRP31 are components and assembly factors of U4/U6.U5 tri-snRNP, respectively ( Figure 2C, Supplementary Table S1). It is likely that METTL16 is recruited to pre-mRNAs via binding to U6 snRNPs and subsequently tri-snRNP assembly.
The identified interactors linked to RNAPI include many pre-rRNA processing factors ( Figure 2B). Contradictory data have been published to date concerning METTL16 binding to rRNAs (44,57,61). METTL5 and ZCCHC4 were recently identified as 18S and 28S rRNA m 6 A MTs, respectively (62,63). We observed METTL16 interactions with the nucleolar pre-rRNA processing factors DIMT1, DDX47, DDX49 and RRP1 ( Figure 2B, C). This agrees with METTL16 localization to the nucleolus (57). Follow up studies will address whether the interaction with these proteins is direct, mediated by pre-rRNAs, or arise from the presence of METTL16 in the nucleolus. In summary, our data indicated that METTL16 is spatially connected to Nucleic Acids Research, 2021, Vol. 49, No. 19 10901 several RNA processing and modification pathways. We hypothesize that cells form distinct subcellular processing and modification bodies assembling specific enzymes to facilitate the processing of diverse coding and ncRNAs.
PCIF1 BioID detects factors involved in RNAPII transcription initiation and co-transcriptional snRNA biogenesis
PCIF1 is the MT responsible for N6-methylation of the 2 -O-methylated adenosine that is directly adjacent to the 5 cap of pre-mRNAs (18,(64)(65)(66). The GO terms analysis of the 57 hits showed that the most significant BP GO terms were connected with RNAPII transcription ( Figure 3A, Supplementary Table S5). Specifically, our BioID results revealed two major links: promoter-proximal pausing and snRNA transcription ( Figure 3B). PCIF1 N6-adenosine methylation depends on prior ribose 2 -OH methylation by CMTR1 (19,20). We observed an interaction between PCIF1 and CMTR1 indicating that both, the ribose methylation and direct interaction with CMTR1 facilitate PCIF1 activity (Supplementary Table S4). PCIF1, alike the m 7 G mRNA capping enzyme, is recruited to nascent transcripts through specific recognition of Ser5-P CTD of the largest subunit of RNAPII (RPB1) (18,67). We observed RPB1 as the most statistically significant hit in our PCIF1 BioID experiment (Supplementary Table S1).
Except for the largest subunit of RNAPII, other interactors of PCIF1 were not yet tackled. Therefore, in parallel we performed affinity purifications (AP) of Strep II-tagged PCIF1 followed by LC-MS/MS analysis in presence and absence of RNase A ( Figure 3C, complete list of significant interactions in Supplementary Table S7). As a background control we used Strep II-tagged eGFP cell line. In agreement with its high affinity to Ser5-CTD of RPB1 (18,21), PCIF1 co-precipitates the whole RNAPII polymerase in an RNA independent manner. Except for RPB6 and 12, we identified the RPB1-12 subunits and GINL1A ( Figure 3D and Supplementary Table S7). Other two RNA-independent factors detected by AP and BioID methods were the yet uncharacterized protein CRML and DNA helicase RECQ5 ( Figure 3D). In addition, PCIF1 Strep II-AP revealed an RNA independent interaction with two proteins not found in BioID, the Ser2 CTD phosphatase RPAP2 and GTPase GPN1 ( Figure 3D and Supplementary Table S7). RECQ5 is a protein with reported roles in transcription and DNA replication and repair (reviewed in (68)). It directly interacts with the cleft of RPB1 of RNAPII and has a negative impact on transcription (69,70).
The BioID PCIF1 interactome revealed more factors involved in transcription regulation. PCIF1 is recruited by RNAPII early during transcription initiation (18), however, RNAPII often pauses early after transcription initiation of protein-coding genes, 20-60 nucleotides downstream of a transcription start site (TSS) (71,72). This promoterproximal pausing represents an early elongation checkpoint regulated by the negative elongation factor (NELF) and the DRB-sensitivity inducing factor (DSIF) and the Integrator complexes (73). Notably, the PCIF BioID revealed an interaction with SPT5 (of DSIF), NELFA (of NELF) and three components of the integrator complex (INT4, INT6 and INT12) ( Figure 3B, Supplementary Tables S1, S3). Im-portantly, immunofluorescence analysis of PCIF1 revealed that it co-localizes with SPT5 in the nucleoplasm (43). In addition, we observed contacts with several chromatin modifiers responsible for marking transcriptionally active chromatin, such as SETD1A and SETD1B (H3K4me3 MTs) and KMT2D (H3K4me1 MT) ( Figure 3B, Supplementary Table S1). It will be interesting to test whether PCIF1 activity plays a role in promoter-proximal stalling of RNAPII and whether the transcripts regulated by this post-initiation mechanism are more frequently carrying m 6 Am at their 5 termini.
The cap-linked m 6 Am modification was found also in RNAPII transcribed snRNAs and some snoRNAs (74). In this respect, we observed interaction with several factors involved in snRNA synthesis. The BioID results revealed ICE1 and ICE2 subunits of the little elongation complex (LEC), the Integrator and PCIF11 ( Figure 3B, Supplementary Tables S1 and S4) and the Strep II AP showed interaction with RPAP2 phosphatase ( Figure 3D, Supplementary Table S7). RPAP2 is recruited to snRNA genes via Ser7-CTD of RPB1 and removes the Ser5 CTD marks (75). This may have a positive effect on LEC which regulates snRNA transcription elongation (76). It is possible that crosstalk between PCIF1, RPAP2 and LEC activities facilitates snRNA synthesis. It is also possible that PCIF1 interacts with the NELF complex at the snRNA locus, where it associates with the Integrator and contributes to snRNA transcription elongation and termination efficiency (77). The role of PCIF1 MT activity on snRNAs is yet to be functionally addressed.
The WW domain of PCIF1 shows high homology to the CTD binding WW domain of PIN1 (43), a CTD binding protein that regulates the binding and release of CTD binding factors during RNAPII transcription (78). This could be even uncoupled from its methylation activity as the Drosophila PCIF1 homologue is inactive but binds Ser5-P CTD of RNAPII in vivo (79). Notably, the list of PCIF1 interactors includes several CTD binding proteins that recognize differently phosphorylated RNAPII CTD, such as the Integrator complex components INT4, INT6 and INT12; CMTR1; SETD2; WAC; SCAF8; PCF11 and RPRD2 (Figure 3B, proteins marked with asterisks; Figure 3E). SETD2 trimethylates H3K36 in the gene body of actively transcribed genes and WAC targets the RNF20/40 ubiquitinprotein ligase complex to active transcription sites to mediate histone ubiquitination (80). Three of these proteins contain CTD-interaction domains (CID): SCAF8, PCF11 and RPRD2. SCAF8, together with SCAF4, suppress the use of early alternative polyadenylation sites on mRNAs (81) and PCF11 is an mRNA and snRNA 3' end processing factor (82). RPRD2 is known to interact with Ser2-P, Ser7-P and Ser2-Ser7 double phosphorylated CTD, but its function remains unknown (83). PCIF1 likely meets these factors when bound to the CTD, however it is surprising that it occurs in the proximity of factors that recognize CTD modifications present during rather diverse stages of transcription. In summary, our results provided a strong evidence of the role of PCIF1 in the synthesis of snRNAs and suggest a potential role in RNAPII stalling ( Figure 3E). It remains an open question whether PCIF1 could also bind Ser7-P or some other forms of modified CTD and whether its recruit- FTO on the crossroad between RNAPII transcription, pre-mRNA processing and DNA repair pathways FTO presents a unique feature among the list of enzymes studied in this work as it targets more than one chemical modification. It can demethylate m 6 A, m 6 Am and m 1 A in mRNAs and some ncRNAs in vitro and in vivo (20,45,84). FTO subcellular localization varies in different cell lines, and its localization was proposed to affect its target specificity (45). The majority of the FTO BioID hits are primarily localized to the nucleus (Supplementary Table S5), which corresponds to FTO nuclear localization in HEK293 (45). The BP GO terms analysis revealed a modest enrichment of general terms connected with transcription, splicing and other RNA metabolic processes ( Figure 4A and Supple- Catalytically activated spliceosome (B*)
Catalytic step I complex (C)
Step II catalytically activated complex (C*)
Post-catalytic spliceosome (P)
Intron lariat spliceosome (ILS) CD2BP2 DHX16 (hPRP2) YJU2 (CCDC94 ) CWF19L2 CRNKL1 (CLF1) Pre-spliceosome (A)
Step 1
Step 2 mentary Table S5). The m 6 A and m 6 Am deposition occur mostly co-transcriptionally (18,(31)(32)(33). The FTO BioID revealed several transcription and chromatin factors indicating that FTO is also recruited to active chromatin loci and acts on nascent RNAs ( Figure 4B).
FTO regulates alternative splicing (AS) in mouse and human cells (30,85,86), however, the mechanism of FTO in AS remains unclear. The BioID results support a direct involvement of FTO in pre-mRNA splicing because we observed contacts with spliceosome components and assembly and disassembly factors ( Figure 4B, C). Among the most significant were the U5 snRNP 52K protein (CD2BP2) (87), two factors important for the first catalytical step of splicing the DHX16 helicase (yeast Prp2) and YJU2 (CCDC94) (88), and CRNKL1 (yeast Syf3) and CWF19L2 (a homolog of yeast Cwf19) ( Figure 4C). Interestingly, in yeast, Cwf19 and Syf3 are part of the excised lariat intron U2/U5/U6 complex (89). The presence of spliceosome factors on the proximity of FTO is in agreement with FTO preferential binding to introns (30,45) and suggests co-transcriptional FTO recruitment to nascent pre-mRNA. Furthermore, it raises the possibility that demethylation of pre-mRNA intronic sites directly modulates AS in vivo (30). FTO could also affect AS via targeting m 6 Am marks at Sm-class snRNA caps (74). In this respect, the BioID results showed FTO interactions with the snRNA transcription factors SNPC4 and ICE1, components of the SNAPc and little elongation complexes, respectively (Supplementary Tables S2 and S3). Although m 6 Am at snRNAs does not alter snRNP assembly (74), FTO activity may affect AS indirectly through regulating snRNA metabolism for example through pre-snRNA export to the cytoplasm (90).
FTO has the potential to target m 1 A sites in tRNAs (45). A recent study reported that the tRNA m 1 G methyltransferase TRMT10A interacts with FTO and can enhance its catalytic activity in vitro (24). In vivo it appears to co-regulate some m 6 A sites in mRNAs (26). The possibility of additional factors mediating the interaction between FTO and TRMT10A was not excluded (24). This could explain why TRMT10A was not enriched on the FTO BioID. However, we observed interactions with three other tRNA modifying enzymes; the dihydrouridine tRNA synthase DUS2L, the guanine dimethyltransferase TRM1 and the PUS TRUB1 (Supplementary Tables S1 and S2). Follow up experiments will tackle the question of whether these enzymes could modulate FTO activity similarly to TRMT10A or whether they simultaneously modify tRNAs.
The most striking result was the identification of proteins involved in DNA replication and repair ( Figure 4B), present in FTO and other baits. There is growing evidence of a functional link between m 6 A RNA modification and DNA damage (91)(92)(93). m 6 A accumulates at DNA damage sites upon ultraviolet irradiation and, importantly, FTO localizes to laser-induced damage sites in U2OS cells (91). Moreover, m 6 A has been recently detected in R loops, a threestranded nucleic acid structure formed by a RNA:DNA hybrid and the non-template single-stranded DNA which can be a source of genome instability for the cells (92). Notably, the most enriched and significant FTO BioID interactor was RADX (Supplementary Table S1), a single-strand DNA-binding protein that is recruited to sites of replication stress to promote replication fork stability (94,95). Along with RADX, we observed other factors involved in DNA double-strand break repair (DSB), such as XRCC4, the E3 ubiquitin-protein ligase RNF8 or the Tyrosyl DNA phosphodiesterase 2 (TDP2) ( Figure 4B, Supplementary Table S1). To study the functional significance of these interactions we evaluated the effect of treating wildtype and FTO-depleted cells with different types of DNA damaging agents ( Figure 4D, E). Similar to RADX depletion (94,95), FTO KO cells displayed accumulation of the DNA damage marker phosphorylated histone H2A variant H2AX (␥ -H2AX) upon treatment with 2 mM hydroxyurea (HU) (Figure 4D). No difference between WT and FTO KO or KD cells was seen upon camptothecin (CPT) treatment ( Figure 4E). HU slows down the initiation of replication and also the progression of replication forks, whereas CPT blocks DNA synthesis and induces DSBs. These results indicated that FTO could be involved in sensing of specific types of DNA replication stress such as collision between transcription and DNA replication. It remains to be addressed whether the catalytic activity of FTO participates in this process. In summary, FTO appears as a multifunctional protein acting at the intersection between RNA transcription, RNA processing and DNA replication and repair.
ALKBH5 interacts with pre-mRNA processing and mRNA export factors ALKBH5 m 6 A DMT was implicated in many parts of mRNA metabolism including splicing regulation, mRNA export and stability of mRNA (23,27,96,97). The BP GO terms enrichment analysis of ALKBH5 in vivo interactome reflected connections to nuclear pre-mRNA processing and mRNA export ( Figure 5A). In addition, we detected several TFs, as well as proteins involved in chromatin remodeling ( Figure 5B) corresponding to the notion that ALKBH5, like PCIF1 and METTL3/14, associates with active chromatin and is co-transcriptionally recruited to the nascent transcripts (35). In contrary to the other baits, ALKBH5 revealed interactions with posttranscriptional mRNA biology ( Figure 5B, C). This indicated that ALKBH5 either remains bound to mRNP particles until their export to the cytoplasm or that it is recruited to (pre)mRNA at multiple steps during its biogenesis.
Immunofluorescence analyses showed that ALKBH5 partially co-localizes with nuclear speckle markers in an RNA-dependent manner (23,97). Accordingly, our ALKBH5 BioID data revealed several nuclear speckle proteins ( Figure 5B, proteins marked with asterisks). Nuclear speckles are nuclear domains enriched in pre-mRNA splicing factors located in interchromatin regions of the nucleoplasm (98). Among ALKBH5 nuclear speckle interactors, there are several alternative splicing factors and nuclear exon-junction complex (EJC) components ( Figure 5B, Supplementary Figure S3A). The assembly of EJC on the mRNA is fully dependent on splicing (99). Specifically, the spliceosomal protein CWC22 interacts with eIF4A3 and serves as a connecting platform between splicing and EJC deposition ( Figure The top scoring hits in our ALKBH5 BioID were however components of the TRanscription and EXport (TREX) complex. The role of ALKBH5 in mRNA export is supported by an earlier observation that ALKBH5 depletion leads to an accumulation of polyadenylated RNA in the cytoplasm (23). ALYREF (also known as THOC4) showed the highest score followed by the other subunits of TREX complex DDX39B, CHTOP, SARNP, POLDIP3 and the THO subunit THOC3 ( Figure 5B, Supplementary Figure S3B, Supplementary Table S1). The TREX complex is recruited to nascent mRNA transcripts during splicing via EJC (104). TREX components, or other export adaptors such as SR proteins, interact with the general mRNA export receptor TAP-p15 complex that transports the mRNP through the nuclear pore. ALYREF and DDX39B accompany the mRNP to the nuclear periphery where they dissociate during translocation through the pore (105). However, the export receptor TAP-p15 complex is not a significant hit in ALKBH5 BioID, indicating that ALKBH5 dissociates from the mRNP at earlier stages of the export process.
Several ALKBH5 BioID interactors possess RNA recognition motifs ( Figure 5B, underlined proteins). To further validate the results and assess which of the interactions are RNA independent, we performed Strep II-tag ALKBH5 AP coupled to LC-MS/MS analysis in the presence and absence of RNase A in 293T cells, respectively ( Figure 5D). This experiment detected stable, RNA independent interactions with the EJC components eIF4A3, RBM8, MAGOH and Pinin (and Acinus in an RNA-dependent fashion) and the RNA export factors ALYREF and CHTOP ( Figure 5E, Supplementary Figure S3, Supplementary Table S7). Other factors found by both analyses were the m 6 A reader and splicing factor YTHDC1 and its interacting partners SRSF9 and SRSF7, the transcription and splicing factor ERH, the ribosome biogenesis factors LAS1L, KRR1, TEX10 and MAK16, and the poly(A) binding and mRNA decay factor ZC3H14 (Supplementary Table S7). Supplementary Table S7.
Cooperation between ALKBH5 and TREX components, export adaptors and RNA-binding proteins can modulate mRNA export. This has been shown for two protein hits from ALKBH5 BioID, YTHDC1 and ZC3H14. YTHDC1 is a nuclear m 6 A reader that plays a dual role in the regulation of alternative splicing and mRNA export (106,107). As for ZC3H14, it binds TREX complex components and together ensures the export of a specific subset of mature and properly processed mRNAs in the mouse brain (108). We hypothesize that ALKBH5 could also be an auxiliary fac-tor of the export machinery. For instance, ALKBH5 could remove m 6 A marks to block YTHDC1 mRNA binding. Alternatively, ALKBH5 could play a scaffolding role independent of its demethylase activity. ALYREF was previously identified as a reader of m 5 C mRNA marks deposited by NSUN2 (109). NSUN2 depletion negatively affected ALYREF binding to m 5 C target sites and inhibited the export of NSUN2-modified mRNAs (109). Currently, the mechanism of ALKBH5 and ALYREF interaction in mRNA export is unclear. It is tempting to speculate that ALYREF coordinates the fate of mRNAs depending on its pattern of modifications. In this regard, the first evidence for functional crosstalk between m 5 C and m 6 A was demonstrated in the regulation of p21 expression (110). The 3' UTR of p21 mRNA bears both m 5 C and m 6 A dependent on NSUN2 and METTL3/14 activities, respectively (110). Silencing of either of these enzymes significantly reduces both modifications concomitantly, and overexpression of NSUN2 and METTL3/14 enhaces p21 protein levels (110). Whether p21 upregulation is due to enhanced mRNA export to the cytoplasm or increased translation was not yet experimentally addressed.
In summary, the interactome analyses revealed that ALKBH5 associates with (pre)mRNAs at multiple levels of their biogenesis ( Figure 5C) and strongly indicated the role of ALKBH5 in mRNA export. Furthermore, in the con-text of other works, it will be exciting to tackle the question of functional crosstalk between different mRNA modifications in gene expression regulation.
m 6 A and m 6 Am MTs and DMTs have distinctive protein contacts in vivo
To tackle whether the MTs and DMTs operate or are coregulated by common factors or pathways, we performed a comparative analysis of the protein networks identified in this study. We first investigated the overlap of enriched BP GO terms between the baits ( Figure 6A). The majority of the enriched BP GO terms (65%) were unique for each bait and 9.8% of the GO terms were shared by all four baits (Figure 6A, white borders). The list of 20 shared terms includes different aspects of (m)RNA processing and splicing (Supplementary Figure S4A). ALKBH5 hits showed the highest significance for these terms, reflecting the tighter connection of ALKBH5 with the mRNA processing machinery compared to the other protein baits. In fact, protein domains analysis uniquely identified a significant enrichment of the RNA recognition motif among ALKBH5 protein hits (Figure 5B, underlined proteins). The BP GO terms analysis between pairs of bait proteins ( Figure 6A, all dashed borders) revealed the highest overlap between PCIF1 and FTO with 22 shared terms mostly linked to transcription regulation ( Figure 6B, terms marked with a red line), which probably corresponds to their shared activity on m 6 Am marks. PCIF1, however, displayed the strongest link with transcription machinery of all the baits tested, revealing a unique enrichment for RNAPII binding, transcription regulation and histone modification (Supplementary Figure S4B, marked with a red line). The second highest overlap between pairs was found between ALKBH5 and METTL16 hits ( Figure 6A, all dashed borders) that share nine GO terms, primarily connected with ribonucleoprotein complexes and ncRNA processing ( Figure 6B, terms marked with a yellow line). This partial overlap could arise from the nucleolar localization of ALKBH5 and METTL16. Overall, these analyses indicated that mammalian MTs and DMTs possess rather distinctive protein networks, suggesting independent regulation of these enzymes in vivo.
Here, we presented a comprehensive analysis of the interactomes of the key RNA adenosine methylases and demethylases. Altogether, our data revealed that these enzymes share a limited number of interactors, pointing to specific molecular mechanisms of their regulation. PCIF1 protein network suggests that it binds nascent RNAs mostly at the transcription loci, whereas ALKBH5 is closely linked to most aspects of pre-mRNA processing and export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome points to the role of this DMT at several levels between RNA transcription, RNA processing and DNA replication and repair.
DATA AVAILABILITY
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (111) partner repository with the dataset identifier PXD021566.
Figure 1 .
1BioID experimental set-up and validation. (A) Schematic representation of the BirA*-fusion protein constructs. (B) Overview of the BioID experimental design: stable inducible 293T cell lines expressing the BirA*-fusion proteins and the control BirA* and NLS-BirA* were prepared. Fusion protein expression and in vivo biotinylation was allowed for 24 h, cell lysates were prepared and biotinylated proteins were isolated by streptavidin-affinity purification. Protein pull-downs were analyzed by LC-MS/MS. Raw MS/MS data were analyzed independently and in parallel by Limma and SAINT analyses. (C) Enrichment (log2 fold change of METTL3-BirA* relative to BirA* control) of the known METTL3 complex auxiliary components on METTL3 C-and N-terminal BirA* fusion proteins affinity purifications. All shown protein hits have adjusted P-values < 0.01. (D) Protein neighbours network of METTL3 identified by BioID. Shared hits from filtered Limma and SAINT data are depicted.
Figure 2 .
2METLL16 BioID reveals proteins involved in the biogenesis of RNAs transcribed by the three nuclear RNA polymerases. (A) Representative biological processes (BP) gene ontology (GO) terms significantly enriched among the protein hits identified by METTL16 BioID. (B) Protein hits components of RNP particles or involved in the processing and/or modification of RNAs transcribed by RNAPI, II and III. (C) Protein neighbors network of METTL16 identified by BioID. Shared hits from filtered Limma and SAINT data are depicted.
Figure 3 .
3PCIF1 interacts with factors involved in RNAPII transcription initiation and co-transcriptional snRNA biogenesis. (A) Representative biological processes (BP) gene ontology (GO) terms significantly enriched among the protein hits identified by PCIF1 BioID. (B) Protein neighbours network of PCIF1 identified by BioID. Shared hits from filtered Limma and SAINT data are depicted. Proteins binding RNAPII are marked with an asterisk. Interactions detected also by Strep II-tag pull-downs are connected by grey lines. (C) Strep II-tag pull-down of PCIF1 or eGFP fusion proteins from whole-cell extracts from 293T cells. Western blot analysis show efficient pull-down and interaction between PCIF1 and Ser5-phosphorylated RNAPII. FT: unbound fraction. (D) Volcano plot representing enrichment (log 2 fold change) versus significance (-log 10 adjusted P-value) of RNAse A resistant interactions identify in LC-MS/MS analysis of Strep II-tag PCIF1 relative to Strep II-tag eGFP control. Complete list of significant interactions is inSupplementary Table S7. (E) RNAPII CTD serine (Ser) phosphorylation pattern during transcription initiation, elongation and termination. Protein hits bound to a specific type of Ser phosphorylation repeat are depicted. The position of RPRD2 on the gene body is only speculative. ment to RNAPII CTD affects the binding of some other CTD interactors.
Figure 4 .
4FTO on the crossroad between RNAPII transcription, pre-mRNA processing and DNA repair pathways. (A) Representative biological processes (BP) gene ontology (GO) terms significantly enriched among the protein hits identified by FTO BioID. (B) Protein neighbours network of FTO identified by BioID. Shared hits from filtered Limma and SAINT data are depicted. (C) Functional states of the fully assembled spliceosome and protein hits identified in FTO BioID. (D) 293T wildtype (WT) and FTO KO cells were treated with 2 mM hydroxyurea (HU) for the indicated hours followed by immunoblotting with antibodies against the indicated proteins (left). Quantification of DNA damage marker ␥ -H2AX signal normalized to tubulin signal from two independent experiments (mean ± S.D.) (right). (E) 293T WT and FTO KO cells (left) and U2OS cells transfected with FTO or non-target (#) siRNAs (right) were treated with 1 M camptothecin (CPT) for the indicated hours followed by immunoblotting with antibodies against the indicated proteins.
5B,C; Supplementary Figure S3A) (100-102). ALKBH5 also interacted with the nuclear EJC auxiliary complex PSAP (SAP18, RNPS1 and Pinin) (103) (Figure 5B, C; Supplementary Figure S3A).
Figure 5 .
5ALKBH5 interacts with mRNA export and pre-mRNA processing factors. (A) Representative biological processes (BP) gene ontology (GO) terms significantly enriched among the protein hits identified by ALKBH5 BioID. (B) Protein neighbours network of ALKBH5 identified by BioID. Shared hits from filtered Limma and SAINT data are depicted. Proteins reported to localize in the nuclear speckles are marked with an asterisk (white, identified by (112); orange, Uniprot database). Proteins containing an RNA recognition motif domain are underlined in red. Interactions detected also in Strep II-tag pull-downs are connected by grey lines. (C) Schematic overview of mRNA co-and post-transcriptional processing (left side). Proteins involved in pre-mRNA processing steps enriched in ALKBH5 BioID are listed (right side). (D) Strep II-tag pull-down of ALKBH5 or eGFP fusion proteins from whole-cell extracts from 293T cells. Western blot analysis show efficient pull-down. FT: unbound fraction. (E) Volcano plot representing enrichment (log2 fold change) versus significance (-log 10 adjusted p-value) of RNAse A resistant interactions identify in LC-MS/MS analysis of Strep II-tag ALKBH5 relative to Strep II-tag GFP control. Proteins detected also in BioID analysis are labelled. Complete list of significant interactions in
Figure 6 .
6m 6 A and m 6 Am MTs and DMTs have distinctive protein contacts in vivo. (A) Venn diagram representing the overlap of enriched BP GO terms for the protein hits of the four studied protein baits. The intersection of the four baits is represented with white borders; three baits with white and dashed borders and two baits with all dashed borders. (B) Enriched BP GO terms for the protein hits shared by two protein baits. Red, shared by PCIF1 and FTO. Yellow, shared by METTL16 and ALKBH5.
ACKNOWLEDGEMENTSWe thank Leona Kledrowetzova, Romana Kaceriakova and Karolina Vavrouskova for excellent technical assistance. We thank Jose Ameijeiras-Alonso and Kristina Gomoryova for the help with R scripts.SUPPLEMENTARY DATASupplementary Data are available at NAR Online.
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) 5-methylcytosine promotes mRNA export -NSUN2 as the methyltransferase and ALYREF as an m5C reader. X Yang, Y Yang, B F Sun, Y S Chen, J W Xu, W Y Lai, A Li, X Wang, D P Bhattarai, W Xiao, Cell Res. 27Yang,X., Yang,Y., Sun,B.F., Chen,Y.S., Xu,J.W., Lai,W.Y., Li,A., Wang,X., Bhattarai,D.P., Xiao,W. et al. (2017) 5-methylcytosine promotes mRNA export -NSUN2 as the methyltransferase and ALYREF as an m5C reader. Cell Res., 27, 606-625.
NSUN2-mediated m5C methylation and METTL3/METTL14-mediated m6A methylation cooperatively enhance p21 translation. Q Li, X Li, H Tang, B Jiang, Y Dou, M Gorospe, W Wang, J. Cell. Biochem. 118Li,Q., Li,X., Tang,H., Jiang,B., Dou,Y., Gorospe,M. and Wang,W. (2017) NSUN2-mediated m5C methylation and METTL3/METTL14-mediated m6A methylation cooperatively enhance p21 translation. J. Cell. Biochem., 118, 2587-2598.
The PRIDE database and related tools and resources in 2019: improving support for quantification data. Y Perez-Riverol, A Csordas, J Bai, M Bernal-Llinares, S Hewapathirana, D J Kundu, A Inuganti, J Griss, G Mayer, M Eisenacher, Nucleic Acids Res. 47Perez-Riverol,Y., Csordas,A., Bai,J., Bernal-Llinares,M., Hewapathirana,S., Kundu,D.J., Inuganti,A., Griss,J., Mayer,G., Eisenacher,M. et al. (2019) The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res., 47, D442-D450.
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INTRODUCTION
Sarcomas are a large category of cancers that arise from mesenchymal cells, which can origin in almost any tissue, including bone, adipose, or muscle (1). For example, bone sarcomas are the most frequent primary solid malignancy of mesenchymal origin characterized by malignant spindle stromal cells that produce bone-like tissue (2). Sarcomas are morphologically heterogeneous, accurate diagnosis of which require an integrated strategy to consider and assess the clinical, molecular, and histologic characteristics of the malignancy (3). In addition, the malignant degree of sarcomas is high, most patients develop metastasis within one year, and the prognosis is rather poor (4). Understanding the molecular mechanisms of sarcoma development is urgent.
Methylation of N6 adenosine (m6A) is the most abundant internal chemical modification in eukaryotic mRNA (5). m6A modification was firstly discovered in 1970s and the detailed functional studies of m6A modification began 2012. Now, m6A modification becomes the most prevalent study of RNA modification that has received increasing attention. A large number of studies have suggested that aberrant m6A modification is the key to tumorigenesis and progression, such as breast cancer, lung cancer, acute myeloid leukemia and hepatocellular carcinoma (HCC) (6)(7)(8). The abundances and effects of m6A modification on RNAs are determined by the complex interactions between different types of regulators, including methyltransferases ('writers'), RNA binding proteins ('readers'), and demethylases ('erasers'). Understanding these different m6A regulators could dramatically increase our knowledge about the role of RNA methylation in the regulation of gene expression and various biological processes (9,10). Recently, lots of studies have demonstrated that m6A regulators were widely perturbed in various types of cancers (11,12). For example, a component of the m6A methyltransferase complex, methyltransferase-like 3 (METTL3), was reported to be associated with translation machinery and promote the translation of oncogenes (RGFR and TAZ) in human lung cancer (13). The overexpression of METTL3 was also observed in HCC and was associated with poorer survival (14). The overexpression of METTL14 was shown to increase the abundance of m6A methylation on primary miR-126, which suppresses metastasis in HCC and breast cancer (15). Aberrant expression of FTO (m6A eraser) was suggested to be favorable for the survival of diverse cancer cells. And the overexpression of FTO could contribute to the proliferation and invasiveness of gastric cancer, squamous cell, and breast cancer cell lines (16)(17)(18). A family of m6A reader proteins IGF2BP1-3 was reported to have oncogenic potential, which was frequently expressed and amplified in cervical or liver cancers (19). All these findings provide strong evidence that m6A regulators play crucial roles in the development and progression of cancers. However, though recent discoveries of the functions and mechanisms of m6A have clarified a new perspective of gene regulation at the RNA level, we still lack a vast amount of knowledge about the functions of m6A regulators in the development and progression of sarcomas.
Therefore, in this study, we emphatically discussed and analyzed the important roles of m6A regulators in sarcomas from a global perspective based on integrative analyses. Firstly, we integrated multi-omics data to analyze the genetic alterations, gene expression and epi-genomics regulation of the of 21 m6A regulators in sarcoma. Secondly, we illustrated the potentials of m6A regulators in tumor immunology, paving the way for the therapeutic strategies of sarcomas based on RNA methylation.
We also investigated the associations between the expression of m6A regulators and sarcoma patient survival and explored the clinical prognostic values of m6A regulators. Importantly, we constructed the regulatory networks for m6A regulators by integrating upstream and downstream regulatory information, including regulatory TFs and non-coding RNAs. And we performed knockdown experiments for YTHDF2 and HNRNPA2B1 to reveal the biological role in the cancer cell invasion and metastasis. Moreover, we also investigated the relationship between m6A regulators and immune cell infiltration in sarcoma patients. Our comprehensive analysis of m6A regulators would provide new insights into their function in the mechanism and development of sarcomas.
MATERIALS AND METHODS
Gene Expression and Genetic Alterations of Sarcoma
The TCGA sarcoma gene expression data and matched clinical data were downloaded from XENA browser (https:// xenabrowser.net/hub/). Based on the data processing pipeline to remove null data, sarcoma-related RNA-seq data containing 265 samples with clinical information were used for further study. TCGA sarcoma genetic alteration data were downloaded from cBioportal for Cancer Genomics. List of m6A regulators were downloaded from the previous study. All m6A regulators related gene expression data, clinical data and genetic alteration data were extracted from above data. The raw clinical data was provided in Supplementary Table S1.
Annotation for m6A Regulators in Sarcoma
In this study, we performed multiple annotation analyses for m6A regulators, such as gene expression comparison, gene correlation and crosstalk network. Gene expression comparison was performed to compare regulator expression in control and tumor groups in TCGA cohorts via gglopt2. Pearson correlation analysis was performed on TCGA expression data via Corrplot. Gene crosstalks were downloaded from String database (https://www.string-db.org/).
Survival Analysis
Hazards Ratio (HR) analysis of m6A regulators in sarcoma was analyzed from GEPIA2 database (http://gepia2.cancer-pku.cn/# survival) based on using Mantel-Cox test. For single gene survival analysis, patients were classified into high-Exp group and low-Exp groups based on mean expression. For multiple gene survival analysis, a risk score model was constructed. The risk score for each patient was computed by linear combination of the gene expression values weighted by the regression coefficient of univariate Cox regression analysis, which was defined as follows: Where, r i is the Cox regression coefficient of gene i in gene set, n is the number of genes in gene set and Exp (i) is the expression value of gene i in corresponding patient. The mean risk score was used to classify patients into high-risk and low-risk groups. Kaplan-Meier survival curve was performed for high-risk and low-risk groups of patients via survival R package. The statistical significance was assessed by log-rank test with a threshold of P < 0.05.
RiskScore = o n i=1 r i Exp i ð Þ
TF-m6A Regulator Regulatory Relationships
To identify upstream TFs of m6A regulators, we collected human enhancers from Fantom database and downloaded gene transcription start site (TSS) from UCSC database. For each regulator, we defined the Fantom enhancers located in 2000 bp~50000 bp far from of the TSS as the enhancers of the regulator. Promoters were defined as +/-2000 bp from regulator TSS. Find Individual Motif Occurrences (FIMO) software was used to scan TF motifs for each regulator's enhancer and promoter at the threshold of P value <1e-4. If a TF located in the enhancer or promoter of the m6A regulator, we considered that this TF could regulate the m6A regulator, which forms a TF-m6A regulator interaction. All TF-m6A regulator interactions were merged to TF-m6A regulator regulatory network and were showed in Cytoscape 3.6.
m6A Regulator-miRNA Regulatory Relationships m6A regulators were demonstrated to play important roles in miRNA maturation and miRNA expression. Thus, matched TCGA miRNA expression profile was downloaded from XENA database and Pearson correlation was conducted to investigate the potential regulatory relationships between miRNAs and m6A regulators. m6A regulator-miRNA network was constructed by merging all positive correlated regulator-miRNA pairs with Pearson correlation coefficients (PCC) >0.4. Pathway enrichment was performed by miEAA.
Immune Cell Infiltration of m6A Regulators in Sarcoma Patients
Infiltration estimation for all sarcoma patients were downloaded from TIMER2 database. The potential role of m6A regulators in cell infiltration was estimated by calculating the correlation between m6A regulator expression and infiltration estimation scores.
Cell Culture
Ewing sarcoma cell lines (A673, SKNMC, TC32, TC71,EW8, TCC446 and EWS502) were used in this study and were cultured in 25 cm 2 cell culture flask (
Wound Healing Assay and Colony Formation Assays
For wound healing assay, cells were cultured in 6-well plates, and the cell monolayer was wounded by sterile 100-mL pipette tips when cells reached approximately 90% confluence. Cells were then rinsed three times with D-Hanks to wipe off the detached cells and were incubated in RPMI 1640 containing 5% FBS for 48 h. For colony formation assay, cells were also cultured in 6well plates for 2-3 weeks. Resulting colonies were calculated following 1% crystal violet staining.
RESULT Genetic Alterations Overview of m6A Regulator
Here, we collected and analyzed 21 m6A regulators in this study, including 8 writers, 2 erasers and 11 readers. Firstly, we viewed the incidence of copy number variations and somatic mutations of the 21 regulators. Results showed that~44% sarcoma patients (117 samples of 265 samples) carried mutations of m6A regulators ( Figure 1A, top). Amplification is the most types of alterations, which could lead to the dysfunctional gene overexpression. It was found that ALKBH5 exhibited the highest mutation frequency of 13% and the patients with high amplification alteration also exhibited the high gene expression ( Figure 1A, bottom and Figure 1B). ELAVL1 also showed the high mutation frequency of 5%. Alterations of ELAVL1 could also determine the ELAVL1 RNA expression. Some reader genes of LRPPRC, YTHDC1 and HNRNPC exhibited low mutation levels. These results demonstrated that genetic alterations could affect the expression of m6A regulators. Genetic alterations were also considered as the risk factor of multiple cancers, these results also implied that m6A regulators might be the driven factors of cancers.
Annotations of m6A Regulators in Sarcoma
Then we viewed the RNA expression levels of m6A regulators in normal samples and tumor samples. Results showed that most of the regulators were high expressed in tumor samples, excepting IGF2P1 (Figure 2A). Particularly, m6A writers showed high upregulated expression in tumor samples, such as METTL3, RBM15, RBM15B and WTAP. To investigate the relationships of m6A regulators, we performed Pearson correlation analysis for m6A regulators based on gene expression, results showed that a high correlation existed among writers, erasers, and readers ( Figure 2B). IGF2BP1 showed low correlations to other regulators. The higher crosstalks were KIAA1429-YTHDF3 and METTL14-YTHDN1. These results implied that writers and readers were worked synergistically. Importantly, we also performed survival analysis for the 21 regulators by risk score model. Results revealed that the 21regulators model had strong prognosis effect in sarcoma ( Figure 2C). Additionally, these writers, erasers, and readers were interacted with each other and formed a close network in String protein-protein interactions ( Figure 2D). We also performed single gene survival analysis for 21 m6A regulators. We mapped all these regulators into GEPIA2 database and yielded the Hazard ratios (HR) of these regulators via Mantel-Cox test. We found that only 4 regulators with HR >1 ( Figure 3A). These results showed that these regulators were high risk factors in sarcoma. Overexpression of the 3 regulators (HNRNPC, HNRNPA2B1 and YTHDF2) could lead to a poor prognosis ( Figure 3B). Furthermore, we also have tested the protein expression of the 3 regulators in control osteoblast cell lines and 2 types of osteosarcoma cell lines, results showed that all these regulators were up-regulated in sarcoma model cells ( Figure 3C).
Upstream Regulation Analysis of m6A Regulators
Based on the above analysis, we found that the expression levels of m6A regulators were dys-regulated between control and tumors.
Thus, here we wanted to investigate the upstream regulators of these m6A regulators. Firstly, we collected all the DNA regulatory elements. Briefly, human enhancers were defined as the DNA regions of 2000bp~50000bp far from of the TSS. Promoters were defined as the regions of +/-2000bp from TSS. According to the results of motif scanning, we found that promoters were occupied more TF binding sites than enhancer (Figures 4A, B). YTHDC1, ELAVL1 and HNRNPA2B1 promoter regions occupied more TF binding sites that other genes. And SP family genes, such as SP1, SP2 and SP4 were the broad TFs for m6A regulators ( Figure 4A). In enhancer perspective, results showed that binding affinity matrix was sparse ( Figure 4B). Only the regulator of IGF2BP1 enhancer occupied more TFs. However, most of upstream TFs showed a negative correlation trend to IGF2BP1, which might explain that the expression of IGF2BP1 was opposite to other m6A regulators. To further uncover the regulatory mechanism of TFs on m6A regulators, we then merged all the TF-m6A regulator pairs (including promoter perspective and enhancer perspective) into a network ( Figure 4C). In this network, we found that IGF2BP1 was the biggest degree node. Some TFs, such as SP1, EGR1 and ZNF263 were the common TFs of multiple m6A regulators. Furthermore, we found that some TF-m6A regulator pairs were both regulated occurred in enhancer and promoter perspective ( Figure 4D). The TFs of SP1 and SP4 were all demonstrated to participate in oncogenesis processes. For example, Aydemir et al. found that SP1 suppressed ADAMTS3 transcriptional activity. SP1 increased type II and III collagen expression and decreased type I collagen expression levels in Saos-2 cells. They provided the first findings for the SP1-related transcriptional regulation of ADAMTS3 and collagen genes in osteosarcoma cell lines (20). SP1 was also demonstrated to regulate lncRNA LMCD1-AS1 and lncRNA ILF3-AS1 to facilitate osteosarcoma progression (21,22). Inhibition of SP family (SP1, SP3 and SP4) could suppress rhabdomyosarcoma cell and tumor growth via non-steroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA) (23). Additionally, we also performed survival analysis for these common pairs. Results showed that all these single genes were not strong prognosis biomarkers ( Figure 4D). However, combining all these genes as a single risk factor could be used as prognosis marker, which suggested the TF-m6A regulator crosstalks had the strong clinical prognostic value.
Downstream Regulation Analysis of m6A Regulators
Previous studies found that m6A regulators were the key players in miRNA processing and maturation, such as METTL3 and HNRNPA2B1 (24,25). Thus, in this study, we investigated the potential regulatory axes between m6A regulators and miRNAs. We calculated all Pearson correlations between miRNAs and m6A regulators ( Figure 5A). Results showed that the reader regulators, such as YTHDF2, HNRNPA2B1, YTHDF1, IGF2BP1 and HNRNPC, were high correlated with multiple miRNAs. These results were coincided with the biological function of m6A readers in RNA processing. We extracted the m6A regulator-miRNA pairs by filtering the pairs at PCC >0.4 ( Figure 5B). We found that HNRNPA2B1 and YTHDF2 were the high-degree nodes in network. Some known pairs, such as HNRNPA2B1-miR-106b, HNRNPA2B1-miR-17 and HNRNPA2B1-miR-93 were identified from this study in sarcoma (24) [5]. We also performed miRNA function enrichment by miEAA. Results showed that multiple cancer-driven pathways were enriched, such as "Ferroptosis", "VEGF signaling" and so on (26,27) ( Figure 5C). Survival analysis revealed that single miRNA could not be used as prognostic marker ( Figure 5D). However, we then integrated m6A regulator-miRNA pair as risk score models to test the prognosis effects of these pairs, results showed that some m6A regulator-miRNA pairs had strong prognostic effects, such a s Y T H D F 2 -m i R -1 0 6 b -5 p a n d Y T H D F 2 -m i R -1 8 6 -5p ( Figure 5E). Importantly, to validate the biological function of m6A regulators in sarcoma, we performed loss of function experiments for two regulators, YTHDF2 and HNRNPA2B1 in osteosarcoma cell line. Results showed that the two regulators were inhibited by siRNA (Figures 6A, E). Inhibition of m6A regulators can affect the downstream miRNAs expression, such as miR-17 and miR-19 families (Figures 6B, F), which were considered as the core downstream miRNAs in Figure 5B. Furthermore, inhibition of m6A regulators expression can lead the tumor cell invasion and metastasis (Figures 6C, D, G, H).
Immune Cell Infiltration of m6A Regulators in Sarcoma
Previous studies found that immune cell level determined the proliferation of cancer cells. In this study, we also investigated the association between m6A regulators and immune cell levels by calculating PCC from TIMER2 data. Results showed that most of m6A regulators were negatively correlated with immune cells ( Figure 7A). Specifically, WTAP exhibited most positive correlation with all immune cells ( Figure 7C). RBM15B showed most negative correlation with all immune cells ( Figure 7D). Additionally, immune cell levels could have significant impact on clinical survival ( Figure 7B). All these results suggested that m6A regulator might regulate cancer progression via controlling immune cell levels in sarcoma patients.
DISCUSSION
Sarcomas are rare malignant tumors that may arise from anywhere of the body, such as bone, adipose, muscle and vascular. However, the conventional pathogenesis of sarcomas has not been found. Therefore, there is an urgent need to identify novel therapeutic strategies and improve prognosis effects for sarcomas. m6A regulation is a novel proposed regulatory mechanism, which was also the most widely (30). Furthermore, m6A regulators, such as RBM15, METTL14 were also demonstrated to act as prognosis markers in multiple cancers, such as pancreatic cancer and hepatocellular carcinoma.
Here, we integrated multi-omics data including genetic alterations, gene expression and epigenomics regulation to systematically analysis the regulatory atlas of 21 m6A regulators in sarcoma. Firstly, we investigated the genetic alterations of m6A regulators and found that~44% TCGA sarcoma patients have genetic mutations. We also investigated the basic annotation of 21 regulators, such as expression correlation and PPI interactions. Then we identified the upstream and downstream regulatory axes of m6A regulators in sarcoma based on motif analysis and mRNA-miRNA expression. These results implied that m6A regulator mediated regulatory axes could be used as prognostic biomarkers. Moreover, we also demonstrated that the expression level of m6A regulators were high related to immune cell infiltration of sarcoma patients. Importantly, we found the expression level of m6A regulators were dys-regulated in sarcoma (Figure 2A). Thus, we wanted to investigate the potential mechanism of m6A regulators. One the one hand, genetic alterations could affect gene expression (Figure 1). On the other hand, epigenetics regulation also determined the gene expression level. We collected the distal enhancer and proximal promoters of all 21 m6A regulators and performed motif scanning to find the TF-m6A regulator crosstalks. As a result, some TFs, such as SP TF families (SP1, SP2 and SP4) were extracted as the key regulators for most of m6A regulators. We also found that promoters occupied more TFs than enhancers and m6A readers were more regulated than others, such as ELAVL1, YTHDC1 and WTAP. Additionally, some TF-m6A regulator crosstalks were both occurred in promoter and enhance perspectives, such as SP1-IGF2BP1 and SP4-ELAVL1, which also exhibited strong prognostic effects than single genes. Recent studies found that m6A regulators were participated in miRNA maturation and processing (31,32). In this regulatory relationship, m6A regulators showed a positive correlation with miRNAs. Thus, we calculated the PCC between miRNAs and m6A regulators. Interestingly, some known regulatory relationships were also identified in sarcoma, such as HNRNPA2B1-miR-106b, HNRNPA2B1-miR-17 and HNRNPA2B1-miR-93. m6A readers were found to positively correlate with most of miRNAs, such as HNRNPA2B1, YTHDF2, YTHDF1, IGH2BP1 and HNRNPC. Notably, m6A regulator-miRNA pairs also showed high prognostic effects. In addition, we also investigated the potential role of m6A regulators in cancer immunology. Results showed that m6A regulators might participate in cancer cell survival and cancer progression by regulating immune cell levels in sarcoma.
In summary, we systematically investigated the regulatory roles of m6A regulators in sarcoma in multi-perspectives and found the potential clinical values of m6A regulators. However, our study also exits limitations. Up to now, the massive RNA modification methylome data of sarcoma was absent. We will integrate the methylation, transcription data to analysis in the future. Furthermore, here we only used the enhancer dataset from Fantom5, which was a common enhancer of human. This is also the limitation of our current study. We will collect more
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
FIGURE 1 |
1Landscape of genetic and expression variation of m6A regulators in TCGA sarcoma. (A) The mutation frequency of 21 m6A regulators in 265 patients with sarcoma in TCGA. Each column represented individual patients. The number on the right indicated the mutation frequency in each regulator. The lower heatmap represents m6A regulators' expression in sarcoma. Three types of regulators were labeled in different colors. (B) The impacts of different genome alterations on gene expression of ALKBH5 and ELAVL1. P<0.01 represents the expression levels were significantly changed in alteration group vs. diploid group.
FIGURE 2 |
2Expression changes and correlations of m6A regulators in sarcoma. (A) The boxplots of expression changes of m6A regulators between controls and tumors. P<0.01 represents the expression levels were significantly changed in tumor groups vs. control groups. (B) Pearson correlations of m6A regulators in sarcoma. Star-labeled nodes represent the higher crosstalks: KIAA1429-YTHDF3 and METTL14-YTHDN1. (C) A Kaplan-Meier survival curve of m6A regulators (risk score model) in sarcoma (P=0.032). (D) PPI interactions of m6A regulators in String database.
FIGURE 3 |
3Prognostic effects of individual m6A regulator in sarcoma. (A) The hazard ratios of m6A regulators in sarcoma. Red marked regulators represent statistically significant risk factors (HR>1). (B) The Kaplan-Meier survival curve of the 3 regulators with high hazard ratios. Low_Exp group and High_Exp group were divided by mean expression. (C) Expression of the 3 regulators in control and model osteosarcoma cell lines. a-Tubulin was used as the reference.
FIGURE 4 |
4Identification of TF-m6A regulator crosstalks in sarcoma. (A) TF motif searching of promoter regions of m6A regulators. Node color represents the PCCs between TFs and m6A regulators. Node size represents the number of TFs that bind to the promoter regions of m6A regulators. (B) TF motif searching of enhancer regions of m6A regulators. Node color represents the correlation score of PCC. Node size represents the number of TFs that bind to the enhancer regions of m6A regulators. (C) Visualization of a TF-m6A regulator crosstalk network. Blue diamond nodes represent m6A regulators and orange circular nodes represent TFs. Green lines represent TFs binding to the enhancer regions of m6A regulators. Pink lines represent TFs binding to the promoter regions of m6A regulators. (D) Upper is the TF-m6A regulator crosstalks that were both regulated via enhancer and promoter. Lower left is the survival p-values of individual genes and combined signature in sarcoma. Lower right is the Kaplan-Meier survival curves of combined signature.
distributed methylation modification in eukaryotic mRNA. Growing evidence have demonstrated that m6A modification played an indispensable role in tumorigenesis. In the field of sarcoma, Zhou et al. demonstrated that knockdown of METTL3 could inhibit the proliferation and invasion of osteosarcoma by regulating ATAD2 (28). Wang et al. found that m6A played a role in the emergence and maintaining of osteosarcoma stem cells and affect the prognosis (29). Miao et al. demonstrated METTL3 promoted osteosarcoma progression by regulating the m6A level of LEF1
FIGURE 5 |
5Identification of m6A regulator-miRNA pairs in sarcoma. (A) The Pearson correlation heatmap of miRNAs and m6A regulators. (B) High-correlated m6A regulator-miRNA pairs. Green circular nodes represent m6A regulators and pink triangle nodes represent miRNAs. (C) Pathway enrichment analysis of miRNAs in network 5B by miEAA. Pathways were ranked based on -log10 (p-value). (D) Survival p-values of individual genes (including single miRNA and single m6A regulator) and combined signature (risk score model) in sarcoma. (E) The Kaplan-Meier survival curves of strong m6A regulator-miRNA pairs.
FIGURE 6 |
6Loss of function experiments of m6A regulators in osteosarcoma cells line. (A) Expression of YTHDF2 in knockdown groups via western blot. Here we used three candidate siRNAs to target YTHDF2. (B) Expression of YTHDF2 and target miRNAs in knockdown groups via Real-time PCR. * represents p<0.05 vs. NC group, N=6. (C) Wound healing experiment results of YTHDF2 knockdown. Here we used siRNA#1 and siRNA#3. (D) Transwell and clone formation experiments results of YTHDF2 knockdown. Here we used siRNA#1 and siRNA#3. (E-H) Expression, wound healing, transwell and clone formation experiments of HNRNPA2B1 knockdown.
FIGURE 7 |
7Immune cell infiltration of m6A regulators in sarcoma patients. (A) The visualization of correlations between m6A regulator expression and TIMER2 immune cell estimation score. (B) The Kaplan-Meier survival curves of between CD4 T cell-enriched patients and other patients. (C) Scatter plots of correlations between m6A regulator expression and TIMER2 immune cell estimation score (positive correlation). (D) Scatter plots of correlations between m6A regulator expression and TIMER2 immune cell estimation score (negative correlation).
Total protein was extracted from cell lines and lysed via RIPA buffer. Degenerated protein concentration was measured by bichinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). In each experiment, 20 mg protein samples were separated in 10% or 15% SDS-PAGE gel and transferred onto nitrocellulose membrane. After 5% non-fat milk blocking, theCorning, NYC, USA) with
Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen,
Waltham, USA) containing 15% fetal bovine serum
(Invitrogen, Waltham, USA) at 37°C, 5% CO 2 environment. x-
treme GENE siRNA (Invitrogen, Carlsbad, USA) were used for
gene knockdown experiments for 24 h. siRNA sequences were
provided in Supplementary Table S2.
Quantitative Real-Time RT-PCR
Total RNA was extracted from cell lines using Trizol reagent
(Invitrogen, Waltham, USA) according to manufacturer's
protocols. cDNA was synthesized by reverse transcription
reagent kit (TAKARA, RR037A, Shiga, JAPAN). Gene
expression was quantified by SYBR Green PCR Master Mix,
and detected using Roche 480 systems. U6 or GAPDH was
served as an internal control for miRNAs and mRNAs,
respectively. 2 -DDCt relative quantification method was used to
show gene expression. Primers are listed in Supplementary
Table S3.
Western Blotting
blots were incubated with primary antibodies including YTHDF2
(1:1000 dilution, #71283, Cell signaling), HNRNPA2B1 (1:2000
dilution, #9304, Cell signaling), HNRNPC (1:1000 dilution,
HPA051075, Sigma) and internal control a-Tubulin (1:2000
dilution, #3873, Cell signaling).
Pang et al. m6A Regulators in Sarcoma Frontiers in Oncology | www.frontiersin.org July 2022 | Volume 12 | Article 911596 enhancer datasets, such as Enhancer Atlas, to validate these results.
Frontiers in Oncology | www.frontiersin.org July 2022 | Volume 12 | Article 911596
AUTHOR CONTRIBUTIONSYH designed this project, BP, DL, BC, WW, and LW processed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.ACKNOWLEDGMENTSWe thanks TCGA database for data supporting. Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.SUPPLEMENTARY MATERIAL
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Introduction
Oesophageal squamous cell carcinoma (ESCC) is a major type of oesophageal cancer and is one of the most common types of cancer, which causes a large number of cancer-related deaths worldwide (1)(2)(3). In recent decades, considerable attention has been paid to treatment innovation through surgical resection in combination with chemo-or radiotherapy; however, the overall survival time of patients with ESCC remains poor due to the high recurrence and metastasis rates (4,5). Thus, it is of great importance to explore the regulatory mechanisms underlying ESCC progression to guide the development of novel diagnostic and therapeutic strategies for ESCC (6)(7)(8)(9).
N6-methyladenosine (m6A) is the most abundant type of methylation, and it serves a crucial role in RNA stability, localization, splicing and translation (10,11). The target genes of m6A are associated with cellular proliferation, organization and transport, as well as cancer-related signalling pathways (10). Methyltransferase-like 3 (METTL3) is the 70-kDa subunit of MT-A, which is part of N6-adenosine-methyltransferase; N6-adenosine-methyltransferase has been implicated in the post-transcriptional methylation of internal adenosine residues in eukaryotic mRNAs, and thus forms m6A (12)(13)(14). It has been widely reported that METTL3 is frequently increased and has an oncogenic role in common types of human cancer, including gastric (15), lung (16), bladder (17), ovarian (18), pancreatic cancer (19), melanoma (20) and breast cancer (21). For example, METTL3 is overexpressed in breast cancer and promotes breast cancer progression through inhibiting the tumour suppressor, let-7g (21). Taketo et al reported that METTL3 promoted chemoresistance and radioresistance in pancreatic cancer cells (19). In contrast, a recent study demonstrated that METTL3 functioned as a tumour suppressor in renal cell carcinoma; METTL3 inhibited renal cell carcinoma proliferation, cell cycle progression, migration and invasion (22). However, to the best of our knowledge, no previous study has focused on the expression pattern and function of METTL3 in ESCC. In the present study, the mRNA and protein expression levels of METTL3 in ESCC were investigated and the clinical significance of METTL3 expression in ESCC was explored. Subsequently, the function of METTL3 in regulating the malignant phenotypes of ESCC cell lines in vitro, and the molecular mechanism behind METTL3 in ESCC were investigated. The findings of this study may provide novel therapeutic strategies for the treatment of ESCC.
Materials and methods
Patient studies. A total of 53 ESCC and paired adjacent tissues were collected from 53 patients with ESCC who underwent resection at The Second Xiangya Hospital of Central South University, China between March 2012 and May 2013. These patients included 30 men and 23 women between 42 and 78 years old. The inclusion criterion was that all patients exhibited primary ESCC, and the exclusion criterion was patients that had received chemotherapy or radiotherapy before surgery. The tissues were stored at -80˚C until required for further experimentation. The follow-up time was 5 years following surgery.
Cell culture and reagents. Normal oesophageal epithelial cell line (HET-1A) and four ESCC cell lines (TE-9, Eca-109, KYSE150 and EC9706) were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and incubated in a humidified atmosphere at 37˚C and 5% CO 2 .
Cell transfection. Lipofectamine ® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cell transfection, according to the manufacturer's protocol. TE-9 and Eca-109 cells (5x10 5 cells/ml) in the logarithmic growth phase were transfected with 100 nM negative control (NC) small interfering RNA (siRNA) or 100 nM METTL3-specific siRNAs (Shanghai GenePharma Co., Ltd.) at 37˚C. Following transfection for 48 h, METTL3 expression levels were evaluated using reverse transcription-quantitative PCR (RT-qPCR).
RT-qPCR. Total RNA was extracted using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reversed transcribed into cDNA using a High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.). The RT conditions were 16˚C for 30 min, followed by 42˚C for 30 min and 85˚C for 5 min. qPCR was subsequently performed using the All-in-One qPCR mix (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: 95˚C for 3 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 30 sec. The following primer pairs were used for the qPCR: METTL3, forward 5'-TTG TCT CCA ACC TTC CGT AGT-3', reverse 5'-CCA GAT CAG AGA GGT GGT GTA G-3'; and GAPDH, forward 5'-CTG GGC TAC ACT GAG CAC C-3' and reverse 5'-AAGTGGTCGTTGAGGGCAATG-3'. Relative METTL3 mRNA expression levels were quantified using the 2 -ΔΔCq method (23) and normalized to the internal reference gene GAPDH.
Western blotting. Total protein was extracted using RIPA buffer (Thermo Fisher Scientific, Inc.). Protein concentrations were determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Proteins (50 µg/lane) were separated via SDS-PAGE on a 12% gel and the separated proteins were subsequently transferred onto a PVDF membrane (Roche Diagnostics) and blocked overnight at 4˚C with 5% milk. The membranes were incubated with primary antibodies against METTL3 (1:500; cat. no. ab66660; Abcam), Bax (1:250; cat. no. ab182733; Abcam), caspase-3 (1:300; cat. no. ab13847; Abcam), Bcl-2 (1:300; cat. no. ab32124; Abcam), PI3K (1:500; cat. no. ab40755; Abcam), phosphorylated (p)-PI3K (1:200; cat. no. ab182651; Abcam), AKT (1:500; cat. no. ab8805; Abcam), p-AKT (1:500; cat. no. ab8933; Abcam) and GAPDH (1:500; cat. no. ab9485; Abcam) for 4 h at room temperature. Following the primary antibody incubation, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no. ab6721; Abcam) for 1 h at room temperature. Protein bands were visualized using Pierce ECL Western Blotting substrate (Thermo Fisher Scientific, Inc.) and protein expression was semi-quantified using ImageJ software (version 1.46; National Institutes of Health) with GAPDH as the loading control.
Cell viability assay. A total of 5x10 3 cells/well of transfected TE-9 and Eca-109 cells were seeded into 96-well plates and cultured in a humidified incubator containing 5% CO 2 for 0, 24, 48 or 72 h. CCK-8 solution (10 µl; Thermo Fisher Scientific, Inc.) was added to each well, and after incubation at 37˚C for 2 h, the absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.).
Colony formation assay. A total of 1x10 3 transfected TE-9 and Eca-109 cells/well were seeded into 6-well plates and cultured in an incubator containing 5% CO 2 at 37˚C for 14 days. Subsequently, cells were fixed with 75% ethanol at room temperature for 1 h and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 5 min. The colonies were visualised and counted using a light microscope (magnification, x10).
Flow cytometric analysis of apoptosis. Transfected TE-9 and Eca-109 cells (1x10 6 cells/ml) were fixed in 75% ethanol at 4˚C for 3 h and subsequently washed with PBS three times. The cells were stained with Annexin V-FITC and propidium iodide using the Annexin V-FITC Apoptosis Detection kit I (BD Biosciences), according to the manufacturer's protocol. Apoptotic cells were subsequently analysed using a FACScan flow cytometer (BD Biosciences) and BD Accuri™ C6 software (version 1.0; BD Biosciences).
Would healing assay. Transfected TE-9 and Eca-109 cells were plated at a density of 5x10 5 cells/well were seeded in 12-well plates and cultured to ≥95% confluence. A 200-µl sterile pipette tip was used to generate the wounds. The cells were washed with Dulbecco's PBS and DMEM (Gibco; Thermo Fisher Scientific, Inc.) was subsequently added. The cells were visualized using a light microscope (magnification, x40) at 0 h and after 24 h incubation at 37˚C. The width of the wounds at 0 and 24 h were determined using ImageJ software (version 1.5; National Institutes of Health).
Matrigel invasion assay. Cell invasion was determined using Matrigel-coated Transwell chambers with an 8-µm pore size membrane (BD Biosciences). In brief, transfected TE-9 and Eca-109 cells (1x10 5 cells/well) and 300 µl serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) were plated in the upper chamber. DMEM (500 ml) supplemented with 10% FBS was plated in the lower chamber. After 24 h at 37˚C, the invading cells on the lower surface of the chamber were stained with 0.1% crystal violet at room temperature (Thermo Fisher Scientific, Inc.) for 5 min and subsequently washed with PBS (Thermo Fisher Scientific, Inc.). Stained cells were counted using a light microscope (magnification, x200).
Statistical analysis. All experiments were repeated at least three times. All data are expressed as the mean ± SD. Statistical analysis was performed using SPSS 19.0 software (IBM Corp.). Differences between groups were determined using Student's t-test or one-way ANOVA followed by Tukey's post hoc test. A χ 2 test was used to analyse the clinical significance of METTL3 expression in ESCC, and Kaplan-Meier analysis and log-rank test were applied for survival analysis. P<0.05 was considered to indicate a statistically significant difference.
Results
Overexpression of METTL3 promotes ESCC progression. The expression levels of METTL3 were determined using RT-qPCR and western blotting in ESCC and adjacent non-tumour tissues. The mRNA expression levels of METTL3 were significantly increased in ESCC tissues compared with adjacent non-tumour tissues (Fig. 1A). Similar results were obtained by western blotting; with METTL3 protein levels increased in ESCC tissues (Fig. 1B). Consistent with the clinical data, the METTL3 mRNA and protein expression levels were significantly higher in ESCC cell lines compared with the normal oesophageal cell line HET-1A (Fig. 1C and D). To study the clinical significance of METTL3 expression in ESCC, the patients with ESCC were divided into high and low METTL3 expression groups based on the median mRNA expression value (2.58) of METTL3. A χ 2 test reported that high METTL3 expression was significantly associated with advanced clinical stage and metastasis of ESCC (Table I).
Kaplan-Meier analysis indicated that patient survival was worse in those patients with high METTL3 levels compared with patients with low METTL3 levels (Fig. 1E). These findings suggested that the upregulation of METTL3 promoted tumour progression and predicted a poor prognosis in ESCC.
Silencing METTL3 gene expression inhibits ESCC cell viability and colony formation, while inducing cellular apoptosis. The role of METTL3 in regulating the malignant phenotypes of ESCC cells was studied. TE-9 and Eca-109 cells were transfected with two METTL3 siRNAs to knockdown the expression of METTL3. Transfection with METTL3 siRNAs significantly decreased METTL3 mRNA and protein expression levels compared with the cells transfected with NC siRNA in both cell lines ( Fig. 2A and B). METTL3 siRNA1 demonstrated a more potent suppressive effect over mRNA and protein expression and was thus selected as the siRNA of choice for further experiments. CCK-8 assays indicated that gene silencing of METTL3 expression significantly suppressed the viability of TE-9 and Eca-109 cells at 72 h compared with NC siRNA-transfected cells (Fig. 2C and D). In addition, the number of colonies formed of TE-9 and Eca-109 cells was significantly reduced in the presence of METTL3 siRNA compared with NC siRNA (Fig. 2E and F).
It was hypothesized that apoptosis may be involved in METTL3-mediated ESCC cell viability. Therefore, the effects of METTL3 inhibition on ESCC cell apoptosis were examined. The apoptotic rate of ESCC cells was significantly enhanced following METTL3 knockdown with siRNA compared with the NC siRNA transfected cells in both cell lines ( Fig. 3A and B), indicating that silencing METTL3 gene expression induced ESCC cell apoptosis. In addition, silencing METTL3 gene expression significantly increased the protein levels of pro-apoptotic Bax and caspase-3 and decreased those of anti-apoptotic Bcl-2 in TE-9 and Eca-109 cells compared with the controls (Fig. 3C and D).
METTL3 knockdown with siRNA suppresses ESCC cellular migration and invasion.
To further study the function of METTL3 in ESCC metastasis, wound healing and Matrigel Transwell assays were used to assess cellular migration and invasion following METTL3 knockdown with siRNA. As shown in Fig. 4A and B, the migratory capacity of TE-9 and Eca-109 cells were significantly inhibited following siRNA METTL3 knockdown compared with the NC siRNA-transfected cells in both cell lines. Similarly, following knockdown of METTL3 expression by siRNA, the invasion of TE-9 and Eca-109 cells was also significantly repressed ( Fig. 4C and D). These findings suggested that the genetic knockdown of METTL3 with siRNA may inhibit ESCC metastasis.
METTL3 inhibition decreases PI3K/AKT signalling pathway activity. The effects of METTL3 knockdown with siRNA on the activity of the PI3K/AKT signalling pathway in ESCC cells was investigated; the PI3K/AKT signalling pathway Table I. Association between METTL3 expression and clinicopathological features in patients with oesophageal squamous cell carcinoma.
METTL3 expression ---------------------------------------------------------------------Variables
Cases Low (n=28) High (n=25) serves a crucial role in cancer cell growth and metastasis (22). In Fig. 5, the quantitative analysis refers to the ratio between total and phosphorylated protein levels. Our data indicated that the phosphorylated protein levels of PI3K and AKT were significantly reduced following transfection with METTL3 siRNA compared with NC siRNA in TE-9 and Eca-109 cells, indicating that METTL3 inhibition decreased the PI3K/AKT signalling pathway activity ( Fig. 5A and B).
Discussion
The expression pattern and function of METTL3 in ESCC has previously not been reported. The present study demonstrated that METTL3 was significantly upregulated in ESCC, which was associated with ESCC progression and poorer prognoses, and that the genetic knockdown of METTL3 with siRNA inhibited ESCC cell viability, colony formation, cellular migration and invasion, induced apoptosis, and decreased PI3K/AKT signalling. The upregulation of, and the oncogenic function of METTL3 has been reported in various types of human cancer (16)(17)(18). For example, METTL3 is upregulated in bladder cancer tissues, and promotes bladder cancer progression via regulation of AF4/FR2 family member 4/NF-κB/MYC signaling (17). METTL3 is upregulated in ovarian carcinoma; it promotes the malignant phenotype of ovarian cancer cells and increases tumour formation in nude mice (18). Regarding the underlying molecular mechanism, a previous study reported that METTL3 promoted mRNA translation of multiple oncogenes, such as the epidermal growth factor receptor and the Hippo pathway effector TAZ, through recruiting translation initiation factors in human cancer cells (11). In addition, Choe et al (24) identified a direct interaction between METTL3 and eukaryotic translation initiation factor 3 subunit H and revealed that this interaction served a crucial role in translation, densely packed polyribosome formation and oncogenic transformation. However, the exact role of METTL3 in ESCC remains to be elucidated. In the present study, it was demonstrated that the mRNA and protein expression levels of METTL3 were significantly increased in tumour tissues and cell lines compared with adjacent normal tissues and the normal oesophageal epithelial cell line, HET-1A. It was further observed that METTL3 upregulation was associated with advanced TNM stage, metastasis and poorer prognoses in ESCC. These findings suggested that upregulation of METTL3 may contribute to the malignant progression of ESCC, and that METTL3 expression may be used as a predictor for worse outcomes of patients with ESCC.
To further clarify the exact role of METTL3 in ESCC, TE-9 and Eca-109 cell lines were selected for further in vitro experiments owing to their high METTL3 expression levels. As METTL3 is upregulated in ESCC cells, TE-9 and Eca-109 cells were transfected with METTL3-specific siRNAs to knockdown its expression. Downregulating METTL3 significantly inhibited ESCC cell viability and colony formation. Similarly, Lin et al (15) reported that METTL3 promoted the proliferation of gastric cancer cells. In addition, knockdown of METTL3 drastically reduced bladder cancer cell proliferation and survival in vitro and tumorigenicity in vivo (17). Overexpression of METTL3 significantly promoted ovarian cancer cell proliferation in vitro as well as tumor formation in nude mice (18). Therefore, the present findings suggested that METTL3 plays a promoting role in ESCC growth. As reduced cell viability may be due to increased cellular apoptosis, the effects of METTL3 downregulation on ESCC apoptosis were studied. The data demonstrated that apoptosis of TE-9 and Eca-109 cells was significantly higher following METTL3 gene knockdown. These findings suggested that METTL3 may inhibit cell apoptosis. Similarly, knockdown of METTL3 also induced breast cancer cell apoptosis (21). To further confirm this, the expression levels of several key factors associated with cell apoptosis, including pro-apoptotic Bax and caspase-3 and anti-apoptotic Bcl2 (25,26) were evaluated. METTL3 inhibition upregulated Bax and caspase-3, and downregulated Bcl-2 in ESCC cells, consistent with the apoptosis assay results. Cancer cell migration and invasion are two key processes of tumour metastasis (27,28); since METTL3 upregulation was associated with ESCC progression in the clinical samples, the function of METTL3 in regulating ESCC cell migration and invasion was also studied in vitro. Gene silencing of METTL3 significantly inhibited the migratory and invasive ability of ESCC cells, suggesting that METTL3 had a promoting role in ESCC migration and invasion in vitro.
It has been well established that the PI3K/AKT signalling pathway serves a crucial role in most aspects of tumour growth and metastasis, and the inactivation of this signalling pathway could effectively inhibit the malignant phenotypes of ESCC cells (29,30). Wang et al (29) reported that the long non-coding RNA, growth-arrest specific 5, suppressed ESCC cell viability and migration through inactivation of the PI3K/AKT signalling pathway. He et al (31) demonstrated that circRNA VRK serine/threonine kinase 1 inhibited ESCC progression and radioresistance through regulating the expression of microRNA-624-3p, in addition to the activity of the PI3K/AKT signalling pathway. The present study reported that METTL3 knockdown significantly reduced the phosphorylation levels of PI3K and AKT, indicating that the PI3K/AKT signalling pathway was inactivated. Similarly, knockdown of METTL3 inhibited the expression and phosphorylation of proteins involved in the PI3K signaling pathway in lung cancer cells (16). These findings suggested that the PI3K/AKT signalling pathway may serve an important role in METTL3-mediated effects in ESCC cells. One limitation of this study is that the clinical sample number was small and more patients should be included in future studies. Moreover, the relationship between METTL3 and AKT should be studied using clinical samples in future studies.
In conclusion, it was demonstrated that upregulation of METTL3 promoted ESCC progression and that inhibition of METTL3 significantly suppressed the malignant phenotype of ESCC cells, at least in part, through downregulating PI3K/AKT signalling. This study may help elucidate the molecular mechanism underlying ESCC progression.
Figure 1 .
1Upregulation of METTL3 promotes ESCC progression. (A and B) mRNA and protein expression levels of METTL3 in ESCC tissues and adjacent tissues, as assessed by reverse transcription-quantitative PCR and western blotting, respectively. ** P<0.01 vs. adjacent tissues. (C and D) mRNA and protein expression levels of METTL3 in ESCC cell lines and the normal cell line HET-1A, as assessed by reverse transcription-quantitative PCR and western blotting, respectively. ** P<0.01 vs. HET-1A cells. (E) Kaplan-Meier analysis of overall survival time of patients with ESCC with high METTL3 expression compared with that of patients with ESCC with low METTL3 expression. METTL3, methyltransferase-like 3; ESCC, oesophageal squamous cell carcinoma.
Figure 2 .
2METTL3 gene knockdown with siRNA decreases ESCC cell viability and colony formation ability. TE-9 and Eca-109 cell lines were transfected with two METTL3 siRNAs or NC siRNA. (A and B) mRNA and protein expression levels of METTL3 were examined following cell transfection with siRNAs against METTL3 compared to NC siRNA in TE-9 and Eca-109 cell lines. (C and D) Cell viability and (E and F) colony formation ability of TE-9 and Eca-109 cell lines transfected with either METTL3 siRNA or NC siRNA were determined. Magnification, x40. ** P<0.01 vs. NC siRNA. METTL3, methyltransferase-like 3; ESCC, oesophageal squamous cell carcinoma; siRNA, small interfering RNA; NC, negative control; OD, optical density.
Figure 3 .
3METTL3 gene knockdown with siRNA induces ESCC cellular apoptosis. TE-9 and Eca-109 cells were transfected with METTL3 or NC siRNA. (A and B) Flow cytometric analysis of the rate of apoptosis of TE-9 and Eca-109 cells transfected with METTL3 or NC siRNA. (C and D) Protein expression levels of apoptosis-related proteins were examined by western blotting in TE-9 or Eca-109 cells transfected with METTL3 or NC siRNA. ** P<0.01 vs. NC siRNA. METTL3, methyltransferase-like 3; ESCC, oesophageal squamous cell carcinoma; siRNA, small interfering RNA; NC, negative control; PI, propidium iodide.
Figure 4 .
4METTL3 gene knockdown with siRNA suppresses ESCC cellular migration and invasion. TE-9 and Eca-109 cells were transfected with METTL3 or NC siRNA. (A and B) Cell migration (magnification, x40) and (C and D) invasion (magnification, x200) capacities were determined in TE-9 and Eca-109 cells transfected with METTL3 or NC siRNA. ** P<0.01 vs. NC siRNA. METTL3, methyltransferase-like 3; ESCC, oesophageal squamous cell carcinoma; siRNA, small interfering RNA; NC, negative control.
Figure 5 .
5METTL3 knockdown with siRNA decreases PI3K/AKT signalling pathway activity. TE-9 and Eca-109 cells were transfected with METTL3 or NC siRNA. (A and B) Protein expression levels of PI3K, p-PI3K, AKT and p-AKT were evaluated by western blot analysis following transfection of TE-9 and Eca-109 cells with METTL3 or NC siRNA. The quantitative analysis refers to the ratio between total and phosphorylated protein levels. ** P<0.01 vs. NC siRNA. METTL3, methyltransferase-like 3; siRNA, small interfering RNA; NC, negative control; p, phosphorylated.
AcknowledgementsNot applicable.FundingNo funding was received.Availability of data and materialsAll datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Authors' contributionsWH designed the study, wrote and revised the manuscript. WL, HL, CZ, MZ and BZ performed all the experiments and the statistical analysis. All authors read and approved the final manuscript.Ethics approval and consent to participateThe study was approved by the Ethics Committee of Second Xiangya Hospital (Changsha, China). Written informed consent was obtained from all patients.Patient consent for publicationAll participants provided written informed consent for publication.Competing interestsThe authors declare that they have no competing interests.This work is licensed under a Creative CommonsAttribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License.
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N6-methyladenosine RNA modification (m 6 A) is one of the most abundant modifications in eukaryotic mRNA, which plays an important role in cancer initiation and progression.
• m 6 A methylation is catalyzed by a multicomponent methyltransferase complex including: METTL3, METTL14, WTAP, METTL16, KIAA1429, RBM15, RBM15B, ZC3H13. WTAP serves as an essential regulatory subunit in methyltransferase which recruits m6A methyltransferase complex to the target mRNA.
• WTAP plays dual roles in cancer either as an oncogene or as a tumor suppressor. It might regulate cancer though m6A methylation or other signaling pathways.
OPEN QUESTIONS
• How does WTAP recruit methyltransferase complex to the target mRNA?
• What determines WTAP localization and in what condition WTAP forms up complexes as WTAP-BCLAF1-THRAP3, WT1-WTAP, or METTL3-METTL14-WTAP?
BACKGROUND
Epigenetics is a branch of genetics that investigates heritable changes in gene expression without changes in the nucleotide sequence [1,2]. Epigenetic regulation has been observed in the context of DNA methylation [3], histone modifications [4], chromatin remodeling [5], transcriptional control [6], noncoding RNAs [7], and cancer immunotherapy [8]. Posttranscriptional modifications, including m1A [9], m5C [10], and m 6 A [11], are abundant and significant, especially m 6 A modifications, because they are considered the most abundant internal modification in eukaryotes [12], with approximately 25% of mRNAs carrying at least one m 6 A site [13,14]. m 6 A modifications can be added not only to mRNAs but also to rRNAs, small nucleolar RNAs (snRNAs), and microRNAs [7,15]. m 6 A modification affects RNA export, leads to spliced pre-mRNAs, and impacts RNA translation and stability [16]. Abnormal regulation of m 6 A has been observed in cancers, and its role as an oncogene or tumor suppressor depends on the cellular environment [17,18].
The main methyltransferases are METTL3, METTL14, and WTAP, which form the m 6 A methyltransferase complex (MTC). The m 6 A level is largely dependent on the MTC. Numerous studies have revealed that the m 6 A level is of great concern in heart failure [19], testosterone synthesis [20], liver steatosis [21], and different cancers [22,23]. The m 6 A modification plays a dual role in cancer biology and is important for the recognition of cancer progression and cancer therapy [24]. To provide a more comprehensive understanding of m 6 A methyltransferase, we focused on WTAP, a constituent of the m 6 A methyltransferase complex.
WTAP was first identified as a splicing factor and then confirmed to be the third component of methyltransferase [14,25,26]. In addition, WTAP fulfils several biological functions, including embryo development, cell cycle progression, cell differentiation, pre-mRNA splicing, and antiviral responses. In this review, we first describe the biological functions of WTAP in detail. Then, we focus on the role of WTAP in cancers either dependent or independent of METTL3-METTL14 methyltransferase and summarize the specific mechanisms of WTAP in tumorigenesis and development.
MOLECULAR MECHANISM OF M 6 A MODIFICATION m 6 A is a widely investigated RNA modification in studies on "epigenetic regulation" [27,28]. The m 6 A RNA modification accounts for 80% of all RNA modifications related to pre-mRNA splicing, miRNAs, lncRNAs, circRNA processing, translation efficiency, and mRNA stability [29]. m 6 A is a dynamic, reversible posttranscriptional modification. The residues of adenosine at the N6 position are localized in the 3ʹ untranslated region (UTR) of the mRNA or close to the termination codon [30,31]. This modification can occur in different biological processes and is mediated by corresponding enzymes termed "writers," "erasers," and "readers" [32].
Methyltransferase-like protein 3 (METTL3) and S-adenosylmethionine (SAM)-binding protein [33] are the most significant components of the methyltransferase complex [34][35][36][37][38]. Methyltransferase-like protein 14 (METTL14) colocalizes with METTL3 in nuclear speckles at a 1:1 ratio [39][40][41][42][43], where it stabilizes the m 6 A methyltransferase complex (MTC) and recognizes specific RNA sequences (RRACH) [30,44]. WTAP recruits METTL3 and METTL14 into nuclear speckles (associated with mRNA export) and is crucial for this unique localization [14,25,26]. Furthermore, RNA-binding motif protein 15 (RBM15) can bind to WTAP and recruit the MTC to specific RNA sites for m 6 A modification [45]. This process is important for the control of m 6 A-promoted X-chromosome inactivation in humans [46]. Zinc finger CCCH-type containing 13 (ZC3H13) interacts with WTAP to retain the MTC in nuclear speckles via its LC domain and thereby promotes its function [47,48]. Other m 6 A writers have been revealed in recent years, including METTL16, METTL5, VIRMA, and ZCCHC4 [49][50][51][52][53].
After the "writers" mark the target mRNA, "reader" proteins, such as YT521-B homology (YTH) domain-containing protein [54][55][56][57][58][59][60][61][62], eukaryotic translation initiation factor 3 (eIF3) [63], the IGF2 mRNA binding protein (IGF2BP) family [64][65][66][67], and the heterogeneous nuclear ribonucleoprotein (HNRNP) protein family [68,69], decode m 6 A methylation to generate signals for nuclear export, translation, RNA splicing, RNA stabilization, and decay [70].
Fat and obesity-related protein (FTO) [71][72][73] and alkB homolog 5 (ALKBH5) [74][75][76] are two essential enzymes for demethylation. "Erasers" are involved in building up the dynamic, reversible modification with "Writers" and "Readers" [77].
In general, m 6 A modification is an abundant and powerful epigenetic modification in eukaryotes. If one key enzyme is disordered, this dynamic modification is disrupted, which impacts human diseases (Table 1, Fig. 1).
OVERVIEW OF WTAP
Structure and cellular localization of WTAP Wilms' tumor 1-associating protein (WTAP) is encoded on human chromosomal region 6q25.3 [78]. WTAP is a 44 kDa protein that contains 396 amino acids and is encoded by the human homolog of FL (2)d [79]. WTAP localizes to both the nucleus and cytoplasm [25,80]. WTAP is a key component in m6A modification, forming a complex with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14 [80]. WTAP contains an extended N-terminal coiledcoil region followed by an unstructured C-terminal part [81] (Fig. 1B). WTAP regulates the localization of the stable heterodimer core complex of METTL3/14 into nuclear speckles through amino acids 5-13 of the nuclear localization signal (NLS) (-PLPKKVRL-to -PLPGGVGL-) at its N-terminus [81]. Notably, the N-terminal coiledcoil region (1-150 amino acids) that contains the NLS is the binding surface of METTL3, which links to the helical structure at the N-terminus of METTL3, called the leader helix (LH) [81]. Although WT1 was found to interact with WTAP, it was confirmed that WT1 was dispensable for the regulation of m 6 A modification by WTAP [25] (Fig. 1B).
Biological functions of WTAP Embryo development. In mice, WTAP plays an essential role in embryonic development. WTAP knockout embryos exhibit proliferative failure [82], and heterozygous mice die at embryonic day 10.5 [83]. In pigs, WTAP knockdown reduced the blastocyst rate and total m 6 A levels [84].
Cell cycle progression and differentiation. Cell proliferation and differentiation are the foundation of growth, development, reproduction, and heredity in organisms [85]. In human umbilical vein endothelial cells (HUVECs), decreased WTAP levels induced cell cycle arrest in the G2 phase. At the same time, the protein levels of cyclin-A2, B1, B2, and CDC20, which are related to the cell cycle [86,87], were significantly decreased [82]. Mechanistically, WTAP stabilizes cyclin-A2 mRNA by binding to its AUUUA motif ACAAAUUAU, which corresponds to the 3ʹ UTR (1526-1534) [82]. These findings indicated that WTAP promotes the G2/M transition in HUVECs (Fig. 2) [82].
WTAP regulates CDK2 mRNA stability, which is related to the G1/S transition [88], in renal cell carcinoma (RCC) and keratinocytes [89]. During RCC cell proliferation, WTAP enhances the stability of the CDK2 mRNA by directly binding to its 3ʹ-UTR ( Fig. 2) [89]. In psoriasis, WTAP not only stabilizes the CDK2 mRNA but also stabilizes the cyclin-A2 mRNA, which promotes the G2/M transition [90]. The binding motif of WTAP in the cyclin-A2 mRNA is ACAAAAUUAU (1526-1534) [82]. Smooth muscle cells (SMCs) proliferate during vascular restructuring and switch to a nonproliferative state when remodeling is complete [91]. The efficiency of WT1 binding to its target promoter is affected by WTAP in the nucleus. Amphiregulin belongs to the epidermal growth factor gene family, which serves as a strong mitogen in SMCs and is regulated by WT1 [92]. When WTAP levels decrease in SMCs, more WT1 bound to the promoter of amphiregulin, switching the cell to a proliferative state. Bcl-2, a protooncogenic apoptosis suppressor, is also activated by WT1 [93]. WTAP was upregulated when SMCs were in a nonproliferative state or the late stage of repair in the intima of injured arteries. Overexpression of WTAP prevents WT1 from binding to the Bcl-2 promoter, thereby downregulating Bcl-2 and activating apoptosis ( Fig. 3A) [94].
pre-mRNA splicing. Alternative splicing of pre-mRNAs plays important roles in cell differentiation and development, and recent studies indicated that most human multiexon genes exhibit alternative splicing [8]. If this process is not highly regulated and accurate, it will lead to mis-splicing events, which may result in proteins with altered function [95].
WTAP interacts with the nuclear splicing factor WT1, forming a splicing complex [96]. Female-specific regulatory protein sex-lethal (SXL) affects sex-specific splicing by regulating the female-specific splicing of transformer (tra) pre-mRNA. Moreover, FL (2)D, the Drosophila homolog of WTAP, forms an RNA-independent complex with SXL [97]. When Fl(2)D was immunodepleted, alternative splicing of transformer pre-mRNA, the target of SXL regulation, was affected [98].
In Drosophila, FL(2)d is distributed throughout the entire eyeantennal imaginal disc and affects retinal development [96] by regulating the alternative splicing of the eye developmental gene Ultrabithorax (Ubx) [99]. In mammalian cells, WTAP and its complex (VIRMA, CBLL1, and ZC3H13) regulate alternative splicing and alternative polyadenylation via inhibitory mechanisms in GCrich sequences [100].
Furthermore, WTAP was found in complexes related to splicing factors, including Snf, U170k, and the two U2AF subunits U2AF38 and U2AF50 [97]. In conclusion, WTAP is closely related to pre-mRNA splicing, but its specific role in this process remains unclear.
Antiviral responses. WTAP is degraded in virus-infected cells through the K48-linked ubiquitination-proteasome pathway upon activation of type I interferon (IFN-I) signaling. IFN-regulatory factor 3 (IRF3) and interferon-alpha/beta receptor subunit 1 (IFNAR1) are two key components involved in IFN-I signaling that are regulated by WTAP in an m 6 A-dependent manner. WTAP maintains the expression of IRF3 and IFNAR1 by enhancing IRF3 translation efficiency via m 6 A modification at its 5'UTR and improving IFNAR1 mRNA stability via m 6 A modification at its 3'UTR at the same time. Following viral infection, degradation of WTAP blocks IRF3 mRNA translation and accelerates IFNAR1 mRNA degradation, which restricts the antiviral immune response and maintains homeostasis (Fig. 3B) [101].
EXPRESSION OF WTAP IN CANCERS
In patient tissue samples, immunohistochemistry results and western blot results have shown that WTAP is highly expressed in dozens of cancers ( Fig. 4 Table 2).
WTAP AS AN M 6 A METHYLTRANSFERASE IN CANCER WTAP in hepatocellular carcinoma (HCC)
The overexpression of WTAP was found to be correlated with a poor prognosis in HCC, and WTAP expression promoted proliferation and metastasis in vitro and vivo [102]. ETS1 is a transcriptional activator that is typically regulated by the Ras/Raf/MEK/ERK pathway [103], and it serves as a tumor suppressor in HCC by downregulating the transcription of p21 and p27 [102]. The expression of ETS1 is regulated by HuR, an RNA-binding protein that binds to and stabilizes m 6 A-modified RNA [104], and WTAP. WTAP was confirmed to increase the m 6 A modification of ETS1 mRNA and interfere with the interaction between ETS1 mRNA and HuR. Thus, WTAP downregulates p21 and p27 expression to promote HCC proliferation (Fig. 5, Table 3) [102,105].
WTAP in osteosarcoma
WTAP was found to be highly expressed in osteosarcoma, and it was a significant independent prognostic factor for overall survival [106]. Chen et al. found that upregulation of WTAP reduces the expression of HMBOX1, an oncogene that inhibits osteosarcoma proliferation and metastasis by downregulating the PI3K/AKT pathway. Specifically, WTAP regulated HMBOX1 in an m 6 A-dependent manner. The m 6 A modification sites in HMBOX1 are in the 3ʹUTR at 2767 and 3080 nucleotides. However, the reader of HMBOX1 m 6 A remains unclear (Fig. 5, Table 3) [106].
WTAP in gastric cancer WTAP was found to be highly expressed in gastric cancer tissues, and its overexpression was correlated with poor prognosis [107]. HK2 plays significant roles in both the Warburg effect, a significant cause of relapse and pathogenesis in gastric cancer [108], and cancer cell immortalization [109]. WTAP promoted the proliferative ability of gastric cancer cells and increased their glycolytic capacity (glucose uptake, lactate production, and extracellular acidification rate) by stabilizing the hexokinase-2 (HK2) mRNA by binding to its 3ʹ-UTR m 6 A site (Fig. 5, Table 3) [107].
WTAP in hematological malignancies WTAP was overexpressed in acute myeloid leukemia (AML) patients, and its expression was related to a poor survival rate. MYC is known as a master transcription factor that regulates genes essential for survival, cell proliferation, and metastasis [110,111] and may act as a downstream regulator of the PI3K/ AKT pathway [112,113]. WTAP downregulates c-Myc expression by increasing the m 6 A modification of its mRNA [114]. Thus, high WTAP expression predicts poor prognosis in AML, and WTAP plays an epigenetic role in AML (Fig. 4, Table 2) [114]. It was also reported that PIWI-interacting RNAs (piRNAs) are related to diffuse large B-cell lymphoma (DLBCL) [115]. piRNA 30473 was highly expressed in DLBCLs, where it promoted proliferation and induced cell cycle arrest. Mechanistically, piRNA-30473 increased WTAP levels to upregulate the global m 6 A level. WTAP increased HK2 expression by enhancing its m 6 A level. The m 6 A reader IGF2BP2 was found to bind to the 5ʹUTR of HK2 mRNA, leading to its stabilization. HK2 is an essential kinase in glucose metabolism that is associated with tumor cell proliferation by enhancing aerobic glycolysis [116][117][118][119]. Overall, the piRNA-30473/ WTAP/HK2 axis contributes to tumorigenesis by regulating m 6 A RNA methylation in DLBCL [115] (Fig. 5, Table 3).
Natural killer/T-cell lymphoma (NKTCL) exhibits high resistance to chemotherapy, which is related to the high expression of ATP binding cassette (ABC) transporter proteins as drug efflux pumps [120,121]. Multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp) are two major proteins in the ABC transporter family that prevent the cellular accumulation of chemotherapy drugs [122]. WTAP was upregulated in NKTCL cell lines. Depletion of WTAP downregulated the expression of MRP1 and P-gp and blocked resistance to cisplatin [122,123]. WTAP also upregulated the expression of dual-specificity phosphatase 6 (DUSP6) by stabilizing its mRNA by increasing the m 6 A modification of its transcript, which induced tumor progression and contributed Fig. 2 The function of WTAP in cell cycle transition. In keratinocytes and renal cell carcinoma cells, WTAP enhances the stability of the CDK2 mRNA by directly binding to its 3'-UTR. In human umbilical vein endothelial cells (HUVECs), WTAP stabilizes cyclin-A2 mRNA by binding to its AUUUA motif ACAAAUUAU, which corresponds to the 3ʹ UTR (1526-1534). These findings indicated that WTAP promotes the G1/S transition and the G2/M transition. Fig. 3 A Model of the mechanism through which WTAP regulates SMC proliferation. The balance between WTAP and WT1 influences the state of SMCs. When the expression of WTAP is reduced, WT1-mediated transcriptional events proceed. Amphiregulin is a direct transcriptional target of WT1 that drives SMC proliferation by upregulating the EGF pathway. Thus, SMCs switch to a proliferative state. When the balance of WTAP and WT1 is reversed, WT1-mediated transcription may be blocked, and the transcription of Bcl-2, which is suppressed by WT1, is activated. SMC apoptosis is increased, and the cells switch to a nonproliferative state. B WTAP in the antiviral immune response. WTAP is degraded in virus-infected cells. After viral infection, degradation of WTAP leads to a decrease in the m 6 A level of IRF3 mRNA and IFNAR1 mRNA, which leads to IRF3 mRNA translation blockade and accelerated IFNAR1 mRNA degradation. This biological process restricts the antiviral immune response and maintains homeostasis.
to WTAP-induced drug resistance via the WTAP/m 6 A/DUSP6 axis (Fig. 5, Table 3) [123].
WTAP in endometrial carcinoma (EC)
WTAP was observed to be upregulated in endometrial cancer cell lines [124,125]. WTAP activated the nuclear factor-κB (NF-κB) pathway by regulating the m 6 A modification of caveolin-1 (CAV-1) mRNA. Reduction of CAV-1 levels by WTAP could enhance the activity of the NF-κB pathway, contributing to the pathogenesis of EC [124,125].
OTHER FUNCTIONS OF WTAP IN CANCER WTAP in cholangiocarcinoma
WTAP shows a tendency toward overexpression in cholangiocarcinoma tissues. In addition, overexpression of WTAP induces the expression of MMP7, MMP28, cathepsin H, and Muc1 [126]. Notably, these enzymes are all involved in the degradation of the extracellular matrix, which can explain the increased invasion of cholangiocarcinoma cells and WTAP overexpression inside lymph nodes or vessels [127][128][129][130]. In addition, Muc1 was shown to regulate EGFR activity [131] to regulate the motility of cancer cells Fig. 4 The function of WTAP in biological process. Immunohistochemistry has been performed in many studies. Strong staining for WTAP was observed in grade IV gliomas, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, and high-grade ovarian carcinoma, with low staining in adjacent normal tissues. Osteosarcoma tumorigenesis Upregulated Oncogene [106] Gastric cancer Upregulated Oncogene [107] Acute myeloid leukemia Upregulated Oncogene [114,132] Natural killer/T-cell lymphoma Upregulated Oncogene [123] Cholangiocarcinoma Upregulated Oncogene [126] Diffuse large B-cell lymphoma Upregulated Oncogene [134] Malignant glioma Upregulated Oncogene [135] Colorectal cancer ? Tumor Suppressor [137] Pancreatic ductal adenocarcinoma Upregulated Oncogene [142] Bladder cancer Upregulated Oncogene [143] Renal cell carcinoma Upregulated Oncogene [89] High-grade serous ovarian cancer Upregulated Oncogene [145] Non-small cell lung cancer ? Oncogene [147] Q. Huang et al. [126]. Therefore, the function of WTAP is an important in cholangiocarcinoma (Fig. 6, Table 4).
WTAP in hematological malignancies
In AML, the molecular chaperone Hsp90 interacted with and stabilized WTAP by decreasing its polyubiquitination, which promoted chemoresistance (Fig. 5, Table 3) [132]. This phenomenon was also observed in diffuse large B-cell lymphoma (DLBCL), a common type of non-Hodgkin lymphoma [133,134] (Fig. 6, Table 4).
WTAP in malignant glioma
WTAP is overexpressed in glioma tissues compared to normal brain tissues. Furthermore, WTAP expression is associated with glioma grade and is an independent prognostic factor for shorter survival in patients with glioma. High expression of WTAP leads to a much lower overall survival rate than low WTAP expression in patients suffering from glioma. Therefore, WTAP may be a novel prognostic marker for glioma (Table 4) [135].
WTAP in endometrial carcinoma (EC)
WTAP also promoted chemoresistance of endometrial carcinoma (EC) cells to cisplatin by facilitating proliferation and repressing apoptosis. Mechanistically, WTAP enhanced the phosphorylation of GSK3β at Ser9, which facilitated the nuclear translocation of β-catenin [136]. Consequently, β-catenin activated the transcription of c-Myc, Survivin, and Bcl-xl to promote chemoresistance to cisplatin [136]. Overall, these results shed light on the strategies to modify the treatment response by altering chemoresistance to cisplatin ( Fig. 6 Table 4) [124].
WTAP in colorectal cancer (CRC)
Carbonic anhydrase IV (CA4) is silenced in colorectal cancer (CRC) [137]. It was recently identified as a preferentially methylated gene that is expressed in normal colon tissues [138] and plays a tumorsuppressive function by inhibiting the Wnt/β-catenin signaling pathway [139,140]. CA4 interacts with WTAP and promotes its polyubiquitination-dependent degradation [137]. WT1 is a negative regulator of the Wnt signaling pathway [141]. WT1 is released from the WT1-WTAP complex by CA4, resulting in the induction of transducing β-like protein 1 (TBL1) and the degradation of β-catenin. A lack of CA4 results in the activation of WNT/β-catenin signaling, which promotes CRC progression [137] (Fig. 6, Table 4).
WTAP in pancreatic ductal adenocarcinoma (PDAC)
The nuclear and cytoplasmic levels of WTAP were much higher in PDAC than in adjacent nontumor tissues [142]. High nuclear levels of WTAP were correlated with a more advanced tumor stage, while cytoplasmic WTAP levels were associated with histological trade and perineural invasion. In addition, high expression of WTAP in the nucleus and cytoplasm differed significantly by sex. Nuclear WTAP levels were identified as an independent prognostic indicator for PDAC and were associated with poor overall survival. Overall, WTAP may be a molecular biomarker in PDAC [142] (Table 4).
WTAP in bladder cancer
Immunohistochemical staining showed that WTAP expression in bladder cancer was significantly higher than that in normal tissues, and high expression of WTAP indicated a poor prognosis [143]. Moreover, both the mRNA and protein levels of WTAP were upregulated in bladder cancer, offering a potential novel approach for the diagnosis and treatment of bladder cancer (Table 4) [143].
WTAP in renal cell carcinoma (RCC)
In RCC, WTAP binds to the transcript of CDK2, a cell cycle-related protein [144], to enhance the stability of its mRNA, thus decreasing the percentage of cells in the G1 phase (Table 4) [89].
WTAP in high-grade serous ovarian cancer (HGSOC) WTAP expression was correlated with a poor prognosis in highgrade serous ovarian cancer (HGSOC) [145]. Mechanistically, WTAP affected migration by regulating proteins related to the epithelialmesenchymal transition (EMT) by decreasing E-cadherin expression and increasing vimentin expression. In addition, WTAP promoted the phosphorylation of AKT, JNK, ERK, and p38, indicating that WTAP might be involved in activation of the AKT and MAPK signaling pathways (Fig. 6, Table 4) [145]. It was also reported that family with sequence similarity 76member A (FAM76A) and HBS1-like translational GTPase (HBS1L) are positively correlated with WTAP according to weighted gene coexpression network analysis (WGCNA), and both were correlated with a poor prognosis [146].
WTAP in non-small cell lung cancer (NSCLC)
High levels of the lncRNA PCGEM1, which is considered to promote cell growth, were detected in NSCLC. PCGEM1 was mostly distributed in the cytoplasm, indicating that it mostly performs its function at the posttranscriptional level. Furthermore, PCGEM1 was found to act as a sponge for miR-433-3p in NSCLC. WTAP is a downstream target of the PCGEM1/miR-433-3p axis. Overall, PCGEM1 plays an important role in NSCLC and can accelerate cancer progression via the miR-433-3p/WTAP axis (Table 4) [147].
WTAP in hepatoblastoma
Hepatoblastoma is a common primary malignant hepatic tumor of infancy and childhood that usually occurs in the first two years of life [148]. Hepatoblastoma susceptibility was correlated with WTAP gene variants. The genotype frequencies of three WTAP single nucleotide polymorphisms (SNPs: rs7766006 G > T, rs9457712 G > A, and rs1853259 A > G) were evaluated in Chinese children, including 313 hepatoblastoma patients and 1446 controls. However, only the rs7766006 GT/TT genotype exhibited a Fig. 6 Other functions of WTAP in cancers. WTAP regulates the differential expression of oncogenes and tumor suppressor genes at the nonposttranscriptional level. WTAP induces the expression of Muc1, which regulates EGFR activity in cholangiocarcinoma. Hsp90 forms a complex with WTAP and stabilizes its protein level to promote chemoresistance in AML. In DLBCL, Hsp90 also stabilizes the WTAP protein, which forms a complex with BCL6. In colorectal cancer, CA4 interacts with WTAP and promotes its degradation in a polyubiquitination-dependent manner so that WT1 is released from the WT1-WTAP complex, resulting in the induction of transducin β-like protein 1 (TBL1) and the degradation of β-catenin, which blocks the Wnt pathway. WTAP was found to facilitate the nuclear translocation of β-catenin and enhance the phosphorylation of GSK3b at Ser9, which induced chemoresistance to cisplatin in endometrial carcinoma by activating the Wnt/β-catenin pathway. Additionally, WTAP was found to regulate the expression of the EMT-related proteins E-cadherin and vimentin. Furthermore, WTAP is involved in the activation of the AKT and MAPK pathways. Overall, WTAP contributes to cell proliferation, apoptosis, invasion, metastasis, and chemo-or radioresistance in different cancers.
significant association with hepatoblastoma risk. Rs7766006 T was associated with a decrease in WTAP mRNA levels. Thus, WTAP SNPs potentially play a role in hepatoblastoma via genetic modification [149].
FUTURE PROSPECTS
WTAP was first reported to be a splicing factor. In the following years, its biological functions have gradually been uncovered, including functions in m 6 A modification, embryo development, cell cycle progression and differentiation, pre-mRNA splicing, and antiviral responses. With the development of techniques for detecting m 6 A modification, WTAP was revealed to be a part of the MTC and to participate in m 6 A modification with both METTL3 and METTL14 and other methyltransferases. In human umbilical vein endothelial cells, WTAP promotes G2/M transition, while in smooth muscle cells, overexpression of WTAP prevents WT1 from binding to the Bcl-2 promoter, thereby downregulating Bcl-2 and activating apoptosis. In renal cell carcinoma, keratinocytes, and psoriasis, WTAP regulates the G1/S transition and G2/M transition by stabilizing specific mRNAs. Thus, WTAP may be a potential biomarker for changes in cell proliferation and differentiation. WTAP is also associated with chemoresistance in hematological malignancies and endometrial carcinoma by upregulating the expression of MRP1 and P-gp and enhancing the phosphorylation of GSK3β at Ser9. These results shed light on the potential of targeting WTAP for the prevention of chemoresistance to cisplatin. During metabolism, WTAP can stabilize the HK2 mRNA, which is associated with aerobic glycolysis and the Warburg effect in diffuse large B-cell lymphoma. The therapeutic schedule can be developed according to this metabolic phenomenon. High expression of WTAP was confirmed in malignant gliomas, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, and ovarian cancer, which is related to progression and poor prognosis (Fig. 6, Table 4), suggesting that WTAP might be a biomarker for the above cancers. In liver cancer, WTAP was observed to increase the m 6 A level of the ETS1 mRNA, thereby facilitating cancer progression. Similarly, WTAP was found to induce the proliferation and metastasis of osteosarcoma by regulating HMBOX1 m 6 A modification. In gastric cancer, WTAP enhanced HK2 mRNA stability through m 6 A modification. In natural killer/T-cell lymphoma, WTAP upregulated DUSP6 expression through m 6 A modification, inducing drug resistance. In acute myeloid leukemia, WTAP downregulated c-Myc expression by increasing the m 6 A modification of its mRNA, making cells resistant to chemotherapy drugs. These cases indicated that the role of WTAP as a methyltransferase is vital in cancer progression. Although no small-molecule inhibitors of RNA methyltransferases and WTAP have been discovered, FTO demethylation inhibitors have been identified. Rhein can bind the FTO catalytic domain to suppress m 6 A demethylation [150]. CHTB, N-CDPCB and meclofenamic acid 2 (MA2) have been revealed to be FTO inhibitors through structurebased virtual screening and biochemical analyses [151,152]. R-2hydroxyglutarate (R-2HG) inhibits FTO activity and increases global m 6 A modification, which has been tested in vitro and in mice [153]. These effects suggest that WTAP-targeted inhibitors may be developed in the future and that a deeper understanding of m 6 A modification is warranted.
CONCLUSION
At present, our understanding of WTAP is insufficient due to a lack of further experiments and additional samples. m 6 A has gradually become a significant focus of cancer research, but the role of WTAP in this process is still at an early stage. Furthermore, the localization of WTAP in nuclear speckles and the formation of a complex with METTL3 and METTL14 need to be further investigated, since this knowledge may be useful for understanding the role of m 6 A modification in cancer biology. In conclusion, many studies have revealed WTAP as a potential biomarker for predicting cancer progression, since it participates in alternative splicing, cell cycle regulation and methylation. Thus, efforts should be made to develop the potential of WTAP for therapies targeting tumorigenesis and tumor development.
DATA AVAILABILITY
The materials that support the conclusion of this review have been included within the article. Q. Huang et al.
Fig. 1
1Mechanism of m6A and fuctional domais in m 6 A methyltransferase. A The dynamic molecular mechanism of m 6 A modification. m 6 A is installed by "writers" (METTL3/14, WTAP, RBM15/15B, VIRMA, and ZC3H13), removed by "erasers" (FTO, ALKBH5, and ALKBH3), and recognized by "readers" (YTHDC1/2, YTHDF1/2/3, IGF2BP1/2/3, HNRNP, and eIF3). B Functional domains in m 6 A writer, eraser, and reader proteins.Q. Huang et al.
Fig. 5
5WTAP serves as a methyltransferase in cancers. WTAP plays a significant role in RNA methylation by recruiting METTL3/METTL14 to form a complex that binds to target RNAs. In this process, WTAP regulates the differential expression of oncogenes and tumor suppressor genes in an m 6 A-dependent manner. It enhances the stability of the HK2 and DUSP6 mRNAs, inducing drug resistance in hepatocellular carcinoma, gastric cancer, and NKTCL. Additionally, WTAP induces the degradation of the ETS1, HMBOX1, and c-Myc mRNAs in an m 6 A-dependent manner, enhancing HCC proliferation and suppressing the invasion and metastasis of osteosarcoma and acute myeloid leukemia.
Table 1 .
1Summary of m 6 A modification enzymes.Components
Enzymes
Intracellular
localization
Biological functions
References
WRITERS
METTL3
Cytoplasm, Nucleus,
Nuclear speckles
m 6 A methyltransferase, DNA damage responses,
DNA-RNA hybrid, Cancer cell proliferation, Cell cycle
progression and survival, Cancer cell resistance to
radiotherapy and cisplatin
[35-38]
METTL14
Nucleus
m 6 A methyltransferase, mRNA degradation or
stabilization, LncRNA stabilization, pre-mRNA splicing,
mRNA exportation, mRNA turnover in tumor
proliferation, Metastasis, Self-renewal and tumor-
initiating capacity
[41-44]
WTAP
Cytoplasm, Nucleus,
Nuclear speckles
m 6 A methyltransferase, Embryo development, Cell
cycle progression and differentiation, Pre-mRNA
splicing, Antiviral responses, Alternative splicing
[78, 82-84, 86, 87, 98, 100, 101]
RBM15/
ZC3H13/
VIRMA
Nuclear speckles,
Nucleus, Nuclear
envelope, Nuclear
membrane
m 6 A methyltransferase, Proliferation, invasion,
migration, and apoptosis, Anchoring the m 6 A
regulatory complex in the nucleus, Controls mouse
embryonic stem cell self-renewal
[45, 48, 51, 70]
ZCCHC4
Nucleus, Cytoplasm
Methylates human 28 S rRNA, Interacts with a subset
of mRNAs, Related to global translation, Cell
proliferation
[52]
METTL5
Nucleus, Cell junction
m 6 A modification of 18 S rRNA, Promotes translation
initiation, S6K activation, and cancer cell growth
[50, 53]
METTL16
Nucleus, Cytoplasm
m 6 A modification of U6 snRNA, lncRNAs, and introns
of pre-mRNAs
[49]
ERASERS
FTO
Cytoplasm, Nucleus,
Nuclear speckles
Demethylation of m 6 A and m1A, Regulation of mRNA
splicing and cell differentiation
[71-73]
ALKBH5
Nuclear speckles
m 6 A demethylation, Participates in the regulation of
mRNA nuclear export and mouse sperm
development, Reduces tumoral proliferative,
migration, and invasion activities
[74-76]
READERS
YTHDF2/3
Nucleus, Cytoplasm
mRNA stabilization/degradation, Regulates mRNA
clearance, Regulates cancer cell proliferation, invasion
and migration
[54, 58, 59, 83]
YTHDC1
Nucleus, Nuclear
speckles
Binds m 6 A-modified pre-mRNAs and mRNAs, and
facilitates exon inclusion, splicing, mRNA nuclear-
cytoplasmic export
[55, 60, 61]
IGF2BP1-3
Cytoplasm, Nucleus
Recognizes m 6 A through K homology domains and
facilitates m6A-modified mRNA stabilization and
protein translation
[64-67]
YTHDC2
Cytoplasm
Regulates mRNA translation or decay and mouse
spermatogenesis
[56]
YTHDF1
Cytoplasm
Selectively recognizes m 6 A-modified mRNA,
Promotes ribosome loading of m 6 A-modified mRNA,
Interacts with initiation factors to facilitate translation
initiation
[57, 62]
hnRNPC/
hnRNPG
Nucleus
Regulates mRNA structure and alternative splicing
[69]
Q. Huang et al.
Table 2 .
2WTAP expression in different cancers.Cancer
Expression
Role
References
Hepatocellular carcinoma
Upregulated
Oncogene
[105]
Table 3 .
3WTAP as an m6A methyltransferase in cancer.Cancer
Biological function
Mechanism
Target
Regulator
References
Hepatocellular carcinoma
Enhance proliferation,
migration
Downregulated the ETS1/
p21, p27 axis in an m6A-
mediated manner
ETS1/p21, p27
/
[105]
Osteosarcoma tumorigenesis
Enhance proliferation,
migration
Downregulated the
HMBOX1/PI3K/AKT axis in an
m6A-mediated manner
HMBOX1/PI3K/
AKT
/
[ 106]
Gastric cancer
Enhance proliferation,
migration
WTAP enhanced the stability
of HK2 mRNA to regulate the
gastric cancer
Warburg effect
HK2
/
[107]
Acute myeloid leukemia
Enhance proliferation
Performed m6A on c-Myc
mRNA and enhanced its
degradation
c-Myc
Cyclins
and Hsp90
[114, 132]
Natural killer/T-cell lymphoma
Promote resistance to
cisplatin
Enhanced m6A on DUSP6
and stabilized its mRNA
DUSP6
/
[123]
Table 4 .
4Other functions of WTAP in cancer.Cancer
Biological function
Mechanism
Target
Regulator
References
Cholangiocarcinoma
Promote invasion,
migration
/
MMP7,
MMP28,
Cathepsin
H, Muc1
/
[ 126]
Diffuse large B-cell lymphoma
Promote proliferation,
counteract etopside-
mediated apoptosis
/
/
Cyclins
and Hsp90
[134]
Colorectal cancer
/
WTAP supports CA4 in
performing its tumor-
suppressive function and
releasing WT1 from the
WTAP-WT1 complex
Carbonic
anhydrase
IV (CA4)
/
[ 137]
Renal cell carcinoma
Promote invasion
proliferation and
migration, accelerate cell
cycle progression
Binds to the CKD2 transcript
to enhance the function of
its mRNA
/
/
[ 89]
High-grade serous
ovarian cancer
Proliferation, migration
and inhibition of
apoptosis abilities
Regulates the epithelial-
mesenchymal transition
(EMT) pathway and AKT and
MAPK signaling pathways
E-cadherin,
Vimentin,
AKT, JNK, ERK
and p38
/
[ 145]
Non-small cell lung cancer
Proliferation, migration
and inhibition of
apoptosis abilities
/
/
PCGEM1/miR-
433-3p axis
[147]
Cell Death and Disease (2022) 13:852
© The Author(s) 2022
AUTHOR CONTRIBUTIONSQH and JM collected the related papers and drafted the manuscript. BZ and ZL participated in the design of the review. XC initiated the study and revised the manuscript. All authors read and approved the final manuscript.COMPETING INTERESTSThe authors declare no competing interests.ADDITIONAL INFORMATIONCorrespondence and requests for materials should be addressed to Zhibin Liao , Xiaoping Chen or Bixiang Zhang.Reprints and permission information is available at http://www.nature.com/ reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/.
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Introduction
Endometrial carcinoma is the most common malignancy of the female genital tract. Histologically, it has long been categorized into two subtypes. [1] Type I tumors (approximately 80%) are endometrioid carcinomas (EECs) with estrogen and progesterone receptors (ERs and PRs) and usually, have a favorable prognosis. However, type II tumors (10-20%), the non-endometrioid carcinomas (NEEC), are not associated with estrogen excess and usually have a poor prognosis. However, approximately 20% of the cases do not fit within this dualistic model; some EECs are aggressive and have poor clinical outcomes. [2] In fact, endometrial carcinoma is a clinically heterogeneous disease, and it is now well recognized that this heterogeneity may be the result of various underlying molecular alterations.
The DNA methylation is involved in numerous biologic events and it concerns approximately 70% to 80% of CpGs in mammalian DNA and associated with transcriptional gene silencing when it occurs in promoters. [3] Studies have indicated that the silencing of the tumor suppressor genes by DNA methylation plays an important role in the pathogenesis of endometrial cancer. Methylation changes to the genome are catalyzed by DNA methyltransferases (DNMT). DNMT3A/3B is de novo methyltransferases with high activity on unmethylated substrates, while DNA methylation is maintained by DNMT1 after the methylation pattern has been established. [4,5] In recent years, our knowledge on the roles of DNMT in DNA methylation has increased substantially. The ESR1/PGR frequently undergoes de novo methylation in a wide variety of tumors, including breast, colon, lung, and brain tumors. [6][7][8][9] However, the mechanisms underlying the loss of expression in endometrial cancer have not been studied extensively. Previously, using immunohistochemistry, we found that DNMT3B protein expression was negatively correlated with ER and PR expression in EEC in a small cohort. [10] The Cancer Genome Atlas (TCGA) provides a wealth of information concerning DNA (somatic mutation, copy number alteration [CNA], and methylation), RNA (transcript level), and particular proteins from thousands of tumor-and case-matched normal tissues. [11] Therefore, in the present study, we collected data from the TCGA database and analyzed the associations between DNMT3A/3B mRNA expression and the methylation and expression patterns of the ESR1/PGR in endometrial carcinomas. We hypothesized that methylation catalyzed by DNMT3A/3B might take part in the ESR1/PGR downregulation in endometrial carcinoma and might possess prognostic utility.
Methods
Study time and design
The TCGA data acquisition Level 3 RNA-Seq, protein RPPA (reverse-phase protein array) and clinical data for uterine cancer patients were obtained from the TCGA data portal (http://cancergenome.nih.gov/). Detailed information concerning the data processing, quality control, and normalization is available on the TCGA open access download directories (https://tcga-data.nci.nih.gov/tcga/ tcgaDownload.jsp). RSEM (RAN-Seq by Expectation-Maximization) expression values were log 2 transformed for statistical analysis.
The TCGA adopted the illumina infinium human methylation 450 (HM450) bead array to assay over 480K CpG sites, and about 99% of RefSeq genes and 96% of CpG islands from UCSC database are included. DNA methylation data were provided as beta values calculated using the formula M/(M+U+100), with M and U representing fully methylated and fully unmethylated intensities, respectively. [12] Mean value was used to stratify these biomarkers as high expression or low expression, hypermethylation or hypomethylation.
The CNA non-linear data were provided as putative copy number variation using GISTIC 2.0 with À2, À1, 0, 1, and 2 representing homozygous deletion, hemizygous deletion, neutral or no change, gain, and high-level amplification, respectively. Mutation data were provided in a mutation annotation manner derived from wholeexome sequencing.
Statistical analysis
Data were analyzed using SPSS19 (SPSS Inc., Chicago, IL, USA). The Kolmogorov-Smirnov method was used to test the normal distribution of measurement data. An unpaired two-sample t-test was performed to compare mRNA or protein expression values. The Chi-squared test or Fisher's exact test were performed to compare categorical variables. A Bonferroni correction was applied for multiple comparisons. Bivariate correlation analysis was performed to investigate the relationship between gene expression values with Pearson's method for data following a normal distribution and Spearman's method for data with an abnormal distribution. Survival analysis was performed using Kaplan-Meier curves, with P-values calculated by the log-rank test. A Cox-proportional hazard regression model was used to perform multivariate analysis. All tests were two-sided, and a P-value <0.05 was considered statistically significant.
Results
Clinical and pathologic characteristics
A total of 544 endometrial cancers with both clinical and gene expression data were obtained from the TCGA database. The clinicopathologic features of all of the patients are summarized in Table 1. A total of 408 EEC (75.0%) and 136 NEEC (25.0%, 114 serous carcinomas and 22 mixed carcinomas) were included in this cohort. The median age of the patients was 64 years (range, 31-90 years). A total of 72.9% of the patients were White, 20.7% were African American, and 3.9% were Asian. The ER and PR methylation data were obtained for 430 cases, and 246 cases had mutation data. CNA data were available for 537 cases. Follow-up information was provided for 542 (99.6%) patients (406 EEC, 136 NEEC), and the median follow-up was 29.0 months (range, 0.1-225.3 months).
DNMT3A/3B is overexpressed in EEC and NEEC and correlated with poor patient survival
The DNMT3A/3B was overexpressed in EEC and was even higher in NEEC (DNMT3A, EEC vs. NEEC: 37.6% vs. 69.9%, t = À7.440, P < 0.001; DNMT3B, EEC vs. NEEC: 42.4% vs. 72.8%, t = À6.897, P < 0.001). Similarly, the expression levels of DNMT3A and DNMT3B increased in EEC (log 2 RSEM = 9.94 ± 0.55 and 8.07 ± 1.01) and became even higher in NEEC (log 2 RSEM = 10.49 ± 0.81 and 8.79 ± 1.16) compared with normal control tissues (log 2 RSEM = 8.85 ± 0.60 and 5.66 ± 0.69), and all the differences reached significance (all P < 0.001) [ Figure 1A]. Among the DNMT3A overexpressed subgroup, as many as 60.5% (150/248) of cases also had DNMT3B overexpression, and the correlation between these two biomarkers was statistically significant (R = 0.265, P < 0.0001; Figure 1B).
In addition, higher DNMT3A/3B expression was associated with poor clinicopathologic variables, including tumor type, tumor grade, lymph node metastasis, and advanced stage with statistical significance [ Table 2]. Survival analyses demonstrated reduced overall survival and disease-free survival for patients with DNMT3A overexpression (P = 0.002 and P = 0.001) [ Figure 1C]. Likewise, DNMT3B overexpression indicated poor overall survival and reduced disease-free months, but the latter was on the borderline of significance (P = 0.026 and P = 0.065) [ Figure 1D]. The combined DNMT3A and DNMT 3B overexpression was significantly correlated with poor patient survival [ Figure 1E]. However, in the multivariate analysis, only DNMT3A remained as an independent prognostic factor (P = 0.013; Table 3).
DNMT3A/3B overexpression was correlated with hypermethylation and reduced expression of the ESR1 and PGR in EEC
Studies have indicated that the ESR1/PGR frequently undergoes de novo methylation and lost expression in a wide variety of tumors, including breast, colon, and lung cancer. [7,9] However, the mechanisms underlying their loss of expression in endometrial cancer have not been studied extensively. Therefore, we next analyzed the relationship between the expression of DNMT3A/3B and the methylation and expression of the ESR1/PGR in EEC and NEEC to assess whether methylation catalyzed by DNMT3A/3B contributes to the low ER/PR expression in endometrial cancer.
In the current study, the mean value was used to stratify ESR1 or PGR (genes encoding ER-a and PR, respectively) as hypermethylation or hypomethylation. The mean value of ESR1 and PGR methylation was 0.68 and 0.36, respectively. Using these criteria, in EEC, 41.1% of ESR1 and 24.8% of PGR were hypermethylated, and in NEEC, the rate increased drastically up to 93.3% and 70.6%, respectively [ Table 4].
Further analyses revealed that in EEC DNMT3A overexpression was significantly correlated with the hypermethylation of the ESR1 and PGR (P < 0.001) and its low expression, both at the transcript and protein level (P < 0.05) [ Table 5]. The hypermethylation (52.1% and 35.5%) and reduced expression (29.4%, 37.7%) of ER/PR occurred more frequently in tumors with DNMT3A overexpression than in those without DNMT3A overexpression (35.8%, 18.4%; 14.1%, 16.9%). The same trend was observed in the DNMT3B overexpression subgroup, and almost all the associations approached statistical significance, except for the correlation between DNMT3B overexpression and ESR1 mRNA downregulation [ Figure 2A and 2B].
However, the above phenomena were not present in NEEC. The DNMT3A/3B expression had no relationship to either ESR1/PGR methylation or expression status; however, DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and ER/PR low expression occurred more often in NEEC than that in EEC [ Table 5].
Hypermethylation was the dominant mechanism resulting in low ESR1/PGR expression in EEC
We found that DNA methyltransferase 3A/3B overexpression was associated with hypermethylation and reduced ER/PR expression in EEC. In addition, limited studies have reported that the downregulation of the estrogen and progesterone receptor in endometrial carcinoma was associated with the methylation of these two genes. For these reasons, we next explored the relationship between ER/PR expression and methylation, CNA and mutation status of these two genes to investigate whether and to what extent methylation contributes to the reduced ER/PR expression in endometrial cancer.
The reduced ER/PR expression occurred in approximately 20% of EEC cases (also stratified by mean value). Among these tumors with low ER/PR expression, as much as 83.1% (54/65) of ESR1 and 59.5% (50/84) of PGR were hypermethylated, while only 7.6% (6/79) of ESR1 and 13.1% (13/99) of PGR was deleted. Both hypermethylation and copy number deletion were significantly correlated with reduced ER/PR expression (P < 0.05) [ Table 4]. Obviously, hypermethylation played a major part in the low expression of the ESR1 and PGR, while copy number deletion played a relatively minor role. The minority of EEC cases had mutations in the ESR1 or PGR; however, it (2) www.cmj.org In NEEC, approximately 70% to 80% of tumors had reduced ER or PR expression. Hypermethylation and copy number deletion of ESR1 and PGR were frequently observed among these tumors (96.3%, 79.6%; 31.3%, and 63.0%, respectively) and were even higher compared with EEC (83.1%, 59.5%; 7.6%, and 13.1%, respectively). However, the mechanism behind low ER or PR expression seemed to be different. The low PR expression was still significantly correlated with the methylation of the PGR (P < 0.001) but this association was lost in the low ER expression subgroup (P = 0.106). By contrast, deletion of the ESR1 was more effective in causing ER low expression. In the low ER expression subgroup, 31.3% of cases showed a deletion in ESR1, which was significantly higher than that in the ER high-expression subgroup (12.8%; P = 0.027). No mutation of ESR1 or PGR was found in NEEC [ Table 4].
Combined DNMT3A/3B overexpression and ESR1/PGR methylation or expression were correlated with poor survival
In univariate analysis, DNMT3A/3B overexpression, ESR1/PGR hypermethylation, and low expression alone or in combination were correlated with poor survival in the whole cohort [ Figures 1C, 3 and 4]. However, as mentioned above, in multivariate analysis, only DNMT3A was an independent prognostic factor of disease-free survival [ Table 3].
Discussion
Taking advantage of the large-scale cancer data sets of TCGA, for the first time, we examined the DNMT3A/3B mRNA expression in 544 endometrial carcinomas. Our data set represents the largest series of endometrial cancer cases assessed for DNMT3A/3B alterations. We found that DNMT3A/3B mRNA was overexpressed progressively from EEC to NEEC compared with normal controls and was significantly correlated with a poor prognosis. The results of this study were not completely consistent with the previous study. Previously, using immunohistochemistry, we found in a small cohort that DNMT3B overexpression was more often associated with EEC than NEEC and was significantly correlated with high tumor grade. [10] Two other groups also observed that DNMT3B expression was higher in EEC than in NEEC. [13] In addition, Xiong et al [14] reported no differences of DNMT3A expression among normal endometrium, EEC, and serous endometrial carcinoma. Together, these discrepancies could be explained by two reasons. One might be due to the limited sample size and different testing methods or score criteria used by different studies. The other might be because the DNMT expression at the transcript level was not completely parallel with the protein level. [13] However, regarding the prognostic value, our findings from the present study were in line with others in that poorly differentiated endometrioid cells expressed higher DNMT3B and high DNMT3A or DNMT3B expression implied a poor prognosis. [14][15][16][17] In addition, in the present study, we found that DNMT3A and DNMT3B overexpression coexisted in most cases and the combination of these two biomarkers correlated well with poor prognosis. A study has shown that the structures and functions of DNMT3A and DNMT3B are very similar. [3] Thus, our findings were consistent with others that showed that DNMT3A and DNMT3B may cooperate and function synergistically in endometrial cancer.
More interestingly, our data indicated that methylation catalyzed by DNMT3A/3B might be the major mechanism resulting in ER/PR downregulation in EEC. First, our results To date, studies concerning the basis of ER/PR downregulation in endometrial cancer are not extensive; however, a link between ESR1/PGR methylation and expression loss in breast carcinoma has been established. [6,18] Early studies reported that the ESR1/PGR was de novo methylated in some ER/PR-negative endometrial cancers, [19][20][21][22] while the other two groups found that the ESR1 was highly refractory to de novo methylation. [23,24] Considering that TCGA adopted the HM450 bead assay to assess over 480K CpG sites covering approximately 96% of CpG islands from the UCSC database, the findings of the present study are robust and support that in EEC methylation-associated transcriptional silencing may account for the great majority of cases of ER/PR low expression, and this may be related to DNMT3A/3B overexpression. A recent study indicated that DNMT could be specifically targeted on particular loci and take part in specific de novo methylation. [3] From this point of view, our findings were consistent with this notion to some extent.
In addition, our data also demonstrated that the mechanism underlying ER or PR low expression in NEEC was distinct from that in EEC. In NEEC, the ESR1 deletion might play a more important role in the low expression of ESR1 compared with hypermethylation. NEEC showed many more ESR1 deletions (25.9%) than EEC (2.7%), and ESR1 deletion occurred more often in the ER low expression subgroup (31.3%) than in the ER highexpression subgroup (12.8%); the difference showed statistical significance. Furthermore, although DNMT3A/3B overexpression (69.9%, 72.8%), hypermethylation (93.3%) and low expression (68.9%) of ER the gene occurred more often in NEEC than those in EEC, their associations were not statistically significant. ESR1 was consistently hypermethylated in the ER low expression subgroup (96.3%) as well as in the ER highexpression subgroup (86.5%). Together, our findings suggest that low ER expression in NEEC might be related to multiple aberrations, and the contribution of methylation catalyzed by DNMT3A/3B could be shielded by other more dominant factors.
For low PR expression in NEEC, our data support that methylation still exerted great effects, while CNA and mutation had little influence. In the low PR expression subgroup, the hypermethylation rate of PGR (79.6%) was significantly higher than in the PR high-expression subgroup (59.5%). However, such methylation was not correlated with DNMT3A/3B overexpression. Recently, one study indicated that PR expression is downregulated at four different levels. [25] In well-differentiated endometrial carcinomas, ligand-induced receptor activation and downregulation are intact. miRNAs mediate the fine tuning of the PR level. As differentiation is lost, PR silencing is mainly at an epigenetic level. Initially, recruitment of the polycomb repressor complex 2 to the PR promoter suppresses transcription. Subsequently, DNA methylation prevents PR expression. Together, all these findings might imply that although methylation is the primary mechanism of PR downregulation in EEC as well as in NEEC, other regulators may be different between endometrial cancer subtypes.
A major limitation of this study is that we used the RNA-Seq expression count of DNMTs from the TCGA to perform the analysis of EC samples without available corresponding protein data to confirm the result; thus, our findings should be interpreted with caution. In addition, data regarding lymphovascular space invasion, recurrence sites, and treatment modalities were not recorded in the TCGA data set, thus limiting the clinical outcome analysis of patients with EC. Nonetheless, the RNA-Seq and its large-scale, uniform accuracy, robust methodology, and high throughput nature, strengthen this study to some extent.
In summary, our findings suggest that DNMT3A/3B overexpression was associated with poor survival in endometrial cancer. DNMT3A/3B might function synergistically. The mechanisms underlying low ER/PR expression may be distinct in EEC vs. NEEC. In EEC, methylation induced by DNMT3A/3B overexpression might play a major role in ER/PR downregulation. Thus, advances in the understanding of the molecular mechanisms of endometrial carcinomas will facilitate the development of novel anticancer therapeutic strategies.
Funding
Figure 1 :
1DNMT3A/3B overexpressed in endometrial cancers and indicated a poor prognosis. (A) DNMT3A/3B overexpressed in EEC and was much higher in NEEC compared with normal controls. * P < 0.001 vs. normal controls or EEC. (B) The expression of DNMT3A and 3B was significantly positively correlated. (C and D) DNMT3A or DNMT3B overexpression correlated with poor survival and the combined two markers also implied unfavorable prognosis (e). DNMT3A/3B: DNA (cytosine-5)-methyltransferase 3A/3B; EEC: Endometrial endometrioid carcinoma; NEEC: Non-endometrial endometrioid carcinoma. these mutations had little effect on their expression (P > 0.05).
n (%). EEC: Endometrial endometrioid carcinoma; ESR1: Estrogen receptor 1; NEEC: Non-endometrial endometrioid carcinoma;
Figure 3 :
3Hypermethylation or low expression of ESR1/PGR was correlated with poor survival. DNMT3A/3B: DNA (cytosine-5)-methyltransferase 3A/3B; EEC: Endometrial endometrioid carcinoma; ESR1: Estrogen receptor 1; NEEC: Non-endometrial endometrioid carcinoma; PGR: Progesterone receptor.
Figure 4 :
4Combination of DNMT3A/3B overexpression with ESR1/PGR low expression and/or hypermethylation indicated poor prognosis in endometrial cancers. DNMT3A/3B: DNA (cytosine-5)-methyltransferase 3A/3B; ESR1: Estrogen receptor 1; PGR: Progesterone receptor.
Figure 2 :
2Hypermethylation (A) and reduced expression (B) of ESR1/PGR occurred more frequently in tumors with DNMT3A/3B overexpression compared with tumors without DNMT3A/3B overexpression in EEC; however, the phenomena were not present in NEEC. DNMT3A/3B: DNA (cytosine-5)-methyltransferase 3A/3B; EEC: Endometrial endometrioid carcinoma; ESR1: Estrogen receptor 1; NEEC: Non-endometrial endometrioid carcinoma; PGR: Progesterone receptor.
Table 1 :
1Clinicopathologic features of 544 endometrial carcinomas Data are presented as median (range) or n (%). EEC: Endometrial endometrioid carcinoma; NEEC: Non-endometrial endometrioid carcinoma.Variables
Values
Age (years)
64 (31-90)
Premenopausal
35 (7.0)
Perimenopausal
17 (3.4)
Postmenopausal
445 (89.6)
Race
White
373 (72.9)
Black or African-American
106 (20.7)
Asian
20 (3.9)
Other
13 (2.6)
Histology
EEC
408 (75.0)
NEEC
136 (25.0)
Grade
1
97 (23.8)
2
118 (28.9)
3
193 (47.3)
Myometrial invasion
Superficial (<50%)
260 (55.3)
Deep (≥50%)
210 (44.7)
Lymph node metastasis
No
291 (77.8)
Yes
84 (22.2)
Stage
I
340 (62.5)
II
52 (9.6)
III
123 (22.6)
IV
29 (5.3)
Chinese Medical Journal 2019;132
Table 2 :
2Data are presented as mean ± standard deviation. DNMT3A/3B: DNA (cytosine-5)-methyltransferase 3A/3B; EEC: Endometrial endometrioid carcinoma; NEEC: Non-endometrial endometrioid carcinoma; RSEM: RNA-Seq by Expectation maximization; SD: Standard deviation.* F values. † t values.Comparison of clinicopathologic features and expression levels of DNMT3A/3B in 544 endometrial cancers
mRNA RSEM (log 2 )
Parameters
DNMT3A
Statistics
P
DNMT3B
Statistics
P
Statistics
P
Age
0.931
*
0.395
2.412
*
0.091
Pre-menopausal
9.96 ± 0.44
7.90 ± 1.12
10.090 † <0.001
Peri-menopausal
9.97 ± 0.59
8.03 ± 1.25
5.793 † <0.001
Post-menopausal
10.10 ± 0.69
8.30 ± 1.10
29.186 † <0.001
Race
0.800
*
0.602
0.163
*
0.959
White
10.09 ± 0.68
8.27 ± 1.06
27.863 † <0.001
Black or African-American 10.09 ± 0.72
8.19 ± 1.17
14.198 † <0.001
Asian
9.89 ± 0.45
8.29 ± 1.10
5.999 † <0.001
Other
10.00 ± 0.41
8.25 ± 1.11
4.937 † <0.001
Histology
À7.440 †
<0.001
À6.897 † <0.001
EEC
9.94 ± 0.55
8.07 ± 1.01
32.757 † <0.001
NEEC
10.49 ± 0.81
8.79 ± 1.16
14.089 † <0.001
Grade
À2.226 †
0.027
À4.358 † <0.001
1 or 2
9.88 ± 0.52
7.87 ± 0.93
27.637 † <0.001
3
10.00 ± 0.57
8.30 ± 1.05
19.777 † <0.001
Myometrial invasion
À1.748 †
0.081
À1.667 †
0.096
Superficial (<50%)
10.01 ± 0.64
8.12 ± 1.10
23.894 † <0.001
Deep (≥50%)
10.12 ± 0.69
8.29 ± 1.06
20.891 † <0.001
Lymph node metastasis
À3.089 †
<0.001
À1.728 †
0.025
No
10.02 ± 0.65
8.22 ± 1.10
23.184 † <0.001
Yes
10.34 ± 0.70
8.53 ± 1.09
13.018 † <0.001
Stage
À3.785 †
<0.001
À2.653 †
0.008
I or II
10.01 ± 0.64
8.17 ± 1.08
28.933 † <0.001
III or IV
10.25 ± 0.70
8.45 ± 1.11
16.952 † <0.001
Table 3 :
3Univariate and multivariate analyses for disease free survivalVariables
Table 4 :
4Associations between methylation, copy number alteration, and mutation of ESR1, PGR, and their expression statusEEC
Table 5 :
5Correlation between DNMT3A/3B expression and the methylation and expression status of ESR1/PGRDNMT3A
Expression
DNMT3B
Expression
EEC
NEEC
EEC
NEEC
Parameters
Low
High
Total
Low
High
Total
Low
High
Total
Low
High
Total
ESR1
Hypomethylation
122 (64.2)
58 (47.9)
180 (57.9)
1 (2.7)
7 (8.5)
8 (6.7)
114 (62.6)
66 (51.2)
180 (57.9)
2 (5.9)
6 (7.1)
8 (6.7)
Hypermethylation
68 (35.8)
63 (52.1)
131 (42.1) 36 (97.3) 75 (91.5) 111 (93.3)
68 (37.4)
63 (48.8)
131 (42.1) 32 (94.1) 79 (92.9) 111 (93.3)
x 2
8.033
À
4.077
À
P
0.005
0.432
0.043
1.000
PGR
Hypomethylation
155 (81.6)
78 (64.5)
233 (74.9) 13 (35.1) 22 (26.8)
35 (29.4)
145 (79.7)
89 (69.0)
234 (75.2) 10 (29.4) 25 (29.4)
35 (29.4)
Hypermethylation
35 (18.4)
43 (35.5)
78 (25.1)
24 (64.9) 60 (73.2)
84 (70.6)
37 (20.3)
40 (31.0)
77 (24.8)
24 (70.6) 60 (70.6)
84 (70.6)
x 2
11.526
0.847
4.621
0.000
P
0.001
0.357
0.032
1.000
ESR1
Low
36 (14.1)
45 (29.4)
81 (19.9)
27 (65.9) 70 (73.7)
97 (71.3)
43 (18.3)
38 (22.0)
81 (19.9)
29 (78.4) 68 (68.7)
97 (71.3)
High
219 (85.9) 108 (70.6) 327 (80.1) 14 (34.1) 25 (26.3)
39 (28.7)
192 (81.7) 135 (78.0) 327 (80.1)
8 (21.6)
31 (31.3)
39 (28.7)
x 2
14.057
0.859
0.842
1.237
P
<0.001
0.354
0.359
0.266
PGR
Low
43 (16.9)
58 (37.7)
101 (24.8) 34 (82.9) 75 (78.9) 109 (80.1)
46 (19.6)
55 (31.8)
101 (24.8) 31 (83.8) 78 (78.8) 109 (80.1)
High
211 (83.1)
96 (62.3)
307 (75.2)
7 (17.1)
20 (21.1)
27 (19.9)
189 (80.4) 118 (68.2) 307 (75.2)
6 (16.2)
21 (21.2)
27 (19.9)
x 2
22.125
0.285
7.985
0.422
P
<0.001
0.593
0.005
0.516
ER-a
Low
55 (27.1)
61 (48.8)
116 (35.4) 26 (78.8) 51 (66.2)
77 (70.0)
53 (28.5)
63 (44.4)
116 (35.4) 22 (71.0) 55 (69.6)
77 (70.0)
High
148 (72.9)
64 (51.2)
212 (64.6)
7 (21.2)
26 (33.8)
33 (30.0)
133 (71.5)
79 (55.6)
212 (64.6)
9 (29.0)
24 (30.4)
33 (30.0)
x 2
15.946
1.734
8.874
0.019
P
<0.001
0.188
0.003
0.890
PR
Low
105 (51.7)
86 (68.8)
191 (58.2) 31 (93.9) 63 (81.8)
94 (85.5)
98 (52.7)
93 (65.5)
191 (58.2) 27 (87.1) 67 (84.8)
94 (85.5)
High
98 (48.3)
39 (31.2)
137 (41.8)
2 (6.1)
14 (18.2)
16 (14.5)
88 (47.3)
49 (34.5)
137 (41.8)
4 (12.9)
12 (15.2)
16 (14.5)
x 2
9.275
À
5.428
À
P
0.002
0.141
0.020
1.000
The data were shown as
n (%). BMI: Body mass index;
DNMT3A/3B: DNA (cytosine-5)-methyltransferase 3A/3B; EEC: Endometrial endometrioid carcinoma; ER-a: Estrogen receptor-a; ESR1: Estrogen
receptor 1; NEEC: Non-endometrial endometrioid carcinoma; PGR: Progesterone receptor; PR: Progesterone receptor (PR is encoded by a single
PGR gene).
Chinese Medical Journal 2019;132(2)
www.cmj.org
The former was also significantly correlated with ER/ PR downregulation, whereas mutations of ESR1 and PGR seemed to have little effect. Second, in EEC, DNMT3A overexpression was significantly associated with ESR1 and PGR hypermethylation and their low expression. The low ER/PR expression (29.4%, 37.7%) and hypermethylation (52.1%, 35.5%) occurred more frequently in the DNMT3A overexpression subgroup compared with the DNMT3A normal expression subgroup (14.1%, 16.9%; 35.8%, and 18.4%, respectively). Similar phenomena were observed in the DNMT3B overexpression subgroups. Third, the combined DNMT3A/3B and ER/PR status or these biomarkers alone were all correlated with shorter survival, and DNMT3A was an independent prognostic factor in multivariate analysis.Chinese Medical Journal 2019;132(2)
www.cmj.org
support that ESR1/PGR silencing occurred primarily at the
epigenetic level. Among the ER and PR low expression
subgroups, 83.1% of ESR1 and 59.5% of PGR were
hypermethylated with statistical significance, with a
minority of cases showing deletion (7.6% for ESR1,
13.1% for PGR) or mutation (8.0% for ESR1, 6.5% for
PGR).
Chinese Medical Journal 2019;132(2) www.cmj.org
This study was supported by a grant from the National Natural Science Foundation of China (No. 81360381).Conflicts of interestNone.
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. D He, X Wang, Y Zhang, J Zhao, R Han, Y Dong, How to cite this article: He D, Wang X, Zhang Y, Zhao J, Han R, Dong Y.
DNMT3A/3B overexpression might be correlated with poor patient survival, hypermethylation and low expression of ESR1/PGR in endometrioid carcinoma: an analysis of the Cancer Genome Atlas. 10.1097/CM9.0000000000000054Chin Med J. 132DNMT3A/3B overexpression might be correlated with poor patient survival, hypermethylation and low expression of ESR1/PGR in endometrioid carcinoma: an analysis of the Cancer Genome Atlas. Chin Med J 2019;132:161-170. doi: 10.1097/CM9.0000000000000054
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Recent findings have identified methylation occurring at the cytosine of mRNA as a new epitranscriptomic mark beyond the methylation of the adenine [1]. These marks open new biological horizons and define the existence of other possible mechanisms that do not correlate with alteration of the genetic sequence, epigenetic modifications or even post-translational modifications. The epitranscriptome identifies methylation occurring at the transcript level, which were once invisible [2], and that could be responsible for the maturation and translational processing of mRNAs. Once more, epi-marks claim their role in cellular processes and further confirm their involvement as key players in cell fate decisions.
Epitranscriptomic modifications have been identified in cancer cells. In particular, methylation at the N6 adenosine of METTLR3 and IKHB5 mRNAs occur in bladder cancer, inhibit ITGA6 expression, and correlate with poor prognosis [3]. Recent advances have highlighted epitranscriptomic processes in pancreatic cancer too. In a study recently published in EBioMedicine, methylation occurring in total cellular mRNA at cytosine 5 decreased in patients affected by pancreatic cancer. The methylation status correlated with clinicopathological parameters such as T stage, proliferation index, tumour recurrence and survival. Furthermore, the authors identified that m5C level correlated with decreased expression of the methyltransferase NOP2/Sun RNA Methyltransferase 6 (NSUN6) [4]. The importance of its activity was recently determined by observing augmented translation of those mRNAs, that were methylated by NSUN6 [5,6]. The emerging role of NSUN6, and m5C methylation mediated by its activity, has been also found in gastrointestinal and head and neck cancers [7,8]. The study of Yang and colleagues showed for the first time the involvement of NSUN6 and its relation to m5C methylation in pancreatic cancer.
The peculiar mechanism of translational processing stands at the crossroads between epigenetic modification (occurring at the genome level), and the interaction between mRNAs and their counterparts with inhibitory function (small-non-coding RNAs). Interestingly, methylation occurring at the adenine and the cytosine of mRNA act differently than in the corresponding DNA. Epigenetic methylation of DNA is responsible for silencing the genetic sequence, to stop transcription. Instead, methylation occurring on mRNA bases promotes nucleic acid translation.
Methylation could offer new insights in terms of interaction between mRNAs, the RNA-induced silencing complex (RISC), and miRNAs, highlighting new regulatory processes of RNA maturation and the translation machinery. This epitranscriptomic mark might not only affect mRNAs and tRNAs, but also small-non-coding RNA and the long-non-coding RNAs, by modulating affinity for their targets, thus amplifying the role exerted by the methylation of RNA. Of note, putative RNA-only methyltransferases have not yet been identified. It has been shown that DNA methyltransferases (DNMTs) and NSUNs are responsible for RNA methylation, but that they are capable of also methylating DNA [4]. Up to now, their dual function of inhibiting transcription and promoting translation is not clearly understood. Further studies are needed to clarify which regulatory processes modulate the functioning of those methyltransferases at the nuclear and cytosolic level.
In the future, the epitranscriptome could be fully integrated in diagnostic tools for the identification of clinicopathological parameters and offer potential therapeutic insight for the treatment of different tumour types.
Declaration of Competing Interest
The author reports no conflicts of interest.
The role of m. X Y Chen, J Zhang, J S Zhu, Mol Cancer. 181103Chen XY, Zhang J, Zhu JS. The role of m. Mol Cancer 2019;18(1):103.
Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. D Dominissini, S Moshitch-Moshkovitz, S Schwartz, M Salmon-Divon, L Ungar, S Osenberg, K Cesarkas, J Jacob-Hirsch, N Amariglio, M Kupiec, R Sorek, G Rechavi, Nature. 485Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 2012;485:201-6.
. H Jin, X Ying, B Que, X Wang, Y Chao, H Zhang, Z Yuan, D Qi, S Lin, Min W , Yang M , Ji W N , EBioMedicine. 47Jin H, Ying X, Que B, Wang X, Chao Y, Zhang H, Yuan Z, Qi D, Lin S, Min W, Yang M, Ji W. N. EBioMedicine 2019;47:195-207.
The RNA methyltransferase NSUN6 suppresses pancreatic cancer development by regulating cell proliferation. R Yang, EBioMedicine. 63103195Yang R, et al. The RNA methyltransferase NSUN6 suppresses pancreatic cancer development by regulating cell proliferation'. EBioMedicine 2021;63:103195.
method for unbiased screening of novel mRNA modification regulators. L Fang, W Wang, G Li, L Zhang, J Li, D Gan, J Yang, Y Tang, Z Ding, M Zhang, W Zhang, D Deng, Z Song, Q Zhu, H Cui, Y Hu, W Chen, 10.1016/j.ebiom.2020.103195Mol Syst Biol. 161110025CIGAR-seq, a CRISPR/Cas-based DOIFang L, Wang W, Li G, Zhang L, Li J, Gan D, Yang J, Tang Y, Ding Z, Zhang M, Zhang W, Deng D, Song Z, Zhu Q, Cui H, Hu Y, Chen W. CIGAR-seq, a CRISPR/Cas-based DOI of original article: http://dx.doi.org/10.1016/j.ebiom.2020.103195. method for unbiased screening of novel mRNA modification regulators. Mol Syst Biol 2020;16(11):e10025.
Sequence-and structure-specific cytosine-5 mRNA methylation by NSUN6. T Selmi, S Hussain, S Dietmann, M Heiß, K Borland, S Flad, J M Carter, R Dennison, Y L Huang, S Kellner, S Bornel€ Ov, M Frye, Nucleic Acids Res. Selmi T, Hussain S, Dietmann S, Heiß M, Borland K, Flad S, Carter JM, Dennison R, Huang YL, Kellner S, Bornel€ ov S, Frye M. Sequence-and structure-specific cyto- sine-5 mRNA methylation by NSUN6. Nucleic Acids Res 2020.
Emerging roles of RNA methylation in gastrointestinal cancers. S Xie, W Chen, K Chen, Y Chang, F Yang, A Lin, Q Shu, T Zhou, X Yan, Cancer Cell Int. 201585Xie S, Chen W, Chen K, Chang Y, Yang F, Lin A, Shu Q, Zhou T, Yan X. Emerging roles of RNA methylation in gastrointestinal cancers. Cancer Cell Int 2020;20(1):585.
Gene signatures of m5C regulators may predict prognoses of patients with head and neck squamous cell carcinoma. M Xue, Q Shi, L Zheng, Q Li, L Yang, Y Zhang, Am J Transl Res. 1210Xue M, Shi Q, Zheng L, Li Q, Yang L, Zhang Y. Gene signatures of m5C regulators may predict prognoses of patients with head and neck squamous cell carcinoma. Am J Transl Res 2020;12(10):6841-52.
| Epitranscriptomics, also known as "RNA epigenetics", is a chemical modification for RNA regulation. Ribonucleic acid (RNA) methylation is considered to be a major discovery following the deoxyribonucleic acid (DNA) and histone methylation. Messenger RNA (mRNA) methylation modification accounts for more than 60% of all RNA modifications and N6-methyladenosine (m 6 A) is known as one of the most common type of eukaryotic mRNA methylation modifications in current. The m 6 A modification is a dynamic reversible modification, which can directly or indirectly affect biological processes, such as RNA degradation, translation and splicing, and can play important biological roles in vivo. This article introduces the mRNA m 6 A methylation modification enzymes and binding proteins, and reviews the research progress and related mechanisms of the role of mRNA m 6 A methylation in the nervous system from the aspects of neural stem cells, learning and memory, brain development, axon growth and glioblastoma. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Main textDiscovery and distribution of m 6 AThe m 6 A is the most common and abundant methylation modification in mRNA[12,13]. In 1974, Desrosie used the polyadenosinic acid (PolyA) structure in eukaryotes, to discover the methylation status of mRNA in hepatoma cells, and found that the main methylation modification in mRNA was m 6 A (approximately 80%)[8]. In addition, | Sarcomas are rare malignant tumors that may arise from anywhere of the body, such as bone, adipose, muscle and vascular. However, the conventional pathogenesis of sarcomas has not been found. Therefore, there is an urgent need to identify novel therapeutic strategies and improve prognosis effects for sarcomas. Methylation of N6 adenosine (m6A) regulation is a novel proposed regulatory pattern that works in posttranscription level, which was also the most widely distributed methylation modification in eukaryotic mRNA. Growing evidences have demonstrated that m6A modification played an indispensable role in tumorigenesis. Here, we integrated multi-omics data including genetic alterations, gene expression and epigenomics regulation to systematically analysis the regulatory atlas of 21 m6A regulators in sarcoma. Firstly, we investigated the genetic alterations of m6A regulators and found that~44% TCGA sarcoma patients have genetic mutations. We also investigated the basic annotation of 21 regulators, such as expression correlation and PPI interactions. Then we identified the upstream and downstream regulatory networks of between transcription factors (TFs)/non-coding RNAs and m6A regulators in sarcoma based on motif analysis and gene expression. These results implied that m6A regulator mediated regulatory axes could be used as prognostic biomarkers in sarcoma. Knockdown experiment results revealed that m6A regulators, YTHDF2 and HNRNPA2B1 participated in the cancer cell invasion and metastasis. Moreover, we also found that the expression levels of m6A regulators were related to immune cell infiltration of sarcoma patients. | Background: Stemness and chemoresistance contribute to cervical cancer recurrence and metastasis. In the current study, we determined the relevant players and role of N 6 -methyladenine (m 6 A) RNA methylation in cervical cancer progression.Methods:The roles of m 6 A RNA methylation and centromere protein K (CENPK) in cervical cancer were analyzed using bioinformatics analysis. Methylated RNA immunoprecipitation was adopted to detect m 6 A modification of CENPK mRNA. Human cervical cancer clinical samples, cell lines, and xenografts were used for analyzing gene expression and function. Immunofluorescence staining and the tumorsphere formation, clonogenic, MTT, and EdU assays were performed to determine cell stemness, chemoresistance, migration, invasion, and proliferation in HeLa and SiHa cells, respectively. Western blot analysis, co-immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter, cycloheximide chase, and cell fractionation assays were performed to elucidate the underlying mechanism.Results: Bioinformatics analysis of public cancer datasets revealed firm links between m 6 A modification patterns and cervical cancer prognosis, especially through ZC3H13-mediated m 6 A modification of CENPK mRNA. CENPK expression was elevated in cervical cancer, associated with cancer recurrence, and independently predicts poor patient prognosis [hazard ratio = 1.413, 95% confidence interval = 1.078 − 1.853, P = 0.012]. Silencing of CENPK prolonged the overall survival time of cervical cancer-bearing mice and improved the response of cervical cancer tumors to chemotherapy in vivo (P < 0.001). We also showed that CENPK was directly bound to SOX6 and disrupted the interactions of CENPK with β-catenin, which promoted β-catenin expression and nuclear translocation, facilitated p53 ubiquitination, and led to activation of Wnt/β-catenin signaling, but suppression of the p53 pathway. This dysregulation ultimately enhanced the tumorigenic pathways required for cell stemness, DNA damage repair pathways necessary for cisplatin/carboplatin resistance, epithelial-mesenchymal transition involved in metastasis, and DNA replication that drove tumor cell proliferation. | The present study aimed to identify potentially critical differentially methylated genes associated with the progression of nasopharyngeal carcinoma (NPC). Methylation profiling data of GSE62336 deposited in the Gene Expression Omnibus database were used to identify differentially methylated regions (DMRs) and differentially methylated CpG islands (DMIs). Concurrently, differentially expressed genes (DEGs) were identified using a meta-analysis of three gene expression datasets (GSE53819, GSE13597 and GSE12452). Subsequently, methylated DEGs were identified by comparing DMRs and DEGs. Furthermore, functional associations of these methylated DEGs were analyzed via constructing a functional network using GeneMANIA prediction server. In total, 1,676 hypermethylated genes, 28 hypomethylated genes, 17 DMIs and 2,983 DEGs (1,655 upregulated and 1,328 downregulated) were identified. Among these DEGs, 135 downregulated genes were hypermethylated; of these, dual specificity phosphatase 6 (DUSP6) and tenascin XB (TNXB) contained DMIs. In the functional network, 154 genes and 1,651 association pairs were included. DUSP6 was predicted to exhibit genetic interactions with other hypermethylated DEGs such as malic enzyme 3 and ST3 β-galactoside α-2,3-sialyltransferase 5; TNXB was predicted to be co-expressed with a set of hypermethylated DEGs, including EPH receptor B6, aldehyde dehydrogenase 1 family, member L1 and glutathione peroxidase 3. The hypermethylated DEGs may be involved in the progression of NPC, and they may become novel therapeutic targets for NPC. | Background: Tumor suppressor epigenetic silencing plays an important role in non-small cell lung cancer (NSCLC) development and progression. Previously, the expression of speckle-type POZ protein (SPOP) has been found to be significantly inhibited in NSCLC. Our research aimed to investigate the molecular mechanisms, clinical significance and epigenetic alteration of SPOP in NSCLC.Materials and methods:Bisulfite sequencing PCR and methylation-specific PCR were performed to test gene methylation. Chromatin immunoprecipitation (ChIP) was performed to detect transcription factor C/EBPα combinations and the promoter of the SPOP gene. Furthermore, we evaluated the effects of C/EBPα siRNA on SPOP expression, tumor cell migration and proliferation via MTT and Transwell assays in vitro and tumor growth in vivo. The relationship between the methylation status of the SPOP gene and clinicopathologic characteristics was investigated.Results: Hypermethylation was found in the CpG island of the SPOP gene promoter in NSCLC tissues, and this methylation was found to be correlated with SPOP expression. SPOP promoter methylation was associated with the pathology grade. The transcriptional activities were significantly inhibited by the hypermethylation of specific CpG sites within the SPOP gene promoter, while 5-aza-2′-deoxycytidine significantly increased SPOP gene expression. C/EBPα also played a key role in SPOP regulation. Five C/EBPα binding sites in the CpG island of the SPOP gene promoter were identified by ChIP. Inhibition of C/EBPα significantly reduced SPOP expression. SPOP mediated the C/EBPα-regulated suppression of invasion, migration and proliferation in vitro and tumor growth in vivo.Conclusions: SPOP function and expression in NSCLS were regulated by DNA methylation and C/EBPα transcriptional regulation combination effects, indicating that the SPOP promoter methylation status could be utilized as an epigenetic biomarker and that the C/EBPα-SPOP signaling pathway could be a potential therapeutic target in NSCLC. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BackgroundCurrently, lung cancer is reportedly the leading cause of tumor-related deaths worldwide, accounting for more than 1 million deaths annually regardless of gender or ethnicity[1]. Nearly 85% of all lung carcinomas are non-small cell lung cancer (NSCLC) [2, 3], consisting of lung adenocarcinoma, lung squamous cell carcinoma, lung large-cell carcinoma and other rare types. The NSCLC prognosis is still poor, and the 5-year survival rate is approximately 15% due to several factors, including the small number of effective drugs, low diagnostic rate during the early stage, and high cancer recurrence and metastasis rates[4]. Although NSCLC is among the most extensively studied disorders over the last few years, it is a very complex process that needs further investigation. Similar to other malignancies, the development and progression of NSCLC are multistage | A) is a widely investigated RNA modification in studies on the "epigenetic regulation" of mRNAs that is ubiquitously present in eukaryotes. Abnormal changes in m 6 A levels are closely related to the regulation of RNA metabolism, heat shock stress, tumor occurrence, and development. m 6 A modifications are catalyzed by the m 6 A writer complex, which contains RNA methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor 1-associated protein (WTAP), and other proteins with methyltransferase (MTase) capability, such as RNA-binding motif protein 15 (RBM15), KIAA1429 and zinc finger CCCHtype containing 13 (ZC3H13). Although METTL3 is the main catalytic subunit, WTAP is a regulatory subunit whose function is to recruit the m 6 A methyltransferase complex to the target mRNA. Specifically, WTAP is required for the accumulation of METTL3 and METTL14 in nuclear speckles. In this paper, we briefly introduce the molecular mechanism of m 6 A modification. Then, we focus on WTAP, a component of the m 6 A methyltransferase complex, and introduce its structure, localization, and physiological functions. Finally, we describe its roles and mechanisms in cancer.Cell Death and Disease (2022) 13:852 ; https://doi. | Pancreatic cancer is associated with a high mortality rate, owing to de novo and acquired drug resistance, thereby leading to highly invasive and metastatic pancreatic cancer cells. Therefore, targeting pancreatic cancer stem cells (CSCs) may be a novel therapeutic strategy for the treatment of pancreatic cancer. Here, we combined a DNA methylation inhibitor (5-aza-2'-deoxycytidine; 5-aza-dC) and ionizing radiation (IR) to improve anti-cancer effects by inhibiting growth and proliferation and promoting apoptosis of pancreatic cancer cells in vitro and in vivo. Importantly, the combinatorial effect of 5-aza-dC with IR on sphere-forming pancreatic cancer cells was preferentially targeted toward CSCs through the downregulation of regulatory factors of self-renewal and CSC surface markers. We next performed the RNA sequencing to understand the underlying cellular mechanisms of the combined treatment with IR and 5-aza-dC in pancreatic cancer cells. Global transcriptome profiling indicated that the expression of the Oct4-centered transcriptional network of genes was significantly downregulated in cells with combination treatment. Our data suggested that combination treatment with DNA methylation inhibitor and IR may be a novel therapeutic strategy for pancreatic cancer. Overall, these findings support the use of epigenetic therapy in combination with radiotherapy to improve therapeutic efficacy by targeting and eradicating pancreatic CSCs. | During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the -class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells. |
463 | Documenting cryptographic operations | We present a new mechanized prover for secrecy properties of cryptographic protocols. In contrast to most previous provers, our tool does not rely on the Dolev-Yao model, but on the computational model. It produces proofs presented as sequences of games; these games are formalized in a probabilistic polynomial-time process calculus. Our tool provides a generic method for specifying security properties of the cryptographic primitives, which can handle shared-and public-key encryption, signatures, message authentication codes, and hash functions. Our tool produces proofs valid for a number of sessions polynomial in the security parameter, in the presence of an active adversary. We have implemented our tool and tested it on a number of examples of protocols from the literature. | In this paper, a new first-order logical framework and method of formalizing and verifying cryptographic protocols is presented. From the point of view of an intruder, the protocol and abilities of the intruder are modeled in Horn clauses. Based on deductive reasoning method, secrecy of cryptographic protocols is verified automatically, and if the secrecy is violated, attack scenarios can be presented through back-tracing. The method has been implemented in an automatic verifier, many examples of protocols have been analyzed in less then 1s. | Application development for the cloud is already challenging because of the complexity caused by the ubiquitous, interconnected, and scalable nature of the cloud paradigm. But when modern secure and privacy aware cloud applications require the integration of cryptographic algorithms, developers even need to face additional challenges: An incorrect application may not only lead to a loss of the intended strong security properties but may also open up additional loopholes for potential breaches some time in the near or far future. To avoid these pitfalls and to achieve dependable security and privacy by design, cryptography needs to be systematically designed into the software, and from scratch. We present a system architecture providing a practical abstraction for the many specialists involved in such a development process, plus a suitable cryptographic software development life cycle methodology on top of the architecture. The methodology is complemented with additional tools supporting structured inter--domain communication and thus the generation of consistent results: cloud security and privacy patterns, and modelling of cloud service level agreements. We conclude with an assessment of the use of the Cryptographic Software Design Life Cycle (CryptSDLC) in a EU research project. | Cryptographic algorithm detection has received a lot of attentions in these days, whereas the method to detect decrypted data remains further research. A decrypted memory detection method using dynamic dataflow analysis is proposed in this paper. Based on the intuition that decrypted data is generated in the cryptographic function and the unique feature of decrypted data, by analyzing the parameter sets of cryptographic function, we propose a model based on the input and output of cryptographic function. Experimental results demonstrate that our approach can effectively detect decrypted memory. | The diagram illustrates the encryption process . | In this paper we report on our efforts in attacking commercial authentication products. The challenge in such attacks is that only very limited information on the implementations are given. We were able to reveal information on the processing sequence during the authentication process even as detailed as identifying the clock cycles in which the individual key bits are processed. To summarize the effort of such an attack is significantly higher than the one of attacking a well known implementation. | Interorganizational workflow systems play a fundamental role in business partnerships. We introduce and investigate the concept of workflow signatures. Not only can these signatures be used to ensure authenticity and protect integrity of workflow data, but also to prove the sequence and logical relationships, such as AND-join and AND-split, of a workflow. Hence, workflow signatures can be electronic evidence useful for auditing, that is proving compliance of business processes against some regulatory requirements. Furthermore, signing keys can be used to grant permissions to perform tasks. Since the signing keys are issued on-the-fly, authorization to execute a task within a workflow can be controlled and granted dynamically at runtime. In this paper, we propose a concrete workflow signature scheme, which is based on hierarchical identity-based cryptography, to meet security properties required by interorganizational workflows. | We propose a PMAC-type mode of operation that can be used as a highly secure MAC (Message Authentication Code) or PRF (Pseudo-Random Function). Our scheme is based on the assumption that the underlying n-bit blockcipher is a pseudo-random permutation. Our construction, which we call PMAC Plus, involves extensive modification to PMAC, requiring three blockcipher keys. The PMAC Plus algorithm is a first rate-1 (i.e., one blockcipher call per n-bit message block) blockcipher-based MAC secure against O(22n/3) queries, increasing the O(2n/2) security of PMAC at a low additional cost. Our analysis uses some of the security-proof techniques developed with the sum construction (Eurocrypt 2000) and with the encrypted-CBC sum construction (CT-RSA 2010). | 463 | Hello,
I made an attempt at solving my IRC chat encryption needs:
~eau/wic
I was wondering how to properly document (descriptive, diagrams?) my usage of crypto primitives to allow an easier and code independent review of the usage of crypto primitives itself including the limitations that led to those choices ?(example: IRC msg are 512 bytes long, ...)
Examples would be more than welcome, feedback if people are curious about my attempt.
Cheers. | Revisiting Cryptographic Accumulators, Additional Properties and Relations to other Primitives ? | I2C protocol, not understanding acknowledge bit | On Six Basic Forms of Confidentiality Cognition | IDEAS To Implement A Way to Make Ciphers have a use and a way to make it fun and challenging to acquire them: | In this paper, we present an efficient revocable signcryption (CP-ABSC) with outsourced unsigncryption scheme based on ciphertext-policy attribute based encryption (CP-ABE) to secure the data sharing in cloud computing that require access control, data encryption, and authentication to ensure message integrity and confidentiality. It can be used to signcrypt a message based on the access rights specified by the message itself. A user can decrypt a ciphertext if and only if it possesses the attributes required by the access structure of the data. Thus CP-ABSC felxible encrypts data based on the access rights of the data specified by the data itself, which differs significantly from the traditional encryption where the user can decrypt is predetermined and must be known by the data source. In addition, the proposed scheme handles attributes revocation with high efficiency and demands low computation overhead on users' device during unsigncryption phase. Our scheme provides collusion attack resistance, message authentication, forgery prevention, and confidentiality. Furthermore, we compare the performance of our scheme with others in terms of the ciphertext, key size, computation cost and functionality.INDEX TERMS Attribute-based encryption, attributes revocation, outsourced decryption, ciphertext-policy. | What is a basic outline of "military-grade" encryption? | AE ERC20 Security question | Cryptographic primitives in blockchains |
464 | The root cause of obesity | Why are poor people obese? | Which are physical health problems that can be caused by being overweight or obese? | What kind of obesity caused by diet? | The more insulin you produce, the more calories you store instead of burn, so the less energy you burn, the more calories you consume, which is why more calories consumed is assumed to be the cause of being fat | In a majority of cases, being overweight is a direct result of poor dieting and exercise. Fat people are fat because of their own actions. I realize there are fringe cases in which someone's weight is out of their control, this is not about them.
Fat people are less attractive, smell worse, take up more space, move slower and always want the AC cranked way up to chill their blubbery sweat flabs. They are a minority(or in the US, majority) that has to be consistently accommodated for. ALL because of their own choices and actions that caused them to become a blob of inconvenience.
We shame smokers. We shame drug users and drinkers. We shame people for bad dental hygiene. Why can't we shame fat people? Why did we have to remove fatpeoplehate?
"Body acceptance" is a joke. People, especially Americans should be encouraged to maintain their health. We should feel bad for some 300 pound narcissistic delusional whale that thinks her weight isn't an issue. | Etiology of Massive Obesity: Role of Genetic Factors | Metabolic syndrome adipose tissue, ultimately promoting visceral adiposity, insulin resistance, dyslipidemia and hypertension, with direct effects on the bone, causing "low turnover" osteoporosis. HPA-axis dysfunction may explain the reported risk indication of abdominal obesity to cardiovascular disease (CVD), type 2 diabetes and stroke. Psychosocial stress is also linked to heart disease. Central obesity is a key feature of the syndrome, being both a sign and a cause, in that the increasing adiposity often reflected in high waist circumference may both result from and contribute to insulin resistance. However, despite the importance of obesity, patients who are of normal weight may also be |
The Current Situation of Obesity and Its Adverse Effects
Obesity, defined as excessive fat deposition, has become increasingly prevalent worldwide. Over the last 40 years, the prevalence rates of obesity in adults (defined as BMI over 30 kg/m 2 ) has been increasing at a rapid pace in both Western societies and developing countries, with the number of obese adults reaching 671 million in 2016 (390 million women and 281 million men) compared to 100 million in 1975 (69 million women and 31 million men) [1]. In addition, the prevalence of childhood obesity has increased steadily in the developed and developing countries [2]. For example, approximately one-third of children in America are overweight or obese [3,4]. A similar situation has occurred in China, with the prevalence of childhood overweight and obesity being about one in five [5,6].
Obesity has become a major public health burden all over the world. It is now well established that obesity is able to progressively lead to and/or exacerbate a wide range of comorbidities [7][8][9], including insulin resistance and type 2 diabetes mellitus (T2DM) [10,11], dyslipidemia [12,13], hypertension [14,15], cardiovascular disease [16,17], nonalcoholic fatty liver disease [18,19], reproductive dysfunction [20][21][22], and cancer [23,24]. As a result, obesity causes adverse effects on the quality of life and has marked economic consequences relating to increased healthcare costs [25,26]. In view of the prevalence of obesity, health consequences, and healthcare costs, there has been substantial interest in identifying effective and safe interventions/strategies to reduce excess body weight/fat in obese people.
Although there are many factors that influence obesity and various interventions to treat it [27], a large body of evidence has demonstrated that dietary interventions/strategies are an effective and safe way to prevent or manage obesity [28][29][30][31]. As one of the micronutrients in the diet, calcium regulates many cellular processes, such as cell proliferation [32], differentiation [33], and bone formation [34]. In addition, dietary calcium has been implicated to be involved in prevention or treatment of obesity [35][36][37][38][39][40]. Onakpoya et al. reported the efficacy of calcium supplementation for management of overweight people and aimed to clarify the treatment effect of calcium supplementation in obese people [36]. Soares et al. mainly demonstrated the effect of calcium and vitamin D on obesity [41]. Barba and Russo's review focused on the association between dairy product consumption and body weight regulation in humans [40].These studies mainly focused on humans or the anti-obesity effect of calcium on overweight people. At present, there are few comprehensive reviews describing the anti-obesity effects of calcium in different models and the underlying mechanisms. In this review, we compile the evidence for the anti-obesity effects of calcium in cell models, animals, and humans and summarize the possible mechanisms by which calcium elicits its anti-obesity effects.
The Anti-Obesity Effects of Calcium Supplementation
Inhibition of Adipogenic Differentiation by Calcium in Cell Models
Expanded fat mass can result from increased adipocyte number (adipogenesis or hyperplasia) and/or increased adipocyte size (hypertrophy) [42]. A decrease in adipogenesis and lipogenesis and/or an increase in lipolysis contribute to fewer adipocyte number and smaller adipocyte size, thus leading to a reduction in fat accumulation. It has been reported that high extracellular calcium ([Ca 2+ ] o , 5 and 10 mM) attenuates adipogenesis in 3T3-L1 preadipocytes [43]. Similarly, 5 mM [Ca 2+ ] o and increased intracellular calcium ([Ca 2+ ] i ) with RyR channel excitomotor caffeine significantly reduced the intracellular lipid content in the primary preadipocytes of mice [44,45]. In addition, increasing [Ca 2+ ] i was able to inhibit early stages of adipogenic differentiation in human preadipocytes [46]. Taken together, these in vitro data indicate that direct treatment of murine and human preadipocytes with calcium or calcium channel regulators could elicit inhibitory effects on adipogenic differentiation.
Anti-Obesity Effects of Dietary Calcium in Animals
Accumulating evidence has demonstrated that dietary calcium supplementation elicits anti-obesity effects on various animals. Our study has indicated that calcium supplementation (0.6% w/w) in drinking water leads to significant decrease in body weight, body fat content, and inguinal white adipose tissue (iWAT) and epididymal WAT (eWAT) index in high-fat diet (HFD)-induced obese mice [47]. In line with our report, Sun et al. found that dietary supplementation of 1.4% and 2.8% calcium significantly decreased the body weight gain and the fat net weight of inguinal fat pad (IFP), epididymal fat pad (EFP), and perirenal fat pad (PFP) in HFD-fed mice [45]. The anti-obesity or body-fat-lowering effects of calcium in mice have also been reported in other studies [48,49]. In rats, it has been shown that, compared with standard chow, calcium-supplemented chow (10 g CaCO 3 /kg of chow) significantly decreased body mass and visceral adipose tissue (VAT) mass in epididymal, retroperitoneal, and mesenteric depots [50]. In agreement, dietary calcium supplementation (10 g/kg) resulted in significant reduction of body mass, trunk fat, and total fat in early weaning Wistar rats [51]. With regard to the brown adipose tissue (BAT), it was reported that calcium-supplemented chow (10 g/kg) had no effects on rat BAT weight compared to standard chow [52]. Collectively, these data suggest that dietary calcium intake could elicit beneficial effects on reducing body fat deposition in murine models.
Anti-Obesity Effects of Dietary Calcium in Humans
Many studies have evaluated the effects of dietary calcium supplementation on body weight/fat loss in humans [38,39,53,54]. According to a survey, people in both developed and lesser developed countries have inadequate calcium intake [35]. The 2011 Institute of Medicine Dietary Reference Intake committee set the recommended dietary allowances at 1300 mg/day calcium for children aged 9-18 years and 1000-1200 mg/day (varying by age) for healthy adults [55]. In fact, most American children do not yet meet these recommendations [56,57]. Thus, increasing the intake of daily calcium is the primary condition for health and may contribute to body weight/fat loss. A meta-analysis revealed the negative correlations between calcium supplementation and weight changes in children and adolescents, in adult men, and either premenopausal or old (above 60 years old) women and suggested that increasing calcium intake could reduce body weight in these subjects [53]. Specifically, it has been demonstrated that each 300 mg increment in regular calcium intake is associated with approximately 1 kg less body fat in children and 2.5-3.0 kg lower body weight in adults [54]. Rosenblum et al. found that calcium and/or vitamin D supplementation contributed to a beneficial reduction of abdominal visceral adipose tissue in overweight and obese adults [58]. In contrast, Winzenberg et al. reported that there was no evidence to support the use of calcium supplementation as a public health intervention to reduce weight gain or body fat in healthy children [59]. It should be noted that vitamin D, which exerts a critical role in calcium absorption [60], plays an important role in influencing the anti-obesity effects of calcium. It has been reported that a deficiency of vitamin D decreases the calcium intake and increases body mass index in children and adolescents [61]. In addition, dietary calcium overdosage has been implicated in some adverse effects, including kidney stones, myocardial infarction, hypercalcemia, and hospitalization with acute gastrointestinal symptoms [62]. Excess (>1200 mg/day) dietary calcium intake is related to higher Framingham Risk Score (FRS), which is generally considered as a tool to assess future cardiovascular risk in humans [63].
The source of calcium may also affect its anti-obesity effects. It has been implicated that the anti-obesity role of calcium intake in children and adolescents might be driven exclusively by dairy calcium [64], implying that dairy calcium might be more effective than calcium supplements. Consumption of a high Ca diet from dairy for 12 weeks was effective in reducing abdominal adiposity in overweight patients with T2DM [65]. Greater intake of high-fat, but not intake of low-fat, dairy products, was found to be associated with less weight gain in middle-aged and elderly women [66]. It was also reported that increasing dairy calcium intake with low-fat milk or yogurt for 12 months had no effect on decreasing body fat or weight gain in overweight adolescent girls [67]. In addition, gender may also influence the anti-obesity effects on dietary calcium. Lee et al. found that consumption of dairy products is associated with reduced risks of obesity and metabolic syndrome in Korean women but not in men [68]. Similarly, Moreira et al. reported an inverse relationship between calcium intake and BMI in only girls (7-9 years old) in Portugal [69]. The discrepancy in the effects of calcium on body weight/fat loss might result from the different subjects, calcium intake amounts, calcium sources, and calcium intake periods. Thus, due to the various influencing factors, the anti-obesity effects of dietary calcium need to be further studied in different subjects.
Possible Mechanisms for Calcium's Anti-Obesity Effects
Effects of Calcium on Adipogenesis
Adipogenesis includes the commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage (preadipocytes) and the terminal differentiation of preadipocytes to mature adipocytes. It is tightly regulated by various signaling molecules and several key adipogenic transcription factors, such as PPARγ and C/EBPα [70]. It has been demonstrated that adipogenesis or hyperplasic adipose expansion is linked to anti-obesity and improved metabolic health [70][71][72]. A large body of evidence has demonstrated that calcium is involved in regulating adipogenesis. Jensen 2+ ] i appeared to exert a biphasic regulatory effect on human adipocyte differentiation, inhibiting the early stages while promoting the late stage of differentiation and lipid filling [46]. We also found that high [Ca 2+ ] o (4 mM) stimulated adipogenesis of porcine bone marrow MSCs (pBMSCs) by increasing the [Ca 2+ ] i level and activating CaMKII and PI3K/Akt-FoxO1 pathways [47]. We further determined that the promotive effects of [Ca 2+ ] o on pBMSCs occurred mainly in the commitment phase but not in the terminal differentiation phase (unpublished data). In line with our results, it has been indicated that high [Ca 2+ ] o enhances adipogenic differentiation of mice BMSCs [73,74] and stimulates adipogenesis of porcine synovium-derived MSCs [75]. These findings imply that calcium stimulates the early stage (commitment stage) and suppresses the late stage (terminal differentiation stage) of adipogenesis. Taken together, calcium may elicit inhibitory or stimulatory effects on adipogenesis in vitro depending on the calcium concentration, cell types, and culture systems.
Compared with the in vitro findings, the in vivo data may better reflect the role of calcium in adipogenesis. In agreement with the enhanced adipogenesis in pBMSCs, we found that calcium supplementation stimulated adipogenesis in mice fed with HFD, with increased adipocyte number and PPARγ expression in inguinal subcutaneous white adipose tissue [47]. Similarly, Zhang et al. found that calcium propionate supplementation in the diet of Wagyu steers could trigger upregulation of PPARγ and CEBPα mRNA expression levels, which could cause long-term activation of adipogenesis [76]. Our unpublished study also demonstrated that dietary supplementation of 1% calcium propionate significantly increased expression of adipogenesis marker genes, such as PPARγ and CEBP/α, in the backfat of finishing pigs. It should be noted that while enhanced adipogenesis in vitro is always accompanied by elevated lipid content, increased adipogenesis (or adipocyte number) in vivo does not mean more fat deposition. In fact, we found that the adipocyte diameter/size in calcium-supplemented mice was much smaller than that of HFD-fed mice. As a result, the WAT index, body fat content, and body weight were significantly reduced by calcium supplementation [47]. In agreement with this, dietary calcium supplementation (10 g/kg) significantly inhibited adipocytes hypertrophy, with a remarkable decrease in adipocyte area in VAT of rats [50]. Similarly, dietary calcium supplementation significantly decreased the lipid droplet sectional area in rat BAT [52]. Therefore, dietary calcium can not only stimulate adipogenesis (or hyperplasia) but also inhibit adipocyte hypertrophy (adipocyte size) in vivo.
Effects of Calcium on Fat Metabolism
Fat metabolism in adipocyte involving the synthesis and degradation of fat (or triglyceride, TG) contributes to the hypertrophy (increase in size) and atrophy (decrease in size) of adipocytes, respectively [42]. Therefore, suppression of fat synthesis and/or promotion of fat breakdown will result in smaller adipocytes and thus less fat deposition. It has been implicated that an increase in dietary calcium intake attenuates diet-induced adiposity by modulating adipocyte intracellular Ca 2+ and thereby coordinately inhibiting lipogenesis and accelerating lipolysis [77]. Sun et al. reported that high [Ca 2+ ] o or [Ca 2+ ] i leads to reduced intracellular lipid content and decreased expression of lipogenesis genes, such as FAS and LPL, and increased expression of lipolysis gene HSL [44,45]. Meanwhile, the store-operated Ca 2+ entry (SOCE) induced the phosphorylation of HSL and increased the pHSL/HSL ratio by the activation of cAMP-PKA pathway in 3T3-L1 cells [78]. Consistent with this, dietary supplement with calcium had a protective effect against HFD-induced obesity in mice by enhancing the expression of HSL [45]. In addition, it was reported that high calcium diet significantly decreased the FAS activity and triglyceride level and increased lipolytic activity with an elevated level of glycerol content in adipose tissue of male Wistar rats, thus leading to lower adiposity index [79]. Furthermore, dietary calcium supplementation during maternal pregnancy and lactation decreased the mRNA expression of FAS and SREBP-1c in the adipose tissue of adult female offspring [80]. Collectively, calcium may elicit its anti-obesity role by modulating fat metabolism, with decreased fat synthesis and increased fat breakdown.
Effects of Calcium on Adipocyte (Precursor) Proliferation and Apoptosis
It has been demonstrated that calcium is involved in regulating proliferation of preadipocytes or MSCs. We observed that the enhanced proliferation of pBMSCs induced by high extracellular calcium was associated with the activation of the calcium-sensing receptor (CaSR) and ERK signaling pathway [32]. Similarly, Rocha et al. found that activation of CaSR elevated proliferation of LS14 preadipocytes [81]. In addition, the pro-proliferation effects of [Ca 2+ ] o have been reported in rat bone marrow-derived progenitor cells [82] and porcine synovium-derived mesenchymal stromal cells [75]. It should be noted that different species, cell types, and/or culture conditions (e.g., calcium concentrations) will cause different proliferative effects of calcium. We found that [Ca 2+ ] o promoted pBMSCs proliferation when [Ca 2+ ] o was greater than or equal to 4 mM [32]. In contrast, Liu et al. reported that the optimal [Ca 2+ ] o for rabbit BMSCs to proliferate was 1.8 mM and that a higher level of [Ca 2+ ] o did not change cell proliferation [83]. In addition, it was shown that low calcium (0.09 mM) greatly enhanced the growth rate and extended the lifespan of human adipose-derived MSCs [84].
Regulation of the adipocyte number by stimulating apoptotic cell death is emerging as a potential strategy for prevention and treatment of obesity. Calcium has been implicated to be linked with apoptosis [85]. It has been shown that a sustained increase in intracellular Ca 2+ triggers apoptotic cell death and that Ca 2+ -mediated apoptosis can be induced in mature adipocytes [86]. Consistent with this, it was reported that high vitamin D and calcium intake activated the Ca 2+ -mediated apoptotic pathway in the adipose tissue of diet-induced obese mice, thus leading to reduced adiposity [49]. In addition, it was shown that calcium caused apoptosis in undifferentiated human adipose tissue-derived MSCs [80]. In contrast, Jensen et al. found that treatment of 3T3-L1 cells with high [Ca 2+ ] o did not significantly affect cell number or viability and did not trigger apoptosis [43]. The distinct effects of calcium on apoptosis in MSCs and 3T3-L1 might be due to the different cell types, culture systems, and calcium concentrations. Besides that, promoting the autophagy of adipocyte cells might also play a part in anti-obesity. The relationship between calcium signals and autophagy was reviewed by Bootman et al. [87]. In this review, they summarized that Calcium (Ca 2+ ) and Ca 2+ channels have been shown to control various stages of autophagic flux. In addition, activation of calcium-sensing receptor (CaSR) induced autophagy in LS14 and SW872 preadipocyte cell lines as well as primary human preadipocytes [88]. Taken together, calcium-induced apoptosis of adipocyte (precursor) might contribute to the beneficial effects of calcium on body weight/fat loss.
Effects of Calcium on Thermogenesis
One of the possible mechanisms by which calcium decreases body fat is enhancing thermogenesis/energy expenditure. Calcium has been implicated to be involved in the regulation of energy balance [89]. It has been well documented that brown adipocytes and beige/brite adipocytes are enriched with uncoupling protein 1 (UCP1) and contributors to thermogenesis/heat production and are thus beneficial for body fat loss [90][91][92]. Conceicao et al. reported that dietary calcium supplementation was able to improve BAT thermogenesis capacity in adult rats that were overfed earlier during lactation [52]. In line with the result, our findings demonstrated that calcium supplementation significantly increased BAT thermogenesis in HFD-fed mice, with higher temperature of interscapular BAT (iBAT) and elevated expression of thermogenesis related genes, such as UCP1 and peroxisome proliferator-activated receptor coactivator 1α (PGC1-α) in iBAT [93]. Accordingly, the induction of thermogenic genes in response to β-adrenergic receptor stimulation was suppressed by reduced intracellular calcium in the brown adipocytes of wild-type mice [94]. In contrast, increased intracellular calcium via transient receptor potential vanilloid 2 (TRPV2) facilitated UCP1 expression and heat production [95]. It has been implicated that the calcium-promoted thermogenic capacity of brown adipocytes may be attributed to the increased mitochondrial fusion and mitochondrial-endoplasmic reticulum contacts induced by calcium [96].
However, Parra et al. found that dairy calcium (12 g/kg diet) intake had no effects on UCP1 expression in BAT and UCP2 expression in WAT of mice and suggested that activation of thermogenesis is not involved [97]. In addition, it was reported that calcium solely after the induction phase of differentiation specifically suppressed gene expression of UCP1, PR domain zinc-finger protein 16 (PRDM16), and PGC1-α [34]. The inconsistent effect of calcium on BAT activation or thermogenesis might be due to the various animal/cell models, calcium doses, and calcium intake durations. Among these factors, calcium dose might be the most important one contributing to the variability.
With regard to calcium and WAT browning, it has been shown that sarco/endoplasmic reticulum Ca 2+ -ATPase 2b (SERCA2b)-mediated calcium cycling can regulate thermogenesis in beige adipocytes [98]. We also found that calcium supplementation in drinking water was able to boost WAT browning, with significantly elevated expression of thermogenesis related genes, including UCP1, PRDM16, and PGC1-α [93]. Taken together, the current evidence suggests that calcium is involved in enhancing thermogenesis by stimulating BAT activation and WAT browning.
Effects of Calcium on Fat Absorption and Fecal Fat Excretion
Decreased fat absorption and increased fecal fat excretion constitute a primary determinant accounting for prevention or treatment of obesity. In animals, it has been demonstrated that high calcium intake depresses fat digestion and absorption in veal calves [99,100]. In addition, at very low concentrations of calcium, the hamster jejunum produced very few chylomicrons, suggesting reduced fat absorption [101]. Furthermore, it has been reported that a high-calcium (2.4%) diet increases fecal excretion of dietary lipid, which might partly contribute to the reduced body fat content in rats [102]. Moreover, Ayala-Bribiesca et al. found that cheddar-type cheeses enriched with calcium led to more abundant calcium soaps, a quantitative index for fecal fatty acids, in rat feces, suggesting elevated fecal fat excretion [103].
In humans, it has been shown that supplementation of calcium decreases fat absorption and increases the fecal excretion of insoluble calcium soaps with fatty acids [104]. Similarly, a short-term increase in dietary calcium intake promoted fecal fat and energy excretion [105]. Increasing calcium intake from low-fat dairy products by 1600 mg/day for seven days doubled the total fat excretion, with no effect on the excretion of bile acids [106]. Christensen et al. estimated that increasing the dairy calcium intake by 1241 mg/day resulted in an increase in fecal fat of 5.2 g/day [107]. In addition, it was reported that supplementation of dairy calcium in conjunction with orlistat augmented fecal fat excretion [108]. Furthermore, oral supplementation of elemental calcium as calcium carbonate dose-dependently increased the percentage of fecal fat secretion to fat intake in men [109]. Moreover, short-term dietary calcium fortification (2200 mg/day total and 550 mg calcium citrate malate) significantly increased dietary saturated fat excreted from 6% to 13% in men [110]. It has been implicated that the formation of insoluble calcium soaps and the alteration of the interfacial organization of hydrolyzed lipids are involved in calcium-induced decreased fat digestion and absorption and increased fecal fat secretion [111,112]. Taken together, the beneficial roles of calcium in decreasing fat absorption and increasing fecal fat excretion might be responsible for its anti-obesity effects.
Effects of Calcium on Gut Microbiota
Emerging evidence has been highlighting an increasingly more important role of gut microbiota in the regulation of obesity [113][114][115][116][117]. It has been indicated that phylum-level changes in gut microbiota composition, decrease in bacterial diversity, and alterations of functional genes and metabolic activities are associated with obesity [118][119][120]. Thus, dietary intervention or modulation of the gut microbiota has the potential to prevent or treat obesity and obesity-related metabolic diseases [121][122][123].
It has been demonstrated that high-calcium diets appear to positively affect gut microbiota composition, favoring the growth of lactobacilli [124]. Similarly, Chaplin et al. showed that calcium supplementation modulates gut microbiota in a prebiotic manner, promoting a healthier metabolic profile, in dietary obese mice [48]. The authors found that calcium supplementation increased the length of the small intestine and the weight of the cecum and cecum feces. Calcium-fed mice exhibited increased levels of Bifidobacterium spp. and Bacteroides/Prevotella and decreased levels of Clostridium coccoides and Clostridium leptum [48]. In line with these results, we found that, compared with HFD-fed mice, supplementation of calcium in drinking water increased the community diversity and specific bacterial abundance in feces [125]. In addition, it has been reported that dietary calcium has a substantial influence on gut microbiota in pigs [126], broilers [127], laying hens [128], and white shrimp [129]. To date, the effects of dietary calcium on human gut microbiota remain largely unknown and need to be further explored. Nevertheless, the current evidence in animals suggests that dietary calcium might interfere with gut microbiota, which partly explains the beneficial effects of calcium on body weight/fat loss.
Conclusions
In this review, we compiled the evidence for the anti-obesity effects of calcium in cell models, animals, and humans. In addition, we summarized the possible anti-obesity mechanisms of calcium, including (a) regulation of adipogenesis, with stimulation on MSCs (or commitment stage) and inhibition on preadipocytes (or differentiation stage); (b) modulation of fat metabolism, with decreased fat synthesis (lipogenesis) and increased fat breakdown (lipolysis); (c) promotion of adipocyte (precursor) proliferation and/or apoptosis; (d) enhancement of thermogenesis, with increased BAT activation and WAT browning; (e) suppression of fat absorption and promotion of fecal fat excretion; and (f) modification of gut microbiota composition and diversity ( Figure 1). In conclusion, the current evidence demonstrates the anti-obesity effects of calcium and suggests the potential application of dietary calcium supplementation for prevention or treatment of obesity. a substantial influence on gut microbiota in pigs [126], broilers [127], laying hens [128], and white shrimp [129]. To date, the effects of dietary calcium on human gut microbiota remain largely unknown and need to be further explored. Nevertheless, the current evidence in animals suggests that dietary calcium might interfere with gut microbiota, which partly explains the beneficial effects of calcium on body weight/fat loss.
Conclusion
In this review, we compiled the evidence for the anti-obesity effects of calcium in cell models, animals, and humans. In addition, we summarized the possible anti-obesity mechanisms of calcium, including (a) regulation of adipogenesis, with stimulation on MSCs (or commitment stage) and inhibition on preadipocytes (or differentiation stage); (b) modulation of fat metabolism, with decreased fat synthesis (lipogenesis) and increased fat breakdown (lipolysis); (c) promotion of adipocyte (precursor) proliferation and/or apoptosis; (d) enhancement of thermogenesis, with increased BAT activation and WAT browning; (e) suppression of fat absorption and promotion of fecal fat excretion; and (f) modification of gut microbiota composition and diversity ( Figure 1). In conclusion, the current evidence demonstrates the anti-obesity effects of calcium and suggests the potential application of dietary calcium supplementation for prevention or treatment of obesity.
Conflicts of Interest:
The authors declare no conflicts of interest.
Abbreviations
BAT brown adipose tissue
Conflicts of Interest:
The authors declare no conflicts of interest.
Abbreviations
Figure 1 .
1The possible mechanisms for the anti-obesity effects of dietary calcium. Calcium may elicit anti-obesity effects through (a) regulation of adipogenesis, with stimulation on mesenchymal stem cells (MSCs) (or commitment stage) and inhibition on preadipocytes (or differentiation stage); (b) modulation of fat metabolism, with decreased fat synthesis (lipogenesis) and increased fat breakdown (lipolysis); (c) promotion of adipocyte (precursor) proliferation and apoptosis; (d) enhancement of thermogenesis, with increased brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning; (e) suppression of fat absorption and promotion of fecal fat excretion; and (f) modification of gut microbiota composition and diversity. Author Contributions: Conceptualization, S.W.; writing-original draft preparation, F.Z. and J.Y.; writingreview and editing, F.Z., X.Z., L.W., G.S., Q.J., and S.W.; project administration, P.G.; funding acquisition, S.W., Q.J., and G.S. Funding: This work was funded by the National Natural Science Foundation of China (31790411, 31672508, 31372397) and the Innovation team project in universities of Guangdong Province (2017KCXTD002).
Figure 1 .
1The possible mechanisms for the anti-obesity effects of dietary calcium. Calcium may elicit anti-obesity effects through (a) regulation of adipogenesis, with stimulation on mesenchymal stem cells (MSCs) (or commitment stage) and inhibition on preadipocytes (or differentiation stage); (b) modulation of fat metabolism, with decreased fat synthesis (lipogenesis) and increased fat breakdown (lipolysis); (c) promotion of adipocyte (precursor) proliferation and apoptosis; (d) enhancement of thermogenesis, with increased brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning; (e) suppression of fat absorption and promotion of fecal fat excretion; and (f) modification of gut microbiota composition and diversity. Author Contributions: Conceptualization, S.W.; writing-original draft preparation, F.Z. and J.Y.; writing-review and editing, F.Z., X.Z., L.W., G.S., Q.J. and S.W.; project administration, P.G.; funding acquisition, S.W., Q.J. and G.S. Funding: This work was funded by the National Natural Science Foundation of China (31790411, 31672508, 31372397) and the Innovation team project in universities of Guangdong Province (2017KCXTD002).
et al. found that high [Ca 2+ ] o (5 and 10 mM) inhibited the adipogenesis of 3T3-L1 preadipocytes compared to controls (1.8 mM [Ca 2+ ] o ), with decreased expression of PPARγ and C/EBPα [43]. Similarly, it has been reported that increased [Ca 2+ ] i with RyR channel excitomotor caffeine significantly suppresses adipogenesis of mice preadipocytes, with decreased lipid content and PPARγ expression [44]. However, Shi et al. demonstrated that increasing [Ca
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465 | [HCG-positive cells in seminoma of the testis]. | Twenty-six tumours of the seminoma type and 54 embryonic carcinomas of the testis were studied with immunoperoxidase, using an anti-beta HCG immune serum and an anti-alpha FP immune serum. The results obtained in first group suggest the possibility of a new type of "pseudo-seminoma" tumours, with high levels of circulating HCG probably due to proliferation of trophoblasts. In the second group, 88% of the tumours gave positive response to one or the other immune sera. However, the anti-alpha FP-positive structures were similar to those of pure vitelline tumours, and the anti-beta HCG-positive structures were syncytiotrophoblastic elements different from those of conventional carcinomas. The combined results of histology, immunohistochemistry and pre-operative biological tests should lead to improved identification of malignant germ cell tumours of the testis. | The management of metastatic seminoma testis. | Abstract The histological, ultrastructural and biological features are described of a spontaneously occurring seminoma in the testis of a male domestic rabbit. The tumour was structurally similar to seminomas in other animal species. | A 52 year-old male with advanced testicular seminoma (stage II N4) underwent right radical orchiectomy, followed by chemotherapy with vinblastine, bleomycin and cis-platinum. This resulted a partial response and he was well for 24 months after the operation. Serial monitoring of serum HCG and HCG-beta has been done, using three kinds of double antibody radioimmunoassays. A substance with immunological similarity to free HCG-beta was detected in sera and tumor extract. The serum level of this substance was reduced in parallel with the cytoreduction of metastasis by chemotherapy. A rational therapeutic approach to advanced seminoma and its serum marker are discussed briefly. | Seminoma Seminoma (also known as "pure seminoma" or "classical seminoma") is a germ cell tumor of the testicle or, more rarely, the mediastinum or other extra-gonadal locations. It is a malignant neoplasm and is one of the most treatable and curable cancers, with a survival rate above 95% if discovered in early stages. Testicular seminoma originates in the germinal epithelium of the seminiferous tubules. About half of germ cell tumors of the testicles are seminomas. Treatment usually requires removal of one testicle. However, fertility usually isn't affected. All other sexual functions will remain intact. The average age of diagnosis is | Immunohistochemical analysis was done on 7 testicular tumors classified as spermatocytic seminoma (SS) and 25 classic seminomas. Except for a few scattered cells, the spermatocytic seminomas were negative for placental-like alkaline phosphatase (PLAP); the classic seminomas were all positive for this enzyme. The SS also were negative for alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (hCG), and leukocyte common antigen (LCA). The ploidy of the seven tumors of SS was as follows: two, diploid; two, near-diploid; one, tetraploid; one, aneuploid; and one, uninterpretable. The essentially negative staining of SS for PLAP was strikingly different from the pattern in classic seminoma. Thus, staining for this enzyme is useful for making the differential diagnosis between classic seminoma and SS. To differentiate between malignant lymphoma and SS, staining for leukocyte common antigen is helpful. | Seminoma is the most common single histological sub-type of testicular carcinoma. Patients usually present with a painless lump and stage I disease. We describe a case of an incidental metastatic seminoma in a 28-year-old man post-renal trauma with a dramatically elevated β-human chorionic gonadotropin (βHCG). His βHCG level has returned to normal post-orchidectomy and chemotherapy. | Dysplastic Development of Seminiferous Tubules and Interstitial Tissue in Rat Hypogonadic (hgn/hgn) Testes1 | 465 | In a retrospective study of 38 patients with pure seminoma, serum and urine levels of human chorionic gonadotropin (HCG) were measured and the cellular origin of HCG-like substance was searched using the technique of indirect immunoperoxidase on step sections of the tumors. Eight of the patients had elevated HCG in serum or urine, and 5 had HCG-positive cells in the sections of tumor specimens. With this technique, two types of HCG-positive cells were identified, syncytiotrophoblastic giant cells (STGC) and mononuclear cells otherwise indistinguishable from seminoma cells. Patients in the present series responded well to conventional radiation therapy or cytotoxic chemotherapy and had a favorable outcome regardless of the presence of STGC or slightly elevated HCG levels. | [Attempt to demonstrate beta-HCG in germinal testicular tumors of the adult by an indirect immunoperoxidase method]. | Chorionic gonadotrophic hormone and transitional cell carcinoma of the bladder. Immunohistochemical characterization of HCG and its alpha and beta subunits | [Immunoperoxidase study of 80 germ cell tumours of the testis in adults (author's transl)]. | Clinical Utility of Oncofetal and Proteins and Hormones as Tumor Markers | Lymphocytes from 10 patients with breast carcinoma were seeded in autologous serum, on autochthonous tumour cells and allogeneic tissue-cultured breast tumour cell lines. In 4 patients, the anti-tumour cell cytotoxicity against at least one of 3 breast tumour cell lines differed significantly from that against autochthonous tumour cells. Further study of these 4 individuals (using their previously frozen lymphoid cells and sera) showed that these differences occurred because serum which decreased (" blocked ") lymphocyte anti-tumour cytotoxicity when applied to one tumour cell line, could either have no effect or potentiate it when applied to another, without any consistent pattern vis-a-vis target-cell susceptibility to these different humoral effects. | The action of methotrexate, actinomycin D, bleomycin, vincristine and hydroxyurea on the production of human chorionic gonadotrophin (hCG) by a choriocarcinoma cell line (BeWo) has been studied. hCG production per unit of cell protein was increased, and this was a continuing process only halted by cell death. | [K-cell activity in patients with germ cell testicular tumors. Effect of cytostatic therapy]. | Immunosuppression in allogeneic murine tumour system. A model for the study of antilymphocyte serum. |
466 | how many phase of minneapolis st paul international airport | been chosen to maximize the capacity at MSP through 2035. It includes three phases through 2020, 2030 and 2035. The final product moves some but not all non-Delta airlines from Terminal 1 to Terminal 2, evens out capacity over the two terminals and will finish with as many as 15 new gates being constructed over both terminals and new parking garages. By 2020 (38 million annual passengers) By 2030 (48 million annual passengers) By 2035 (54 million annual passengers) Total 2035 LTCP Recommended Development Cost ~$2.54 billion Minneapolis–Saint Paul International Airport Minneapolis–Saint Paul International Airport , also less commonly known | and 34,368 checked bags screened. For the event, the TSA brought in more than 100 additional agents and 20 canines to MSP for the expected number of passengers. Minneapolis–Saint Paul International Airport has two terminals with a total of 131 gates, both of which were named for famous Minnesotans: the Lindbergh Terminal 1 (named after the aviator Charles Lindbergh) and the smaller Humphrey Terminal 2 (named for former US Vice President Hubert Humphrey). Both terminals are on different sides of the airfield and not interconnected; one who wishes to transfer between terminals must take the free Light Rail. The larger | Minneapolis–Saint Paul International Airport is open to all passengers for a fee. It is located in Terminal 1 on the mezzanine level of the Airport Mall. PGA MSP Lounge is a golf-themed lounge, open to all passengers for a fee. It is located in Terminal 1 at the north end of the Airport Mall's mezzanine level at the intersection of concourses C, D & E. United Airlines has a United Club in Terminal 1 between gates E6 and E8. InterContinental Hotels operates a full service on-site hotel at the airport with 291 rooms on 12 floors. Originally intended to be open for Super Bowl | Pauls Valley Municipal Airport Pauls Valley Municipal Airport is a city-owned, public-use airport located two nautical miles (4 km) south of the central business district of Pauls Valley, a city in Garvin County, Oklahoma, United States. It is included in the National Plan of Integrated Airport Systems for 2011–2015, which categorized it as a "general aviation" facility. Although most U.S. airports use the same three-letter location identifier for the FAA and IATA, this airport is assigned PVJ by the FAA, but has no designation from the IATA. Pauls Valley Municipal Airport covers an area of 480 acres (194 ha) at | the airport at one point on a seasonal basis from Minneapolis/St. Paul (MSP) during the winter months and continues to serve Harlingen seasonally with Boeing 737-800 jets. Delta Airlines started seasonal service to Harligen in 2013 from its Minneapolis/St. Paul (MSP) hub using Airbus A320's and continues to fly the seasonal route using CRJ-900's. Valley International Airport covers at an elevation of . It has three asphalt runways: 17R/35L is 8,301 by 150 feet (2,530 x 46 m); 13/31 is 7,257 by 150 feet (2,212 x 46 m); 17L/35R is 5,949 by 150 feet (1,813 x 46 m). In 2011 | History of Minneapolis its debut in Minneapolis with the opening of the Blue Line on June 26, 2004. This line, part of the METRO system, starts in downtown Minneapolis and progresses southeastward along Minnesota State Highway 55 (also known as Hiawatha Avenue), passes Minnehaha Park on the west side, and serves the Minneapolis–Saint Paul International Airport before it terminates at the Mall of America in Bloomington. In 2014, service began on the Green Line which connects downtown with the University of Minnesota and downtown St. Paul. As industry and railroads left the Mississippi riverfront, people gradually became aware that the riverfront could be | Tower Municipal Airport is a city-owned public-use airport located one nautical mile (2 km) northwest of the central business district of Tower, a city in Saint Louis County, Minnesota, United States. It is located on Lake Vermilion and is also known as Tower Municipal Airport & Seaplane Base.
Facilities and aircraft
Tower Municipal Airport covers an area of at an elevation of 1,369 feet (417 m) above mean sea level. It has two runways designated 8/26 with a 3,400 x 75 ft (1,036 x 23 m) asphalt surface. It also has one seaplane runway landing area designated 14W/32W which measures 5,000 x 200 ft (1,524 x 61 m).
For the 12-month period ending April 30, 2007, the airport had 3,700 general aviation aircraft operations, an average of 10 per day. In March 2017, there were 15 aircraft based at this airport: 14 single-engine and 1 ultralight.
References
External links
Airports in Minnesota
Seaplane bases in the United States
Buildings and structures in St. Louis County, Minnesota
Transportation in St. Louis County, Minnesota | Valley International Airport the airport had 43,731 aircraft operations, averaging 119 per day: 36% general aviation, 34% military, 22% airline, and 8% air taxi. 32 aircraft were then based at the airport: 88% single-engine, 9% multi-engine, and 3% jet. On April 2, 2012, United Express flight 4128 made an emergency landing at Corpus Christi due to unknown reasons when it suffered damage to its front landing gear and also experienced a flat tire. The flight originated in Harlingen and was heading to George Bush Intercontinental Airport in Houston. There were 37 passengers on board and there were no injuries. The aircraft was an | 466 | been chosen to maximize the capacity at MSP through 2035. It includes three phases through 2020, 2030 and 2035. The final product moves some but not all non-Delta airlines from Terminal 1 to Terminal 2, evens out capacity over the two terminals and will finish with as many as 15 new gates being constructed over both terminals and new parking garages. By 2020 (38 million annual passengers) By 2030 (48 million annual passengers) By 2035 (54 million annual passengers) Total 2035 LTCP Recommended Development Cost ~$2.54 billion Minneapolis–Saint Paul International Airport Minneapolis–Saint Paul International Airport , also less commonly known | how many phases of minneapolis saint paul international airport | what is the name of the lounge at minneapolis saint paul airport | how many passengers will be travelling to mopa airport by 2035 | how many passengers did macarthur airport have in 2005 | how many terminals are there at pyongyang airport | Sustainable Development and Airport Surface Access: The Role of Technological Innovation and Behavioral Change | Literature Table at PSP Airport | who is responsible for managing bartow municipal airport |
467 | what is one of the defining characteristics of somiedo natural park | Soğuksu National Park Hot springs and cold water sources are found in these areas. The hot springs are evaluated as spa. In the northern part of the national park, petrified woods are found, which were formed by volcanic activation about 10–12 million years ago. A preserved petrified tree trunk of length has diameter. The forests in the national park, which exhibit the characteristics of European-Siberian vegetation, cover an area of . Dominant species include coniferous trees such as Scots pine (65%), larch (24%) and fir (6%). Other notable trees include broadleaves such as oak (5%), alder, aspen ("Populus tremuloides"), maple, dogwood ("Cornus") and | The vegetation of Voyageurs National Park, Minnesota, U.S.A., was investigated, in part to establish a plant community classification system that would be useful to park managers and naturalists, and to evaluate short-term changes within plant communities. Samples from 120 stands were ordinated and classified on the basis of synecological coordinates, a system of ecological coordinates relating floristics to physical site conditions. Along indirect gradients of moisture and nutrient conditions, 12 ecological types were distinguished. Within each ecological type, it is expected that stands of diverse cover types will tend to converge, over a period of 150–200 years, on an edaphically constrained “climax” community characteristic of that ecological type, given present management practices and disturbance regimes. Permanent plots were established in 46 stands to permit future testing of these projections. Ecological types, named for expected dominants and for a typical undergrowth species or genus, include (... | The appearance of logistic parks is due to the inherent demand of the quick development of logistic industry. The reasons that the logistic parks are extended and developed rapidly are the recognition, advocacy and aegis of our government. At present, logistic industry in our country is in the underway phase, the two main themes of the development of logistic industry in our country are what foster the corporations of the third party and mark out modern logistic parks. This article introduces concisely the development and genre of logistic parks. Based on it, the emphases of this article anatomize the developing stage and relate problems of logistic parks in our country, then use for reference with them. | Isiboro Sécure National Park and Indigenous Territory Bolivia is a major site for observing wildlife. It is accessed by water, entering through the Black arroyo from the Sécure river during high water season, or by land on foot or horse from the communities of Dulce Nombre or Limoncito. The water route lacks a formal port from which tourists may embark. The land route is by way of the road through the southern colonized area of TIPNIS from Isinuta to Aroma. TIPNIS has experienced substantial deforestation, particularly in the region of the park outside the red line, known as Polygon 7, where agricultural colonization has taken place since | The transformation of the natural landscape, triggered by human activities, such asagriculture, cattle range, tourism, urban growth, quarrying, and the use of trails in ConservationUnits, represent negative impacts to the ecosystem functionality, as well as risk to the economicdevelopment, through tourism. Therefore, this article addresses research work carried out byLAGESOLOS (Laboratory of Environmental Geomorphology and Soils Degradation),regarding two Conservation Units (UCs): Serra da Bocaina National Park (PNSB) and Serra doMar State Park (PESM). These two environments were created to guarantee the wholeprotection to the flora, fauna, natural beauties, as well as to guarantee the use for educational,leisure and scientific purposes, of the two Parks, which are suffering with the urban growth,disorganized tourist activity, quarrying, agriculture and cattle range, causing soil degradationand biodiversity loss. | Stolby Nature Sanctuary Stolby Nature Sanctuary (Russian:запове́дник «Столбы́»), (in English, "The Pillars") is a Russian strict ecological reserve located 10 km south of the city of Krasnoyarsk, on the northwestern spurs of the Eastern Sayan Mountains. The site is known for its dramatic complexes of rocks; 3.5% of the reserve is open to hikers seeking to visit and climb the rocks. Over 200,000 visitors per year are recorded. The park was founded in 1925 by citizens the picturesque Syenite Buttes and surrounding rocky landscape. The park’s area is 47,219 hectares. Stolby has been nominated to be on the list of | Why is zion national park important? | Shrub Preference and Utilization by Big Game on New Mexico Reclaimed Mine Land | 467 | Somiedo Natural Park Somiedo in a World Heritage Site, for which transhumance would be one of the defining characteristics. The park is a stronghold of the Cantabrian brown bear. In 2009 the Spanish newspaper "El País" referred to Somiedo with its 30 bears as the Spanish Yellowstone. The Fundación Oso Pardo (FOP) opened an interpretation centre in October 2011 called “Somiedo y el Oso” in Pola de Somiedo. The total number of bears in Spain is small and the government classes them as endangered. The bears' future is put at risk by fragmentation of their habitat in the Cantabrian Mountains. 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468 | A new model describing the kinetics of micelle formation from chemical relaxation studies | Abstract An equation, which describes the dependence of critical micelle concentration (XCMC) with temperature, has been derived on the basis of Δ G 0 =−RT ln K , linear behavior of the enthalpy of micellization with temperature, and compensation phenomena in which the enthalpy and the entropy of micellization change linearly with each other. The new equation has yielded excellent fitting results of XCMC(T) for various surfactant systems. More interestingly, it yields d=2, irrespective of surfactant system, in the power-law description of XCMC(T), |X CMC −X CMC ∗ |= const |T−T ∗ | d with the minimum CMC, X CMC ∗ , and the temperature, T* at X CMC ∗ . The value of d=2 is confirmed from the fits to reported literature data. | A new way to determine the critical micelle concentration (CMC) based on the mobilities of system peaks is presented. A general approach for the CMC determination is based on the change of the slope or on finding the inflection point in the plot of a physical property of solution as a function of surfactant concentration. The determination of CMC by system peaks in CE utilizes a "jump" instead of a continuous change in the measured quantity. This phenomenon was predicted by the program PeakMaster, which was modified for simulation of micellar systems. The simulation of the steep change in mobilities of the anionic system peaks showing the CMC value was verified experimentally in a set of measurements, where the concentration of the surfactant was varied while the ionic strength was kept constant. The experimental work fully proved our model. A comparative electric current measurement was carried out. The proposed method seems to offer easier CMC determination as compared to the standard methods. | Effects of cetyltrimethylammonium bromide (CTABr) micelles on second-order rate constants (knobs) for nucleophilic reactions of amines (piperidine and n-butylamine) with ionized phenyl salicylate (PS-) reveal a nonlinear decrease with the increase in [Dn] (where [Dn] = [CTABr]T − cmc) at a constant [NaBr] and 35 °C. The observed data, at a constant [NaBr], fit reasonably well to a pseudophase model of micelles, and such a data fit gives kinetic parameters such as CTABr micellar binding canstant (KS) of PS-. The effect of [NaBr] upon KS is explained with the empirical relationship KS = KS0/(1 + ψ[NaBr]), where ψ is an empirical parameter. | Micelle–monomer equilibria in solutions of ionic surfactants and in ionic–nonionic mixtures: A generalized phase separation model | Degree of Counterion Binding of Alkylpyridinium Halide Micelles: Its Relation to Micelle Shape and Size | Abstract A combinational kinetic model was employed for the first time to determine the quantitative values of some kinetic parameters, including induction period (IP), critical reverse micelle concentration (CMC), and maximum attainable concentration of lipid hydroperoxides (PV max ) in a range of edible oils peroxidised at 60 °C. On the basis of the calculated coefficients of variation (CV), the model was able to estimate the kinetic parameters much better than the conventional tangent method. The potency of the combinational kinetic model to provide the values of markedly lower variability caused the small differences among the experimental results to be appeared statistically. | An all-atom 5 nanosecond molecular dynamics simulation of a water-solvated micelle containing 60 sodium dodecyl sulfate monomers was performed. Structural properties such as the radius of gyration, eccentricity, micellar size, accessible surface area, dihedral angle distribution, carbon atom distribution, and the orientation of the monomers toward the micelle center of mass were evaluated. The results indicate a stable micellar system over the duration of the simulation. Evaluation of the structure and motion of the sodium counterions show (1) a long equilibration time (1 nanosecond) is required to achieve a stable distribution of counterions and (2) approximately 25% of the sodium ions are located in the first shell and 50% are located in the first two shells of the micelle during the course of the simulation. The structure of the micelle oxygen−sodium ion radial distribution function reveals two distinct peaks which divide the counterions into those close to the micelle (first shell) those far from the ... | The dynamics, structural properties, and energetics of hydration water around a sodium dodecyl sulphate micelle have been investigated using molecular dynamics simulation. A clear revelation of the slow dynamics of the hydration water has been made by separate measurements of the rotational and translational properties. Calculated diffusion coefficients fall within the range of experimentally observed quantities. The water-micelle head group (MHG) hydrogen bond is more stable (by an amount approximately 7.0 kcal/mol) compared to the water-water hydrogen bond. The difference in stability of the water monomers forming different numbers of hydrogen bonds (n=0,1,2) with the MHG has clearly been shown from the analyses of their rotational relaxation, residence times, as well as the energy of interaction with different components of the system. The singly hydrogen-bonded water species is the most abundant and stable. The entropy plays the key role in controlling the relative abundance of the different species. | 468 | Kinetic models of micelles formation | Polymer chemistry: Micelles inside out | what kind of micelles are used as drug carriers | In this work, the microemulsion polymerization modeling problem is addressed with an integrodifferential approach. The procedure was applied to experimental data, previously presented, on the microemulsion polymerization of hexyl methacrylate (C 6 MA) and styrene (STY). It was found that: (i) the nucleation rate is not linear with time, as assumed before, (ii) a vitreous effect is observed even in reactions where the polymer's glass transition temperature is lower than the reaction temperature, (iii) radical entry to polymer particles and coagulation among particles are negligible, (iv) the rate decrease interval is also caused by a reduction of active sites, (v) a mechanism in which micelles provide monomer to living particles was detected, and (vi) a simple three-parameter mechanistic model was obtained, capable of describing the studied systems. | A kinetic study of anion effects in the deamination of dodecylamine micelles | Morphology Transformation of a Polymeric Micelle Induced by Two Encapsulated Particles | Modeling the Self-Aggregation of Small AOT Reverse Micelles from First-Principles | what is the relationship between micelles and bulk solvents | Heating-Induced Micelle to Vesicle Transition in the Cationic−Anionic Surfactant Systems: Comprehensive Study and Understanding |
469 | Free registration for book worm delux? | I am aware of the piracy subreddits, but they usually just direct you to websites where you can download the books. I’m looking more for a subreddit where User A requests a certain book, then User B can email the book directly or maybe send a link to a dropbox where they may have the book. | Curious on the ways you can use the Internet to properly promote a free online book in pdf file format ? | Where can you free book reports online? | I rented the Kindle ebook version of this. I was told by my Professor that the textbooks were supposed to come with a registration code for InQuizitive, but the ebook does not seem to come with this? None of the pages have an access code, yet the textbook states in the "Preface for Teachers" that it offers InQuizitive, so why didn't I get a registration code from Amazon??? | Were can you download microsoft word for free? | every time you make a free account you get a token for a book then Use the mobile app and download the books onto your phone and you keep the books even if you cancel and you can download from multiple accounts to the same phone and keep all of the books. Then cancel your membership and they often offer more free audio books for not canceling or a free month | Tucker Max has some funny stories and when I heard that this was free I was like "why the heck not!" Now I want to get his actual books. | Hopefully, the title makes sense. I am looking for sites and resources related to releasing an E-Book for free from the get-go (not charging for the book). I know I could release chapters on Reddit, or submit to websites such as Wattpad and Inkitt, but was looking to see if there are other recommendations. I am looking to create a resource list to utilize in the future for areas that free E-Books/Anthologies could be released for anyone to read for free. Any suggestions are greatly appreciated! | 469 | How do you register Bookworm Deluxe? | Bookworm Challenge Un-Unlocked | Deluxe edition not very deluxe | I need help regarding deluxe edition. | The Stuffie is advertised that in comes with a book ... | If you plan on buying the books get the Deluxe editions! | Deluxe codes no longer valid. | How do you get a free miley world account? | How do you get free membership on adventure quest world? |
470 | Mutagenic Analysis of the F0Stator Subunits | ATP5E The ATP5F1E protein weighs 5.7 kDa and is composed of 51 amino acids. The protein is a subunit of the FF ATPase, also known as Complex V, which consists of 14 nuclear and 2 mitochondrial -encoded subunits. The nomenclature of the enzyme has a long history. The F fraction derives its name from the term "Fraction 1" and F (written as a subscript letter "o", not "zero") derives its name from being the binding fraction for oligomycin, a type of naturally-derived antibiotic that is able to inhibit the F unit of ATP synthase. The F particle is large and can | Abstract ::: A strain of Escherichia coli with the fol gene deleted and a kan gene inserted in its place was created for use in cloning and isolation of mutant dihydrofolate reductase. Southern blot analysis and dihydrofolate reductase enzyme assays confirmed the delta fol::kan genotype. A thyA mutation accompanied the fol deletion and is required for survival of a dihydrofolate reductase-deficient strain. | The fluoride-resistant phenotypes of butyrylcholinesterase (BChE) are determined by two different alleles: F1 (BCHE*243M) and F2 (BCHE*390V). As the frequencies of these mutations had not yet been estimated for random population samples, we studied the frequency of the F2 mutation in a sample (N = 1184) of blood donors (96% Whites) from a southern Brazilian city (Curitiba). The plasma samples were first screened for the fluoride-resistant phenotype by an inhibition method (1), and then the BCHE UF samples were examined by direct DNA analysis. PCR was followed by digestion with the HphI restriction enzyme, according to (2). The population frequency of the F2 mutation was 0.34% ± 0.12%. This frequency characterizes F2 as a non-polymorphic (less than 1%) allele. | The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-d-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 μM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s-1. Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of ... | FokI The enzyme Fok1 (Fok-1), naturally found in "Flavobacterium okeanokoites", is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves, without further sequence specificity, the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site. Its molecular mass is 65.4 kDa, being composed of 587 amino acids. The recognition domain contains three subdomains | Alpha-tubulin N-acetyltransferase of the cell such as in the cytoskeleton, cytoplasm, or the clathrin coated-pit in the membrane. This is closely related to one of its main functions which is the catalysis of microtubule acetylation. ATAT1 might tend to undergo a process known as mutagenesis according to which, a genetic mutation is produced. This may occur spontaneously or, on the other hand, due to the action of mutagens. It is possible to classify the different results of mutagenesis depending on which of the 421 aminoacids have been changed. If glutamine (Q), which occupies the 58th position in the sequence of aminoacids is | Mutagenic effect of AF-2 in higher plants was examined with heterozygotic maize and soybean. Mutagenicities of AF-2 were observed with higher concentrations than 0.0001μg/ml and 0.0005μg/ml in soybean and maize, respectively. | Chromosomal mutants of Escherichia coli deficient in the expression of F-plasmid functions were selected by mutagenizing F- cells, introducing an F' plasmid into the mutagenized cells by conjugation, and identifying transconjugants resistant to the donor-specific bacteriophage Q beta by a simple spray test. All but 1 of 25 mutants were defective in an extracellular stage of Q beta infection, suggesting that they fail to elaborate F-pili. At least six of these were also deficient as deoxyribonucleic acid donors. More than half of the mutants appear to be altered in peviously undetected chromosomal genes required for the expression of F-related cellular functions. | 470 | The a and b subunits constitute the stator elements in the F0 sector of F1F0-ATP synthase.Both subunits have been difficult to study by physical means, so most of the information onstructure and function relationships in the a and b subunits has been obtained using mutagenesisin combination with biochemical methods. These approaches were used to demonstrate thatthe a subunit in association with the ring of c subunits houses the proton channel throughF1F0-ATP synthase. The map of the amino acids contributing to the proton channel is probablycomplete. The two b subunits dimerize, forming an extended flexible unit in the peripheralstalk linking the F1 and F0 sectors. The unique characteristics of specific amino acid substitutionsaffecting the a and b subunits suggested differential effects on rotation during F1F0-ATPaseactivity. | ROTATIONAL COUPLING IN THE F0F1 ATP SYNTHASE | Subunit 4 of ATP synthase (F0F1) from yeast mitochondria. Purification, amino-acid composition and partial N-terminal sequence | Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer108 in loop 2 and ϵTyr114 in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits. | Assembly of F0 Sector of Escherichia coli H+ ATP Synthase INTERDEPENDENCE OF SUBUNIT INSERTION INTO THE MEMBRANE | The proton pore of the F 1 F o ATP synthase consists of a ring of c subunits, which rotates, driven by downhill proton diffusion across the membrane. An essential carboxylate side chain in each subunit provides a proton-binding site. In all the structures of c-rings reported to date, these sites are in a closed, ion-locked state. Structures are here presented of the c 10 ring from Saccharomyces cerevisiae determined at pH 8.3, 6.1 and 5.5, at resolutions of 2.0 Å, 2.5 Å and 2.0 Å, respectively. The overall structure of this mitochondrial c-ring is similar to known homologs, except that the essential carboxylate, Glu59, adopts an open extended conformation. Molecular dynamics simulations reveal that opening of the essential carboxylate is a consequence of the amphiphilic nature of the crystallization buffer. We propose that this new structure represents the functionally open form of the c subunit, which facilitates proton loading and release.npg | Two types of cDNA clones encoding a precursor of the delta-subunit of the human mitochondrial F0F1 ATP synthase complex (EC 3.6.1.34) have been isolated from a human cDNA library. Both clones contain a 504 basepair open reading frame that encodes a polypeptide with a presequence 22 amino acids in length and a mature protein 146 residues in length. The difference between the two types of cDNA clones is the presence of a 296 basepair insert in the 3' untranslated region of the delta-subunit cDNA in one of the types. | H+ -ATPases catalyze the synthesis of ATP from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. The enzyme consists of two parts: the hydrophobic, membrane integrated F0-part is involved in proton transport and contains the subunits a, b and c with a likely stoichiometry ab2c12 for the Escherichia coli enzyme. The nucleotide binding sites are located in the hydrophilic F1-part with subunit composition α3β3γδe [1]. During ATP hydrolysis intersubunit rotation of the γ-subunit in the F1-part (which was dissociated from the holoenzyme) was observed with single fluorescence labeled enzymes in realtime using enhanced videomicroscopy [2 - 4]. ::: ::: ::: ::: ATP synthesis and hydrolysis occur at the catalytic binding sites of the β-subunits. In the crystal structure of the F1-ATPase three different conformations of the three β-subunits were detected [5]. From these structural data a detailed mechanistic model of the F1-ATPase as a ‘stepped rotatory motor protein’ was developed [6]. According to this model, large conformational changes in the lower part of the β-subunits are expected during catalysis. ::: ::: ::: ::: We investigated the diffusion of the F1-part before and after binding of ATP with fluorescence correlation spectroscopy (FCS). The β-subunit was specifically labeled with a newly synthesized sulfonyl fluoride derivative of Sulforhodamine G. The labeled amino acid is not known yet. Preliminary FCS measurements with EF1 are shown (Fig.1.). Upon ATP binding the translational diffusion time is decreased by about 15 percent due to changes in size and shape of the enzymes during the catalytic cycle. ::: ::: ::: ::: Similar results were obtained for EF1-ATPase with a fluorescence label on the γ-subunit [7]. These FCS measurements are supported by new electron microscopy data, which show a significant shrinking up of F1-part of the enzyme upon binding of the non-hydrolysable ATP derivative AMPPNP. The diameter of the F1-part decreased mainly in the upper half of the F1-part upon binding of AMPPNP [8]. | Mutations at Glu-32 and His-39 in the epsilon subunit of the Escherichia coli F1F0 ATP synthase affect its inhibitory properties. |
471 | Rust bullet does a great job on rust | How do you get rid of rust on cars? | Best product for what I needed. Rust conversion. | Can steel rust faster? | This product Is excellent for rusty parts. I've been using this product for 30 plus years ever since my grandpa introduced me to it as kid working on old bicycles. Now I use it in industrial manufacturing and it's a time/ money saver! | Guns, Kinfes, scissors, tools, anything with a hinge. This is hands down the best rust protectant on the planet.
As soon as I get a new product with any of the above the first thing I do is apply Eezox.
Don't get the spray it over sprays, makes a mess and wastes too much. | Great stuff. A little goes a long way.
Bye bye rust.
A little tip... I went to the local hardware store and bought a spray bottle and put the spray top on this bottle. Fit perfect! Works great. | Have used this product in the past. Very good for what it is designed for: a primer over rusted metal. | It worked very well on the rust stains. It was not strong enough to cut through whatever is building up under the rim of the toilet. I'll have to find something stronger for that. | 471 | Rust bullet does a great job on rust. I used a toothbrush and it was an easy task. The bad news is the fumes. Do not get it on your hands or use it in an enclosed area. No free lunch on rust removal! | This stuff did not remove any rust... ... | How do I get rid of rust quickly? | It worked very well on the rust stains. It ... | How do you remove rust from the fridge body and how to save against it? | Will lemon juice remove rust? | How to remove rust from a metal hob? | If I get my gun cerakoted will that remove rust? | Is there a way to remove rust from inside a bike frame? |
472 | Synthesis, antiproliferative, and antiviral activity of certain 4-substituted and 4,5-disubstituted 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyrimidines | A new convenient synthesis of pyrrolo[3,2,1-ij]quinolin-4-one derivatives is described. In this method, methyl-7-hydroxyquinoline-2-ones are the starting materials onto which the third pyrrolo ring is condensed directly, yielding dehydrogenated methyl-9-hydroxypyrrolo[3,2,1-ij]quinolin-4-ones | A series of functionalized pyrido[2,3-d]pyrimidin-7(8H)-ones were prepared by a KF/Al2O3 mediated condensation of 4-(alkylamino)-2-(methylthio)pyrimidine-5-carbaldehydes and phenyl acetic acid ester derivatives. | 6-Substituted 5-amino-2-hydrazino-4-phenylthieno[2,3-d]pyrimidines (2a–c) were synthesized and used as key intermediates for the synthesis of new thienopyrimidotriaz\oles and pyrazolylthienodipyrimidines. | A series of substituted pyrrolo[2,3-e][1,3,4]thiadiazine 4,4-dioxides is synthesized from 1-substituted-2-amino-3-cyanomethylsulfonyl-4,5-dimethylpyrroles. | Abstract 6-( p -Bromophenylthio)-5-methyl-2,4,7-triaminopyrido [2,3-d]-pyrimidine was found to be active on P388 and L1210 leukemias while 6-alkylthio derivatives were inactive. | Abstract A synthetic pathway to reach easily the 4-thio-D-ribofuranose is described. Some corresponding pyrimidine α and β 4′-thioribonucleosides have been synthesized and evaluated as antiviral agents against various viruses. | Abstract A series of 2,4,6-trisubstituted pyrimidines ( 3a – o ) was synthesized and evaluated for their in vitro antimalarial activity against P. falciparum . Out of the 15 compounds synthesized 11 compounds showed MIC in the range of 0.5–2 μg/mL. These compounds are in vitro several folds more active than pyrimethamine. | A method for preparing the amino, alkoxy, and alkylsulfanyl derivatives of pyrano[4″,3″:4′,5′]pyrido-[3′,2′:4,5]thieno[3,2-d]pyrimidines based on 8-chloro-2,2-dimethyl-5-pyrrolidin-1-yl-1,4-dihydro-2H-pyrano[4″,3″:4′,5′]pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine was developed. The anticonvulsant activity of the compounds synthesized here was investigated. | 472 | The synthesis of selected 4-substituted and 4,5-disubstituted pyrrolo[2,3-d]pyrimidine nucleosides in which the sugar was replaced by a (1,3-dihydroxy-2-propoxy)methyl (DHPM) group is described. We report herein the synthesis and evaluation of such compounds for antiproliferative and antiviral activity | Antiviral Activity of Inhibitors of Pyrimidine De-Novo Biosynthesis | New method for the synthesis of pyrrolo[2,3-b]dihydroquinolines | Synthesis and Anticonvulsive Activity of 5-Pyrrolidin-1-Ylpyrano[4″,3″:4′,5′]Pyrido-3′,2′:4,5]Thieno[3,2-D]Pyrimidine Derivatives | Abstract A novel, two-step, facile route for the synthesis of pyrrolo[2,3- b ]quinoxalines via 2,3-dioxopyrroles, enhanced by microwave irradiation, is presented. The newly synthesized 2,3-dioxo-5-halophenyl pyrrolo precursors 4a – c as well as the non-aromatized ethyl 2-(4-halophenyl)-1-methyl-2,4-dihydro-1 H -pyrrolo[2,3- b ]quinoxaline-3-carboxylates 6a – c and the aromatized ethyl 2-(4-halophenyl)-1-methyl-1 H -pyrrolo[2,3- b ]quinoxaline-3-carboxylates 7a – c were evaluated for their antioxidant, cytostatic, and antiviral properties. Most of them proved to be potent hydroxyl radical scavengers and inhibited in vitro lipid peroxidation. The compounds showed moderate antiproliferative activity, while 6a inhibited vaccinia virus at an EC 50 value of 2 μM, and 4c and 6c inhibited Sindbis virus at EC 50 values of 4 μM. | Novel Nucleoside Analogues: First Synthesis of Pyridine-4-Thioglycosides and Their Cytotoxic Evaluation. | Antimalarial activity of 2,4,6-trisubstituted pyrimidines. | Summary The compound (±)-2-amino-3,4-dihydro-7-[(1α, 2α, 3β, 4α)-2,3-dihydroxy-4-(hydroxymethyl)-1-cyclopentyl]-7H-pyrrolo[2,3-d]pyrimidine-4-one, referred to as 54.247-RP, is a new nucleoside analog with anti-herpes properties. At 25 μg/ml (MIC), the antiviral activity appeared to be selective, since the compound inhibited viral DNA synthesis to a greater extent than cellular DNA synthesis. However, at 100 μg/ml, host cell DNA synthesis was also markedly reduced. 54 247-RP, administered intraperitoneally in multiple treatments at doses of 10–20 mg/kg, significantly increased the survival time of mice infected with either HSV 1 or HSV2. | Synthesis and antiproliferative activity of imidazo[1,2-a]pyrimidine Mannich bases. |
473 | Does anyone know where I can get a FLAC rip of Soup? | I sent this product to my sister she's been a widow for a short time anyway. I always talk about Amazon good ideams so I sent these soup flake she's so please she's going to order more. Thank You | My family loves this soup. We grew up on it and now enjoy it as adults. Since we cannot find in the the markets, using Amazon is the only way to buy it. | i've used lipton dry soup for years. i'm always looking for new ways to include it in my recipes. | What is a good soup? | a retail line of "heat-n-serve" soups would be available in May at select grocery stores. There were five different variations available made by SoBe Beverages and supervised by Al Yeganeh. The soups were packaged in 15 oz. ‘Grab-N-Go’ clear packages. Since its launch in May 2005, "The Original SoupMan" line of soups is sold in 14 states and over 7,000 grocery stores across the United States and Canada. In May 2017, Robert Bertrand, the chief financial officer of The Original Soupman, was arrested and charged with income tax evasion for failure to pay Medicare, Social Security, and federal income taxes | Bowling for Soup Songs? | What is split-pea soup? | This soup used to be worth the money, but somehow the recipe changed and it is tasteless now. Too bad! I rarely like packaged soup - this one was good. Hope they will realize and change their receive back. | 473 | I can't afford to shell out the $400 for soup on vinyl so I'm just looking for the highest quality recording of it I can find. | Best quality vinyl releases? | What is the best/cheapest vinyl pressing company to press my album. | What are the best sounding albums pressed to vinyl? | Looking for a list of the definitive vinyl pressings for albums - or at least a list of pressings to avoid | Theoretically, What is the data capacity of a vinyl LP record? | Need your help on affordable vinyl records online | Best value for money pressing vinyl | Essentially vinyl - W records |
474 | I just got home to Cincinnati after the game this weekend | There will be a large group coming into Cincinnati for a weekend soon. I'm looking for ideas we can do to explore Cincinnati a bit/have a fun time. Dinner is already covered, just looking for things we can do during the day. Any ideas?
Edit: Preferably low-cost activities but all ideas are welcome. | Where do you guys park at when you are going to the Vanderbilt game? | **GAME** | Cincinnati](#f/cincinnati)Cincinnati @ [ECUECU
--:|:--
**Location** | ECU Dowdy-Ficklen Stadium
**Time** | 7:00PM ET
**Watch** | **TV:** CBSSN
**Odds** | *Spread:* Cincinnati -24 - *Over/Under:* 48
|| Flair]( ¦ [Merch]( ¦ [Chat
||Made with the /r/CFB Game Thread Generator
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### LET'S TALK FOOTBALL! | Hey so I'm heading up to Oakland for a few days with a bunch of my friends. We're trying to catch the game tonight at a bar somewhere near where we're staying in South Oakland. We're all college kids so we just want somewhere with a good atmosphere for the game but also hopefully cheap beer. | I'm in town on business and I'm staying within walking distance of the park and thought about catching the game tomorrow night. I got to see a game at Camden about 15 years ago when I was a kid and loved it. Hit me up and we can take in the game together and we can talk baseball. | And will you attend more this year now that the team made the postseason?
I usually try to get to 5-10 games a year. I also probably watch 80% of the evening games on TV, check scores for the weekday day games. | Just thought I'd pass it along in case someone on /r/detroit has ~~hoop~~ pitch (?) dreams.
From the DCFC facebook page]( they're holding tryouts tonight (Feb. 6th) at 11pm at [Oakland Yard Athletics in Waterford.
**Edit**: ALSO, as mentioned by /u/PolskaPrincess, everyone is welcome to hang out and watch the tryouts. I've heard they might even have *beer*. | Tonight! Come watch Cincinnati's finest and funniest standup comedians craft their material on the Video Archive's beautiful patio. Live comedy, craft cocktails, and secret video-doors. What else could a person want in life?
Wanna try your hand at standup? Sign ups are at 7. Show is at 7:30. Bring your friends and get ready to laugh! | 474 | It was my first game in Baltimore and I had a blast. Your pre-game festivities and the noise in the stadium are heads and tails above Paul Brown Stadium; it’s like the Ravens and bengals play in a different league. Everyone I met in the city was great. Had dinner at a little place named Maisey’s and loved it. My fiancé set this trip up for me because she knows I love the Ravens. She set up a meeting with TJ Weist (her coworker and him grew up together) and he gave me a used, official ball. I was on top of the world all weekend. The crab cakes were amazing, Ft. McHenry was super cool, and the inner harbor was fun. Thanks Baltimore, will see the Ravens when they come to Cincy to whip some ass! | | Any advice to a visiting Ravens fan this weekend? | Going to the away game at Baltimore: what's the stadium like and how easy is it to move up to nicer, empty seats? | This one tied up the package on the Baltimore series but it was boring.. Everything worked out perfectly for everyone and ... | I've lived in Chicago, San Francisco, San Diego, and currently Sacramento. What's Baltimore like? | Baltimore Pinball League Meet and Greet: Tomorrow, 5/28 | Where to throw a happy hour in Baltimore? | Places to Celebrate in Baltimore? | Anyone go to the race in Baltimore last year? |
475 | Which us state pays its governor the most money? | State that pays the governor the most? | The state that pays its governor the highest salary and amount of it? | What is the state governor's salary? | What is the salary of a state governor in georgia? | What states does the us owe money to? | Alaska Governor's salary? | What three states have the lowest tax rate? | What are the five largest state and local and government expenditures? | 475 | What state pays its governor the highest salary? | What is salary of alaska governor? | What is salary of Tennessee's governor? | what is the average salary of a us governor | What US state has the highest average salary? | What is the us state pays its govenor the most money? | Tennesse governor's salary? | How much is minimum pay of the state of Michigan? | How muchdid the us pay for hawaii? |
476 | Investigation of surface reactions by the static method of secondary ion mass spectrometry: V. The oxidation of titanium, nickel, and copper in the monolayer range | Summary Electrochemistry has played a significant role in many research fields. Owing to its sensitivity and selectivity, in situ electroanalysis has been widely used as a fast and economical means for achieving outstanding results. Although many spectroscopic techniques have been used in electrochemistry, the challenges to capture short-lived intermediate species as a result of electron transfer in the buried solid electrode and electrolyte solution interface remains a grand challenge. In situ imaging mass spectrometry (IMS) recently has been extended to capture transient species in electrochemistry. This review intends to summarize newest development of IMS and its applications in advancing fundamental electrochemistry. | The application of static SIMS in characterising the chemistry of surfaces in basic and applied surface science is briefly demonstrated. First the ability of SSIMS to monitor the chemical structure and coverage of the adsorbed state is demonstrated with reference to CO and ethene adsorption on single crystal metals. The use of time-of-flight SSIMS to monitor the chemical state of the surface of a organic gas detector phthalocyanine is outlined. Finally a microfocussed liquid gallium beam is used to probe the mechanism of localised oxide scale growth on a steel alloy used in nuclear reactor construction. | Time-of-flight secondary ion mass spectroscopy (ToF SIMS) analysis was performed on 316L stainless steel polarized potentiodynamically up to different potentials in NaCl solution. The surface film thickness increased with the ending potential of the potentiodynamic polarization, when estimated by the oxygen ion depth profiles. The chloride ion intensities at the film/metal interface were correlated with the ending potentials during the potentiodynamic polarization and cumulative anodic charge densities that govern the pit initiation. The results and analysis support the passivity breakdown mechanisms considering the role of chloride ions at the metal/film interface, rather than at the film/solution interface. | This contribution deals with the systematic investigation of different surface finishes (electropolished, polished, ground, sandblasted) on the oxidation behaviour of chromium steels. The specimens were oxidized in a H 2 -2.5%H 2 O atmosphere at 872 K for 1 h to 100 h. Depth profiles were recorded by secondary neutral mass spectrometry (SNMS) to determine the elemental composition of the oxide scale and the diffusion profiles below the scale. The surface finish was found to influence both the thickness of the oxide scale and the depletion of the selectively oxidized elements. In the case of austenitic steels also the composition of the oxide scale is dependent on the surface finish: Electropolished samples oxidized for 100 hours exhibit an iron and nickel-rich oxide film, whereas on polished, ground and sandblasted specimens chromium and manganese-rich scales are found. | The desorption kinetics of halogens from Nb, Ta, Mo and W polycrystalline surfaces are studied, at low coverage (Θ ⩽ 10−2 of a monolayer) and high temperature (1800–2400 K), using a pulsed ionic beam method. For a relatively high energy incident positive ion beam (ϵ = 2500 eV), the diffusion kinetics of implanted Br and I ions towards the surface are observed. At low energy (ϵ < 500 eV), a first order kinetic corresponding to the desorption of halogens from these surfaces is found. Preliminary results are given on the desorption energy and the preexponential factor of halogens adsorbed on Nb, Mo, Ta and W polycrystalline surfaces. The main result of this systematic study is that the mean adsorption lifetime for a given temperature, as the desorption energy decreases for F through I on all of the studied surfaces. A similarity between Nb and Ta, as between Mo and W surfaces for the desorption energies of halogens is also found. | Computer simulation of possible distributions of H ad and D ad atoms developed on nickel surface as a result of partial reactions of anodic hypophosphite oxidation and cathodic proton (deuteron) discharge from water respectively was used for modeling of the catalyst surface state according to on-line electrochemical mass spectrometry data. The simpliest lattice-gas model gives a probable qualitative description of the catalyst surface state and allows the genesis of electrocatalytic properties of nickel with the electrode potential to be followed. Location of anodic and cathodic half-reactions at special types of sites was evidenced, leading to formation of non-equilibrium H 2 , HD and D 2 mixtures with the lower HD content than that predicted theoretically. | Abstract The density functional formalism is used to investigate theoretically the underpotential adsorption of Tl on Ag(111). In agreement with experiment, a relatively compact structure is predicted for the adsorbed monolayer. While the work function of the substrate covered by a monolayer of adsorbate is very close to that of the pure adsorbate metal, the position of the effective image charge differs appreciably for both systems. The second harmonic generation response of the system is also analyzed in the quasi-static limit. | Abstract The electronic processes of Cu(Ag, V, Rh)(0 0 1) surface oxidation are comparatively analyzed based on the recent ‘chemical bond–valence band–potential barrier’ (BBB) correlation mechanism [C.Q. Sun, Prog. Mater. Sci. 48, 521–685 (2003)], which allows reaction formulae for all the observed phases with identification of individual atomic valence and the binding kinetics at the surfaces with the same geometry. It is consistently understood that the forming kinetics of the primary oxide tetrahedron and its derivative on the valence density-of-states (DOS) are intrinsically common for all these analyzed systems, though the patterns of observation in terms of morphology and crystallography vary from situation to situation. However, the lattice size and electronegativity of the host surfaces determine extrinsically the site selectivity of the oxygen, the order of bond formation and the orientation of the oxide tetrahedron. | 476 | Abstract By means of the static method of secondary ion mass spectrometry the oxidation of a polycrystalline titanium, nickel, and copper surface cleaned by ion bombardment, was investigated in the oxygen dose range up to 1200 L at room temperature. On titanium and nickel two different oxide phases were found, one covering the other. The thickness of the oxide layer on copper did not exceed one monolayer. | A fast and low-cost method using electrolysis for sample preparation of carbon steel present in weld electrodes aiming to achieve quantification of heavy metals by inductively coupled plasma mass spectrometry (ICP-MS) was developed. Conditions of the electrolysis, such as pH and electrical charge were investigated to improve the solubility and concentration of the analytes in the electrolyte. The method showed high reproducibility, with a relative standard deviation (RSD) of less than 3.05%, and the recovery from 88.6 to 108.9% for the analytes demonstrates the accuracy of the developed method. | The use of a cupric ion-selective electrode as end-point detector in precipitation titration of oxalate | Burning of Mixtures of Copper Oxide with Titanium | Washed human erythrocytes were incubated with titanium dioxide (TiO2) particles at 37°C for 1 hr and hemolysis was determined by the percentage of hemoglobin released (optical density at 540 nm; OD540) from the cells. Effects of TiO2 on OD540 were corrected and dose-response curves were analyzed by the Hill plot. Judging from the estimated dose to cause 50% hemolysis, the anatase form of micron-scale (<5000 nm) particles was 73 and 11 times more potent than the amorphous (<50 nm) and rutile (<5000 nm) forms, respectively, whereas it was 1.3 times more potent than the nano-scale (<25 nm) anatase particles. Plasma abolished the hemolysis due to anatase and rutile forms. Thus, hemolytic effects of TiO2 can be greatly different depending on the polymorph but not on the primary size (nano- or micron-scale) of particles. TiO2-induced hemolysis is unlikely to occur in vivo because of the presence of plasma. | Evaluation of A Case of Battery Ingestion with Inductively Coupled Plasma-Mass Spectrometry Metal Analysis | Paint Spray is developed as a direct sampling ionisation method for mass spectrometric analysis of additives in polymer-based surface coatings. The technique simply involves applying an external high voltage (5 kV) to the wetted sample placed in front of the mass spectrometer inlet and represents a much simpler ionisation technique compared to those currently available. The capabilities of Paint Spray are demonstrated herein with the detection of four commercially available hindered amine light stabilisers; TINUVIN® 770, TINUVIN® 292, TINUVIN® 123 and TINUVIN® 152 directly from thermoset polyesterbased coil coatings. Paint Spray requires no sample preparation or pre-treatment and combined with its simplicity -requiring no specialised equipment -makes it ideal for use by non-specialists. The application of Paint Spray for industrial use has significant potential as sample collection from a coil coating production line and Paint Spray ionisation could enable fast quality control screening at high sensitivity.Abstract: Paint Spray is developed as a direct sampling ionisation method for mass spectrometric analysis of additives in polymer-based surface coatings. The technique simply involves applying an external high voltage (5 kV) to the wetted sample placed in front of the mass spectrometer inlet and represents a much simpler ionisation technique compared to those currently available. The capabilities of Paint Spray are demonstrated herein with the detection of four commercially available hindered amine light stabilisers; TINUVIN ® 770, TINUVIN ® 292, TINUVIN ® 123 and TINUVIN ® 152 directly from thermoset polyester-based coil coatings. Paint Spray requires no sample preparation or pre-treatment and combined with its simplicity -requiring no specialised equipment -makes it ideal for use by non-specialists. The application of Paint Spray for industrial use has significant potential as sample collection from a coil coating production line and Paint Spray ionisation could enable fast quality control screening at high sensitivity. | Secondary Ion Mass Spectrometry (SIMS) extracts chemical, elemental, or isotopic information about a localized area of a solid target by performing mass spectrometry on secondary ions sputtered from its surface by the impact of a beam of charged particles. This primary beam sputters ionized atoms and small molecules (as well as many neutral particles) from the upper few nanometers of the sample surface. The physical basis of SIMS has been applied to a large range of applications utilizing instruments optimized with different types of mass analyzer, either dynamic SIMS with a double focusing mass spectrometer or static SIMS with a Time of Flight (TOF) analyzer. Here, we present a short review of the principles and major applications of three different SIMS instruments located in Switzerland. | Laser desorption ionization mass spectrometry imaging for the study of metal surfaces |
477 | GRASP with path relinking for the symmetric Euclidean clustered traveling salesman problem | We present a new symmetric traveling salesman problem tour construction heuristic. Two sequential matchings yield a set of cycles over the given point set; these are then stitched to form a tour. Our method outperforms all previous tour construction methods, but is dominated by several tour improvement heuristics. | We study the traveling salesman problem (TSP) in the 2-dimensional Euclidean plane. The problem is NP-hard in general, but trivial if the points are in convex position. In this paper, we investigate the influence of the number of inner points (i.e., points in the interior of the convex hull) on the computational complexity of the problem. We give two simple algorithms for this problem. The first one runs in O(k!kn) time and O(k) space, and the second runs in O(2 k k 2 n) time and O(2 k kn) space, when n is the total number of input points and k is the number of inner points. Hence, if k is taken as a parameter, this problem is fixed-parameter tractable (FPT), and also can be solved in polynomial time if k=O(log n). We also consider variants of the TSP such as the prize-collecting TSP and the partial TSP in this setting, and show that they are FPT as well. | This paper discusses the κ-BENDS TRAVELING SALESMAN PROBLEM. In this NP-complete problem, the inputs are n points in the plane and a positive integer κ, and we are asked whether we can travel in straight lines through these n points with at most κ bends. There are a number of applications where minimizing the number of bends in the tour is desirable because bends are considered very costly. We prove that this problem is fixed-parameter tractable (FPT). The proof is based on the kernelization approach. We also consider the RECTILINEAR κ-BENDS TRAVELING SALESMAN PROBLEM, which requires that the line-segments be axis-parallel.1 Note that a rectilinear tour with κ bends is a cover with κ-line segments, and therefore a cover by lines. We introduce two types of constraints derived from the distinction between line-segments and lines. We derive FPT-algorithms with different techniques and improved time complexity for these cases. | In this paper we consider the Asymmetric Quadratic Traveling Salesman Problem (AQTSP). Given a directed graph and a function that maps every pair of consecutive arcs to a cost, the problem consists in finding a cycle that visits every vertex exactly once and such that the sum of the costs is minimal. We propose an extended Linear Programming formulation that has a variable for each cycle in the graph. Since the number of cycles is exponential in the graph size, we propose a column generation approach. Moreover, we apply a particular reformulation-linearization technique on a compact representation of the problem, and compute lower bounds based on Lagrangian relaxation. We compare our new bounds with those obtained by some linearization models proposed in the literature. Computational results on some set of benchmarks used in the literature show that our lower bounding procedures are very promising. | In this paper, we present a polynomial time approximation scheme (PTAS) for a variant of the traveling salesman problem (called segment TSP) in which a traveling salesman tour is sought to traverse a set of n ∊-separated segments in two dimensional space. Our results are based on an interesting combinatorial result which bounds the total number of entry points in an optimal TSP tour and a generalization of Arora's technique5 for Euclidean TSP (of a set of points). The randomized version of our algorithm takes O(n2(log n)O(1/∊2)) time to compute a (1+∊)-approximation with probability ≥l/2, and can be derandomized with an additional factor of O(n2). | A tour τ of a finite set P of points is a necklace-tour if there are disks with the points in P as centers such that two disks intersect if and only if their centers are adjacent in τ. It has been observed by Sanders that a necklace-tour is an optimal traveling salesman tour. | In this chapter we deal with the problem of solving symmetric TSP (STSP) instances to optimality. Of course, STSP instances are particular cases of asymmetric TSP (ATSP) instances, those for which the distance between any two cities is irrelevant of the direction. Therefore we could transform any instance of the STSP to an asymmetric one and use the results of Chapter 4 to solve it. In fact the techniques of Chapter 4 do not perform well when the costs of the arcs (i,j) and (j,i) only slightly differ. Progress in the solution techniques for the STSP is such that it is common to transform an ATSP into a symmetric one to solve it to optimality (see [474]). | Inspired by an application in the field of on-demand public transportation, we perform a Monte Carlo simulation study on the probability distribution of the length of Traveling-Salesman-Problem (TSP) tours between small numbers of random locations. We consider a fixed convex region, where we generate a fixed number of random locations from a known probability distribution and find the corresponding euclidean TSP tour for them. We simulate this process extensively and perform both quantitative and qualitative analyses of the resulting experimental distribution for the TSP tour length. We show that, under certain assumptions on the shape of the region and the probability distribution of locations, the length of the TSP tour is well-approximated by a normal distribution, even for as few as five locations. Furthermore, we propose experimental models for estimating the mean and standard deviation of the tour length. | 477 | In this paper, we propose new heuristics using several path-relinking strategies to solve the Clustered Traveling Salesman Problem (CTSP). The CTSP is a generalization of the Traveling Salesman Problem (TSP) in which the set of vertices is partitioned into clusters and the objective is to find a minimum cost Hamiltonian cycle such that the vertices of each cluster are visited continuously. A comparison among the performance of the several different adopted path-relinking strategies is presented using instances with up to 2000 vertices and clusters varying between 4 and 150 vertices. Also computational experiments were performed to compare the performance of the proposed heuristics with an exact algorithm and a Genetic Algorithm. The obtained computational results showed that the proposed heuristics were able to obtain competitive results related to the quality of the solutions and computational execution time. | A probabilistic traveling salesman problem (PTSP) is essentially a traveling salesman problem (TSP) in which the number of nodes to be visited in each problem instance is a random variable. One can define, as well, other probabilistic problems in the context of network optimization. In this paper we give an overview of some results obtained on the PTSP and on a probabilistic version of the shortest-path problem. | Generating Travelling-Salesman Problems with Known Optimal Tours | Among numerous NP-hard problems, the Traveling Salesman Problem (TSP) has been one of the most explored, yet unknown one. Even a minor modification changes the problem’s status, calling for a different solution. The Generalized Traveling Salesman Problem (GTSP) expands the TSP to a much more complicated form, replacing single nodes with a group or cluster of nodes, where the objective is to find a minimum-length tour containing exactly one node from each cluster. In this paper, a new heuristic method is presented for solving singlevehicle single-depot GTSP with the ability of controlling the search strategy from conservative to greedy and vice versa. A variant algorithm is then developed to accommodate the multi-vehicle single-depot condition, which is modified afterwards to accommodate the multi-vehicle multi-depot GTSP. | In the standard version of the traveling salesman problem (TSP), we are given a set of customers located in and around a city and the distances between each pair of customers, and need to find the shortest tour that visits each customer exactly once. Suppose that some of the customers are located in the center of the city. Within a window of time, center city becomes congested so that the time to travel between customers takes longer. Clearly, we would like to construct a tour that avoids visiting customers when the center of the city is congested. This variant of the TSP is known as the time dependent TSP (TDTSP). We review the literature on the TDTSP, develop two solution algorithms, and report computational experience with our algorithms. | A Novel Metaheuristic for Travelling Salesman Problem | A multivariant travelling-salesman problem | A comparative analysis of several asymmetric traveling salesman problem formulations | A Fast Composite Heuristic for the Symmetric Traveling Salesman Problem |
478 | what is the term for a flow of water upstream of a traffic bottleneck | Traffic flow at a highway bottleneck is as follows: A spontaneous traffic breakdown occurs, where there are free flows both upstream and downstream of the bottleneck before the breakdown has occurred. In contrast, an induced traffic breakdown is caused by a propagation of a congested pattern that has earlier emerged for example at another downstream bottleneck. Empirical data that illustrates the set of fundamental empirical features of traffic breakdown at highway bottlenecks as well as explanations of the empirical data can be found in Wikipedia article Kerner’s breakdown minimization principle and in review. The generally accepted classical fundamentals and methodologies of traffic | Bottleneck is a common term used to describe the process/operation/person that constrains the performance of the whole system. Since Goldratt introduced his theory of constraint, not many will argu ... | Details of traffic evolution were studied upstream and downstream of a freeway bottleneck located near a busy on-ramp. It is shown that on certain days the bottleneck became active upon dissipation of a queue emanating from somewhere further downstream. On such occasions, the bottleneck occurred at a fixed location, approximately one kilometer downstream of the merge. Notably, even after the dissipation of a downstream queue, the discharge flows in the active bottleneck were nearly constant, since the cumulative counts never deviated much from a linear trend. The average bottleneck discharge flows were also reproducible from day to day. The diagnostic tools used in this study were curves of cumulative vehicle arrival number versus time and cumulative occupancy versus time constructed from data measured at neighboring freeway loop detectors. Once suitably transformed, these cumulative curves provided the measurement resolution necessary to observe the transitions between freely flowing and queued conditions and to identify some important traffic features. | effects on speed, especially through bottlenecks. The authors began by discussing previous approaches to traffic flow theory. They note that at the time there had been some experimental work, but that “theoretical approaches to the subject [were] in their infancy.” One researcher in particular, John Glen Wardrop, was primarily concerned with statistical methods of examination, such as space mean speed, time mean speed, and “the effect of increase of flow on overtaking” and the resulting decrease in speed it would cause. Other previous research had focused on two separate models: one related traffic speed to traffic flow and another related | Bottleneck literally refers to the narrowed portion (neck) of a bottle near its opening, which limit the rate of outflow, and may describe any object of a similar shape. The literal neck of a bottle was originally used to play what is now known as slide guitar.
Metaphorically, the term may also be used as an analogy for any of the following implications of rate limitation or function restriction:
Computing
Bottleneck (network), in communication networks using max-min fairness
Bottleneck (software), a software component that severely affects application performance
Internet bottleneck, when high usage slows the performance on the Internet at a particular point
Von Neumann bottleneck, a limit of throughput between a computer's processor and memory
Interconnect bottleneck
Geography
Bottleneck (K2), a mountain feature near the top of K2 mountain
Choke point, a feature that reduces passability of terrain
Free State Bottleneck, a quasi-state that existed in Germany during the time of the Weimar Republic
Other
Bottleneck (engineering), where the performance of an entire system is limited by a single component
Bottleneck (production), where one process reduces capacity of the whole chain of processes
Nocturnal bottleneck hypothesis to explain several mammal traits
Population bottleneck, an evolutionary event that drastically reduces a population
Traffic bottleneck, a local disruption in a transportation network
See also
Liebig's law of the minimum
Reverse salient | Demand exceeding the capacity of a bottleneck will create congestion upstream of that bottleneck. Once this congestion occurs, the maximum flow through this bottleneck decreases (capacity drop). By limiting the flow towards the bottleneck, one can prevent or postpone the capacity drop and the accompanying congestion. In case the bottleneck is caused by an on-ramp, a common approach is to meter the on-ramp flow. For metering to be effective the algorithm has to be tuned carefully. Normally, the parameters of a metering algorithm are fit for the situation. However, traffic is dynamic and external factors might change, which both lead to changes in parameters of the traffic process. This paper studies how these parameters can be updated dynamically in the control algorithm. It considers various ramp metering algorithms and introduces methods to adapt their parameters. They are tested with simulations using the METANET model. This shows that parameter adaptation improves traffic state. Gains in travel time due to parameter adaptation are typically several percent compared to non-adaptive ramp metering. Road authorities can use these findings to improve ramp metering algorithms and reduce delays. | Bottleneck (engineering) link, a data processing software, etc. In computer programming, tracking down bottlenecks (sometimes known as "hot spots" - sections of the code that execute most frequently - i.e. have the highest execution count) is called performance analysis. Reduction is usually achieved with the help of specialized tools, known as performance analyzers or profilers. The objective being to make those particular sections of code perform as fast as possible to improve overall algorithmic efficiency. In a communication network, sometimes a max-min fairness of the network is desired, usually opposed to the basic first-come first-served policy. With max-min fairness, data flow between | Traditional micropipelines based on handshaking mechanisms are simple and reliable, but their throughput is limited by the round-trip flight time between two consecutive micropipeline stages. We propose an RSFQ implementation of a micropipeline with simple credit-based flow control that can hide the round-trip latency and significantly improve the throughput. In this paper, we present numerically calculated and experimentally measured throughput for several types of RSFQ credit-controlled micropipelines (including the special case of a micropipeline with only one credit), and their critical comparison. | 478 | upstream from a traffic bottleneck (shockwave). Congestion shockwaves will vary in propagation length, depending upon the upstream traffic flow and density. However, shockwaves will generally travel upstream at a rate of approximately 20 km/h. Traffic on a stretch of road is said to be stationary if an observer does not detect movement in an arbitrary area of the time-space diagram. Traffic is stationary if all the vehicle trajectories are parallel and equidistant. It is also stationary if it is a superposition of families of trajectories with these properties (e.g. fast and slow drivers). By using a very small hole in | Traffic wave | TIME SCALES, FLUCTUATIONS AND CONSTANT FLOW PERIODS IN UNI- DIRECTIONAL TRAFFIC | The macroscopic determination of speed and flow of a highway traffic stream | Observation and Characteristics Analysis of Traffic Flow in Nanjing | Long-tail traffic | the mechanism for long tail traffic is based on feedback from | This paper employed cross correlation in order to estimate wave velocities between several successive detector stations during congested periods over a large data set. Given homogeneous vehicles and drivers, the macroscopic model of Lighthill, Whitman and Richards (LWR), predicts that for a convex flow-density relationship all waves should propagate upstream during congested periods. The analysis first employed cumulative arrivals - a functional flow - and then the local traffic speed as the detections stations. It was then shown that the flow-based analysis yielded mixed results, with many measurements being consistent with earlier research, but many more measurements falling outside of the typical range of measured wave velocities from the literature. But vehicles are not homogeneous and it was also shown that flow and occupancy depend on effective vehicle length as well as the local traffic speed. Because the vehicle lengths travel downstream with the vehicles, this information will hinder the attempts to extract wave velocities propagating against the flow traffic when using flow-based measures. | Some aspects of the theory of traffic flow |
479 | I am new to this plague of bed bugs, and have some questions. | How do you know bed bugs are gone? | They are able to crawl and move short distances within an infected area, and slowly spread to other rooms in the home or business. Another way bed bugs move around is by finding their way into purses, backpacks, suitcases, briefcases, clothing, and jackets. | According to this...\nhttp://pmo.umext.maine.edu/factsht/bedbugs.htm\nControl of bed bugs can be difficult.especially in homes that have many cracks and crevices, loose wallpaper, etc. Three actions need to be considered for quick relief and control....\n1.) A good vacuum cleaning \n2.) Use a space spray(bug bomb) to penetrate an infested area.\n3.)After space spraying, use a light application of an approved aerosol spray on mattresses, stuffed chairs, clothes, etc., to kill surviving bed bugs.\nAir everything out afterwords...\nA fresh paint job and caulking to seal the cracks\nmight help, too.\nOr call a better exterminator...\nhttp://www.globalexterminating.com/bed-bug-infestation.html | Last week, I found out I had bed bugs
This means it is a spell cast by humans that makes bed bugs not exist | I bought it 3weeks ago and so far 90 percent of those creepy bed bugs are gone along, used along with some damicticus earth (almost how you spell it ) and two shots of spray. Tried the hooont plug in and didn't work at all for bed bugs! | Fleas are not usually found infesting a homeowner's beds and bedding. ... The more likely situation is that flea eggs, larvae and pupae are living under the bed or, even more likely, are living in the bed and bedding of the household pet(s). | ITS OK, NOT ANY REAL BIG HELP, LIST TO YOUR PEST CONTROL COMPANY IF YOU EVER NEED A BED BUG TREATMENT. | Bed Bugs: Clinical Relevance and Control Options | 479 | I had been getting bites on my arms starting 3 or so weeks ago, and thought maybe my indoor cat had somehow picked up fleas. I treated the cat, and was still getting bitten but thought maybe it was still some stray fleas, so i started doing some deeper cleaning in areas the cat mostly hung out. When I was vacuuming the windowsill area in my bedroom, I saw something run across my bed spread. I squished it with a tissue, and started searching the internet to identify what it might be. Sure enough, discovered it was a bed bug. I then pulled the sheets and mattress cover back to check along the seams of the mattress and saw more. This all happened late in the evening, so I had to wait until morning to call an exterminator.
The exterminator came yesterday morning and confirmed that I do indeed have bed bugs, but it appears they are confined to my bedroom, and as far as he could tell only in the bed. He assured me that my infestation rated about a 2 on a scale of 10, and shouldn't be too difficult to eradicate. He gave me a list of preparations, and said they would call today to schedule the first of three treatments. He left my mattress and box springs turned up on their sides, and told me to leave it that way until they came back. I am working on preparing the room for them to spray, but I have some questions.
When I vacuum thoroughly in there, how should I clean out my vacuum to not risk spreading them to other areas of the house? I have a really shitty old bagless vacuum, but it has three or four filters. Could the bugs get into the filters? I had just purchased a fancy new vacuum, but I am not even going to take it out of the box until the bugs are gone.
I know from reading here that I should not be sleeping elsewhere, but the exterminator said I would need to until they came back for the first treatment. I bought encasements, and they are going to put them on for me when they do the treatment and then I will return to sleeping in my room. In the meantime, I am cleaning, running clothing in the dryer and bagging. What precautions should I take entering and leaving the room to prevent spreading the bugs? Am I being too paranoid, that they might be clinging to my feet when I walk in and out of the room? I feel like I am going crazy with worry.
My bedroom is the master bedroom of a three bedroom house, and he checked all other rooms of the house and didn't find bugs in any other furniture. I live with my adult daughter and 2 year old grandson, and don't want to risk their bedrooms. Please help me! | Exterminators can't find my bed bugs but I am being bit + found them biting me | I just moved in to the garage and I'm having an issue with bugs on my mattress what is the best way spray my bed? | How do you kill fleas in home and carpet? | Totally infested with fleas, please help | How to keep roaches or water bugs away from my room and bed? | Flea infestation, but they're only biting me so my roommate won't let me bomb the whole house. What do? | Bed Bugs, Fleas, Ticks and Dust Mites Disappear | I got bit by a bed bug and then I killed it outside my dorm. Is my dorm infested? |
480 | What does Hypersmooth 2.0 make ReelSteady Go redundant? | In a recent paper, we proved that a large class of spacetimes, not necessarily homogeneous or isotropous and relevant at a cosmological level, possesses a preferred codimension 1 submanifold, i.e., the past cosmological horizon, on which it is possible to encode the information of a scalar field theory living in the bulk. Such bulk-to-boundary reconstruction procedure entails the identification of a preferred quasifree algebraic state for the bulk theory, enjoying remarkable properties concerning invariance under isometries (if any) of the bulk and energy positivity and reducing to well-known vacua in standard situations. In this paper, specializing to open Friedmann–Robertson–Walker models, we extend previously obtained results and we prove that the preferred state is of Hadamard form, hence the backreaction on the metric is finite and the state can be used as a starting point for renormalization procedures. Such state could play a distinguished role in the discussion of the evolution of scalar fluctuati... | I use a profile with Time Context with repeat every 15 minutes to change my wallpaper. Does it consume too much battery? | Hi everyone! I am currently familiarizing myself with PCA and I am wondering what effects it will have on the eigenvalues and eigenvectors, if you change somethin in the underlying data set. Does it have any effect?
Thanks for any help | For example, before update, Carbide had this in the support slot which gave the main commander 30% increased reload speed. Unless I missed something, I haven't seen anyone having this anymore. | Whenever I join a Frag Shack server it sets my cl_updaterate to 0 and cl_cmdrate remains at 128. I always have it set to 128 in console;however, a majority of their servers default it to 0 for me. Valve DM sets it at 64 and other non Frag Shack community servers such as PBFortress-Retakes keep cl_updaterate at 128. Is this a problem for anyone else on Windows 10? Having cl_updaterate set to 0 makes eliminating anyone that is not stationary almost impossible. | Just had a quick read over the patch notes.
Still no fix for Montagne and Pulses forced toggle.
Is this ever going to be added? It's not fair that we're forced to play by other peoples requirements, when there are optional selections for things like gadget deployment and drone controls.
Has Ubi ever made comment on this? | I ran into this problem mid run when I would sleep and just after waking up it would turn on a motion blur that would mess up your depth of field unless you used the all round stamina booster coffeeTM or a supplement using the hard to use resource caffeine. Do you think the devs would get rid of this annoying feature? | For those of you interested in a Profile Setting for the new Freestyle Feature for nvidia Graphic Cards here we go:
I. Contrast
I.1 Gamma - 70
I.2 Contrast - 50
II. Color
II.1 Vibrance - 50
II.2 Color Enhancer - 20
III. Details
III.1 Sharpen - 20
III.2 Clarity - 70
III.3 HDR Toning - 75
III.4 Bloom 0
IV. Exposure
IV.1 Exposure - 60
IV.2 Highlights - 50
IV.3 Shadows - 70
Have fun seeing things again as a Killer after the Engine "Update" a year ago
edit. Tweaked the values a little bit to make it not look that extremly unnatural. | 480 | Of course there are a ton of videos comparing HS2.0 vs ReelSteady. My question is: "What about HS2.0 *AND* Reelsteady? I know RS works great with the Hero 5, 6 & 7. But with HS2.0 being so much better than HS1.0, does RS still make a noticeable difference *on top* of HS2.0? Sorry if this has been discussed before. I just can't seem to find any answers anywhere. | Reasons why ds1 is better (or not) than the others. | Can anyone change my mind, or at least give me a more balanced view, on the HS2 project? | DH2 and DH1: why is the former considered better? | How is Skyward Sword pacing in comparison with TP/OoT and ALTTP? | Which SC2 Race is Most Similar Overall to its SC1 Version? Please share your opinion | but better than S1 Vol | Claptrap(borderlands) vs R2D2(Starwars) | To everyone saying "Please compare D3 to D2 classic, not D2:LoD" ... |
481 | The ‘‘effective’’ sound absorption of unique variable acoustic devices in Jensen Concert Hall, Pocatello, Idaho | Sound Absorption at Low Frequencies: Room Contents as Obstacles | This paper examines how the individual variations of chair type, row spacing, as well as the presence of occupants and carpet, combine to influence the absorption characteristics of theater chairs as a function of sample perimeter-to-area (P/A) ratios. Scale models were used to measure the interactive effects of the four test variables on the chair absorption characteristics, avoiding the practical difficulties of full scale measurements. All of the test variables led to effects that could lead to important changes to auditorium acoustics conditions. At mid and higher frequencies, the various effects can usually be explained as due to, more or less, porous absorbing material. In the 125 and 250 Hz octave bands, the major changes were attributed to resonant absorbing mechanisms. The results indicate that for accurate predictions of the effective absorption of the chairs in an auditorium, one should use the P/A method and reverberation chamber tests of the chair absorption coefficients to predict the absorp... | Ando's book joins one of a handful written since 1900 on the science of concert hall acoustics. It contains seven chapters packed with physical acoustics, the behavior of the physical hearing system, the response of the nervous system, psychoacoustic judgments, the composition of a sound field in a hall, prediction of subjective preferences in halls based on the sound field, and design studies for concert halls. This paper attempts to relate Ando's teachings to the practical experience the author has had with the acoustics of a number of concert halls. Areas in which the book fits these examples well and in which further research seems indicated will be presented. | Sound absorption of wood-based materials | My wife and I are in search of practical, aesthetic sound absorbance in our new home. It is a large room, all hard surfaces, with an 18 foot ceiling. Any suggestions, whether diy or manufactured products, are appreciated. Thank you! | This paper investigates sound levels on stage and in the audience from field measurements and the literature for small and midsize amplified venues for various entertainment types. The effect of sound levels are also briefly examined in regard to hearing and comfort of patrons and employees. Furthermore, reduction in sound level internally is examined as a community noise control measure relative to soundproofing. | There is no sound absorption and the quality of stainless steel seems to be very poor the joints are not properly done. Very disappointed for being a Kohler. | ABSTRACTThe absorption coefficient (α) of a few indigenous acoustic materials has been measured in a small reverberant room, after mounting the material in different ways in the empty room with and without diffusing elements. It has been found that, depending upon the mounting conditions of the material, the absorption of sound is sometimes greater than the absorption effected by the area of the material alone due to diffraction at the edges of the material and the diffuse sound field created from the placement of the material. As such, whenever a studio is treated, considering only the absorption due to the area of the materials, the reverberation time of the finished studio is much less than the optimum.The value of α for a few materials under different mounting conditions are given in this report and how and when these values should be used in planning acoustic treatment of a small studio has been discussed. | 481 | The results of acoustical measurements taken at a new 1200 seat concert hall with variable acoustics demonstrate that significant reverberation control can be obtained with strategically located, but rather unusual, ab‐sorptive devices. These unique elements yield a change in RT60 of 1 second, with insignificant reduction in room volume and minimally exposed absorptive area. Various measures for estimating RT60 are compared, along with CATT‐Acoustic modeling, to assess the best characterization of the influence of these devices, including an ‘‘effective’’ sound absorption coefficient. | Optimized design for an anechoic-reverberant-type listening room | Equal Reverberance Contours for Synthetic Room Impulse Responses Listened to Directly: Evaluation of Reverberance in Terms of Loudness Decay Parameters | The use of high‐energy measurement signals combined with digital multichannel recording and postprocessing leads to a highly efficient collection of impulse responses in concert halls. During measurements in two similar concert halls, the Christchurch Town Hall, New Zealand, and the Hong Kong Cultural Centre (both described in companion papers), responses were collected in all seats in some areas and in different positions in some single seats, in addition to the usual sampling of all areas. An omnidirectional loudspeaker source was used, and the measurements were repeated in several source positions. The distribution of various parameters in these areas are compared to the results for the whole hall. Based on this comparison the use of mean and standard deviation as measures of location and spread for the parameters is discussed, and the variation of parameters with varying source locations is demonstrated. Approaches toward the reduction of parameter sensitivity to insignificant details in the early sou... | How do I evaluate the sound system at a venue? | Acoustic feedback considerations in simulations of sound systems in rooms | Effects of room shape and diffusing treatment on the measurement of sound absorption coefficient in a reverberation room | The effect of stationary diffusers in the measurement of sound absorption coefficients in a reverberation room: An experimental study | Comparative analysis of absorption coefficients’ effect on room acoustic parameters determined by simulation software |
482 | when was give me back my man written | the "Billboard" Hot 100, except for 1982's "Change," climbing into the Billboard R&B Top 20 (#12). His label venture was exacting a heavy financial cost on White, so he concentrated on mostly touring and finally folded his label in 1983. After four years he signed with A&M Records, and with the release of 1987's "The Right Night & Barry White", the single entitled "Sho' You Right" made it to the "Billboard" R&B charts, peaking at #17. In 1989 he released "The Man Is Back!" and with it had three top 40 singles on the "Billboard" R&B charts: "Super Lover", which | My Man (Jade Ewen song) "My Man" is a song by English singer Jade Ewen. It was written by Ina Wroldsen, and produced by Harry Sommerdahl and Kalle Engstrom for Ewen's debut studio album. The song was released as a digital download in the United Kingdom on 17 September 2009. Musically, "My Man" is a pop and contemporary R&B and song backed by electro and R&B beats and a synthesizer. It is notably different from her previous single "It's My Time", which was composed by Andrew Lloyd Webber for the Eurovision Song Contest 2009. "My Man" was positively reviewed by | My Man (Tamar Braxton song) can't believe that you're with her / I just can't believe she stole my man." Braxton discusses the affair further by singing: "She called me 'bout her man, Well, I didn't understand, she was talkin' 'bout my man (heifer)." "Billboard"'s Sadie Bell described "My Man" as an "emotional ballad", and said the lyrics are about infidelity and its negative impact on a marriage. Libby Hill of the "Los Angeles Times" called it a "fiery torch song about a man who done her wrong". According to Mikael Wood of the same publication, the single's central message was to "never trust a | My Man (Jade Ewen song) Nick Levine of Digital Spy gave the song a four out of five star rating and called it "very good contemporary pop" and a "complete U-turn" from "It's My Time". Philip Ellwood of Entertainment Focus praised the song's chorus, in addition to Ewen's vocal performance. He also wrote that the end result is "a monster hit waiting to happen and a single that is better than it has any right to be". Oikotimes.com commended the song as "instantly catchy and memorable". Vicki Lutas of BBC described "My Man" as "sexy, strong and ferocious". She applauded the chorus, but admitted that | peaked at No. 38 on the "Billboard" Hot 100, and it spent a total of seven weeks on the chart. It also peaked at No. 6 on "Billboard"s Easy Listening chart. I'm a Better Man "I'm a Better Man" was written by Burt Bacharach and Hal David. It was a hit for Engelbert Humperdinck in 1969. It was a follow up to the previous release, "The Way It Used To Be". The record was released in the United States on Parrot 40040. In the 1980s, the song ended up on an Engelbert compilation "Release Me" which included other songs such | She's My Man "She's My Man" is a song by the Scissor Sisters, released on 5 March 2007 as the third single from their second studio album "Ta-Dah". The song became another UK hit for the group, peaking at number 29. Lukas Ridgeston appears as the cover model on artwork for the single. The song's music shares some similarities with Elton John's rhythm structure for "I'm Still Standing", although with a slower beat and lower pitch. The single's video was directed by Nagi Noda, and shot in Tokyo. It uses the Kuroko technique, where the band members act out a | Bettye LaVette a local record producer. In 1962, aged sixteen, she recorded a single, "My Man — He's a Lovin' Man", with Matthews, which became a Top Ten R&B hit after Atlantic Records bought distribution rights. This led to a tour with rhythm and blues musicians Clyde McPhatter, Ben E. King, Barbara Lynn, and then-newcomer Otis Redding. She next hit the charts with "Let Me Down Easy" on Calla Records in 1965. This led to a brief stint with The James Brown Revue. After recording several singles for local Detroit labels, LaVette signed to the Silver Fox label in 1969. She cut | Man! I Feel Like a Woman! and it pays homage to Robert Palmer's "Addicted to Love" and Tone Lōc's "Wild Thing" music videos, featuring Twain dancing with buffed and blank-eyed male models. The song was the opening song on both Twain's Come On Over Tour and the Up! Tour as well as Twain's headline on the Super Bowl XXXVII Halftime show. It was also used to comic effect in a 2004 Chevrolet Colorado TV commercial, as well as being on the soundtrack of Brazilian telenovela "Laços de Família". The song was also performed by "American Idol" winner Carrie Underwood during the fourth season, and by Britney | 482 | Give Me Back My Man "Give Me Back My Man" is a song written and recorded by the American rock band The B-52's. It was released as the second single from their 1980 album "Wild Planet" and is one of many solo vocal performances from Cindy Wilson in the band's earlier years. "Give Me Back My Man" was a staple in The B-52's concerts in the 1980s and was usually one of the first few songs played. Early on, it was played just as it was on the record, with Schneider playing extra synth and glockenspiel. After the release of | who sings i'm the man of the year | what's the song i want you back by nsync | who sings i've got your back | what was the b-side of get back | what is the name of the song i'm a man | who sings the song give in to me | when was my old man by zac brown released | what album is i'm back from |
483 | Currently proven methods that are used to obtain devices with high-quality graphene on silicon wafers involve the transfer of graphene flakes from a growth substrate, resulting in fundamental limitations for large-scale device fabrication. Moreover, the complex three-dimensional structures of interest for microelectromechanical and nanoelectromechanical systems are hardly compatible with such transfer processes. Here, we introduce a methodology for obtaining thousands of microbeams, made of graphitized silicon carbide on silicon, through a site-selective and wafer-scale approach. A Ni-Cu alloy catalyst mediates a self-aligned graphitization on prepatterned SiC microstructures at a temperature that is compatible with silicon technologies. The graphene nanocoating leads to a dramatically enhanced electrical conductivity, which elevates this approach to an ideal method for the replacement of conductive metal films in silicon carbide-based MEMS and NEMS devices. |
graphene transistors on the SiC wafer below, and metallic vias patterned through the Si wafer for 3d interconnection between the two electronic platforms. This contrasts with most Si/graphene integration schemes [19][20][21] where graphene-and Si-device areas are implicitly designed side by side on the same plane. The Si wafer transfer solution described below in detail presents several advantages. The transfer can be realized in principle on the wafer scale (Si to SiC transfer at the wafer has been already realized [22] ) and the resulting double-wafer is compatible with silicon-VLSI. The top monocrystalline Si surface present the quality required for CMOS, that was difficult to obtain by growing Si on SiC by chemical vapor deposition, molecular beam epitaxy or electron beam evaporation [22] . The transfer relies on Si to EG/SiC wafer bonding that is based on the silicon-on-insulator (SOI) technique, a mature industrial process in silicon technology. In our case for Si to EG/SiC bonding we have adapted the process by adding an Al 2 O 3 layer to assist bonding. Epitaxial graphene is grown on the crystalline SiC wafer [13] prior to the Si-SOI transfer, therefore the high temperature graphene on SiC growth is not limited by the lower Si melting point, allowing very good quality (nanostructured) graphene, and any post-processing if required. Moreover, the graphene layers/nanoribbons remain untouched on their growth substrate. This ensures that graphene's integrity, interface and nanostructure properties are preserved. Moreover, access to the graphene structures from above provides significant architectural flexibility for graphene device interconnects. Finally, the often-quoted [23] drawback of the epitaxial graphene is the SiC substrate cost (currently about $20/cm 2 and decreasing) that deserves to be addressed upfront. Considering, that high-end consumer electronics processors currently cost more than $1000, it is clear that if a SiC substrate were to be used in those, the SiC cost would amount to only a few percent of the total price, which is very reasonable, especially if unsurpassed performance is achieved. interconnected by metal pads. This process can clearly be generalized to wafer size (SiC wafers are now commercially available up to 150mm diameter). We next discuss some of the process steps in more detail.
One of the key steps is the Si to EG/SiC wafer bonding (step 7). Si-wafer size bonding has been an industrial process for two decades [24] , but there are only few reports on SiC wafer to Si wafer bonding [22,[25][26][27] , and none of Si on graphitized SiC. The primary challenge was to realize bonding to the SiC substrate coated with graphene that is well known for its non- sufficiently robust to withstand the stress of the smart-cut process. It should be noted that bonding of small wafer dies like those used here (3.5x4.5mm 2 ) is particularly challenging and requires much higher bonding energy and much cleaner interfaces than for wafer scale bonding. For instance, for a 4-inch Si wafer, particles as small as 1 m diameter typically result in a 5 mm diameter unbonded area [28] , which is the size of SiC dies. Therefore thorough cleaning is required: contaminant particles, mostly found at the edges due to dicing and handling must be removed. Figure 3c The Si wafer transfer method proposed here preserves the structural quality of EG. A key point in the process is to selectively grow alumina at specific locations by atomic layer deposition (ALD) (step 6). In the process alumina selectively coats the prepared graphenefree regions (that are obtained by growing sub-monolayer graphene or by removing locally graphene by plasma patterning). The selective coating is realized by depositing ALD -Al 2 O 3 directly with no pre-seeding, in contrast to the deposition of dielectric for graphene field effect transistors where special treatments are use to force Al 2 O 3 to cover graphene (see for instance [29,30] ).
In the example of Figure 4, sub-monolayer graphene was grown on the C-face of 4H-SiC.
Raman spectroscopy is used to identify graphene regions (characteristic 2D and G peaks, see for instance Fig. 4c) from bare SiC. Fig. 4a shows an AFM image of the surface after ALD-Al 2 O 3 direct deposition. The dark area is a single layer EG layer draped over the SiC substrate steps. The graphene layer is recognized also by its surface pleats (white lines) as is usual for graphene on the C-face [9,13] . As is clear from the AFM image graphene is clean from alumina.
Alumina coats preferentially the surrounding bare SiC substrate, as shown by the surface roughness contrasting with that of graphene (AFM line profile of Fig. 4b). Here we use to our advantage the non-wetting properties of graphene, that is in general problematic when growing dielectric on graphene for top-gating (a functionalized or seed layer is required [29,30] ).
As alumina is deposited, the uncoated graphene becomes lower than the Al 2 O 3 -coated SiC.
This prevents EG from making direct contact with the Si wafer die in the following bonding step because bonding happens only between the Al 2 O 3 coated areas. The Raman spectra of EG/SiC (Fig. 4c) show that the characteristic G and 2D peaks of graphene remain unchanged before and after ALD-Al 2 O 3 deposition and no D peak indicating of disorder is seen in either case.
The successful bonding indicates that graphene is not involved in the bonding process (graphene on the contrary delaminates easily). In order to connect the top (Si) and bottom In this study, successful Si wafer die bonding has been realized on two types of EG samples: C-face SiC substrates coated with a sub-monolayer graphene layer and on an array of nanoscopic graphene ribbons grown by the templated growth method [9,12] on the Si-face, as demonstrated now. Figure 5 shows Si to structured EG/SiC integration. As can be seen in the optical image of Fig. 5a-b, successful bonding is obtained between Si wafer die and structured EG/SiC. In this example arrays of 200 parallel graphene ribbons (100nm x 100 µm) were selectively grown on the sidewalls of trenches patterned in the 4H-SiC substrate (Si face) [9,10,12,13] . The 50nm deep vertical trenches dry-etched in SiC (Fig. 5c) recrystallize into welldefined crystallographic facets upon annealing around 1500˚C resulting in 100nm wide sidewall templates. Because graphene growth rate is slower on the Si (0001) face, graphene ribbons are first formed on the sidewall facets. By adjusting the growth conditions and time, ribbons can be selectively grown, as seen in the electrostatic force microscopy (EFM) image of Fig. 5d. It is important to note that in this case graphene nano-structuring is realized prior to substrate bonding. There is therefore no temperature limitation to produce high quality, smooth edged graphene nanostructures. It was also demonstrated that sidewall graphitization is not limited to lines and the etched SiC substrate acts as a template for graphene growth [9,13] .
The main goal of the Si to graphene integration is to interconnect the graphene device platform to the Si-CMOS technology on the same wafer (steps 9 and 10). Graphene is not disrupted by the bonding process. A finite resistance of a few hundreds ohms is measured between any 2 leads, as shown in Figure 6c. (iii) Exposed and Si covered graphene have a similar resistivity R sq = 200-300 /sq, typical for highly doped single or few layer graphene [31,32] , and a maximum contact resistance R C~6 00 .µm, which is in the range of published values for metal to graphene contacts [33] . The graphene quality and good metal connection to the top silicon wafer die have been further tested by applying a large current through the leads. The IV characteristics are linear and current density, as high as 1.5mA/ m, can be reversibly applied on leads connecting Si-covered and exposed graphene, with no observable degradation of the leads or of graphene.
We have demonstrated here the critical step of a graphene -silicon integration scheme to produce a monolithic integration of two wafers acting as interconnected parallel electronic platforms. The process is quite flexible and we envision the development of electronic devices on both platforms. CMOS technology can be implemented on top of the silicon wafer, which surface is entirely free for device processing. The smart-cut technique [24] allows to choose the thicknesses of the Si layer (5 nm to 1.5 µm) and of the SiO 2 oxide (5 nm to typically 5 µm).
Ion implantation, epilayer growth and standard lithography techniques can be safely
implemented to the top Si layer, and even more so when the graphene is protected during processing, i.e. if the windows or vias are fabricated as the last step. Epitaxial graphene is in any case very robust to chemical treatments (Figure 4d). Moreover EG on SiC can safely withstand temperatures up to 450°C in air and 1000°C in vacuum, since these annealing steps are used routinely to clean graphene from contaminants (as demonstrated in the AFM image and Raman spectra of Figure 7). The effect of air annealing on graphene is shown.
These studies show that fully developed graphene devices and interconnects on the SiC surface can be produced prior to bonding and that they survive the bonding process. Particle contamination was the main impediment to successful monocrystalline substrate bonding in our case. However, this study was done with small dies (~15 mm 2 ), in a non-stringent cleanroom environment. Despite these drawbacks, the successful bonding achieved here together with the large scale device integration demonstrated for epitaxial graphene [12,30] indicates that this process has an industrial potential. Compared to graphene transfer or printing, this graphene to Si integration method takes full advantage of the crystallinity of the substrate and of epitaxial growth process (continuous high quality 2D sheet, well defined and reproducible interface, well known industrial grade substrate, no potentially damaging transfer required).
Beyond graphene for electrodes, this integration is envisioned for high performance electronics for instance in ultra high frequency electronics [29,30] , spintronics [34] , optoelectronics. We have indicated that graphene sidewall nanoribbon arrays can be integrated to Si with the same process. We believe that the recently discovered exceptional electronic and transport properties [8,10,11] of sidewall graphene ribbons grown directly on SiC [8,9,12] will become an important direction for nanoscale electronics.
In conclusion, we have developed a unique monocrystalline silicon transfer method to fabricate monolithic integration of graphene on SiC /silicon 3d stacked layers, that is fully compatible with VLSI technology and preserves graphene integrity and nano-structuring.
Instead of the conventional graphene transfer technique, thin monocrystalline silicon layers are transferred onto EG/SiC wafer dies using well-established SOI wafer bonding and smartcut techniques. The transferred crystalline silicon layer can serve as the basis of silicon-CMOS devices, and is connected to EG layer by metallic leads. High quality graphene nanostructures grown at high temperature are integrated with no degradation.
Methods
(1) A 300nm thick oxide was grown by thermo-oxidization on a p-doped (10 15 cm -3 ) Si wafer.
(2) Hydrogen ions (140 keV, dose 8.5×10 16 /cm 2 ) were implanted in the Si wafer at depth of 900nm, according to the implantation simulation (TRIM package). The temperature (15˚C) was controlled during implantation to avoid wafer blistering.
(3, 6) For bonding, 30nm Al 2 O 3 was deposited directly by atomic layer deposition in a Savannah 100 ALD system, at 160˚C, using TMA as a precursor. No graphene seeding layer was used, contrary to graphene transistors, such as in refs. [29,30] .
(4-5) Submonolyer graphene was grown on the C-face of insulating 4H SiC by the confinement controlled sublimation method [13] at 1500˚C. For the ribbon array, patterned SiC (Si-face) trenches were etched in SF 6 /O 2 plasma, using Poly (methyl methacrylate) (PMMA) as a mask. After CCS growth at 1450˚C, the 50nm deep sidewalls recrystallize at 29 degree from the (0001) orientation, providing a 100nm wide facet for ribbon growth. Raman spectroscopy and EFM clearly identifies graphene on the sidewalls.
(7) After Al 2 O 3 deposition, samples were stored in DI water for more than 24 hours in order to improve their hydrophilic properties. The wafers dies were first bonded in DI water to avoid particle contaminants from air, then transferred to a pressure module. Stronger bonding strength is achieved by subsequent annealing.
(8) The bonded dies were heated up to 400˚C in air so that the resulting H 2 pressure splits the Si wafer dies along the H implantation plane. For this, a fast ramping (10°C/min) from room temperature to 300°C was followed by a slow ramping (5°C/min) from 300C to 400°C. The bonded dies were kept at 400°C for 60 min, then naturally cooled down to room temperature. showing that the transferred Si layer is 1.2µm thick. showing the characteristic 2D and G Raman peaks of graphene. The SiC substrate Raman spectrum was subtracted. Note that the graphene 2D peak has a single Lorentzian shape as typical for MEG [9] . The extremely small D peak reveals the high structural quality of MEG that is not affected by the annealing in air. The AFM images in the inset (scale bar: 1µm)
show a patterned MEG graphene cross, before (left) and after (right) 400°C annealing in air.
The white dots are residues from the resist used for patterning. The graphene cross is cleaner after anneal, that doesn't visibly change graphene. The same white line (graphene pleats) are observed and the roughness on graphene decreases from 1nm (before) to 0.1nm (after) annealing. Note that the SiC outside the graphene cross remains quite contaminated.
Figure 2
2shows a process flow of the proposed Si to EG/SiC integration. (1) silicon oxide is grown by thermo-oxidization on a commercial monocrystalline Si wafer. (2) Hydrogen ions are implanted in the oxidized-Si wafer. (3) 30 nm thick aluminum oxide is deposited by atomic layer deposition on the SiO 2 /Si dies (4-5) EG is grown on SiC. Non graphene covered areas are managed on the wafer, either growing submonolayer EG on the C-face, or by plasma etching graphene in patterned area, or by growing graphene only on the sidewalls of trenches etched in 4H-SiC (Si-face). (6) 30 nm ALD-Al 2 O 3 is deposited on EG/SiC. Because of growth selectivity, Al 2 O 3 growth is confined in SiC regions not covered with graphene. (7) The Al 2 O 3 /SiO 2 /Si and Al 2 O 3 /EG/SiC wafers are bonded together using Al 2 O 3 as a bonding interface. (8) Upon heating the bonded wafers to (400˚C), the Si wafer splits at the ion implantation depth (smart-cut), leaving a thin monocrystalline Si layer bonded to the EG/SiC wafer. (9) Windows are opened by standard microelectronic patterning and etching processes to expose some area of the buried EG layer. (10) EG and the top crystalline silicon layer are
sticking properties. Our solution consists of adding an intermediate alumina layer between the Si wafer utilizing graphene free regions of the SiC wafers. This solves also two of the mains challenges of wafer bonding. One is the stress during thermal treatment because of the different thermal expansion coefficients between Si and SiC. The second is that the two facing surfaces have to be smooth and flat. Significant SiC surface step bunching during EG growth can be a limiting factor.
Figure 3a
3aEG/SiC). Gold color indicates strong bonding contrasting with weaker bonding in the blue (or green) areas that are located mostly at the sample edge.Figure3b shows the optical image of the 2 halves of a bonded wafer after smart-cut splitting (step 8 above). On the left is the SiC die with the Si layer bonded to it (Si/SiO 2 /Al 2 O 3 -Al 2 O 3 /EG/SiC stack). The darker area is where crystalline Si has transferred from the Al 2 O 3 /SiO 2 /Si wafer shown on the right. The shape of the transferred silicon layer (left) matches precisely the bright area on the Si wafer die (right), which shows the success of the smart-cut transfer. The profilometer scans of Figure 3d on the transferred wafer (black trace) and on the Si wafer (red trace) wafers show that in this example a Si/SiO 2 layer 1.2 µm thick was transferred. The successful Si smart-cut transfer shown in Fig 3a-b demonstrates the wafer bonding strength. The wafer splitting is caused by the formation of molecular hydrogen blisters at the specific depth of proton implantation in the Si wafer. The SiC/Si wafer bond needs to be
is a scanning electron microscope (SEM) image of the bonded interface between transferred silicon and SiC. The image is taken with a tilt angle at the edge of the Si layer and shows the section of the SiO 2 coated Si bonded to Al 2 O 3/ SiC. The image shows that the interface is clean and sharp with no gaps or cracks.
(
graphene) electronic layers, openings are etched in the bonded Si wafer, dry and wet etching is used to open the vias for metallic 3d connection between the Si and graphene layers. The Raman spectrum of Fig. 4d shows that graphene is not significantly affected by the optimized etching process used to open the large windows of Fig. 4a-b through the Si/SiO 2 /Al 2 O 3 layer (etching will certainly be further optimized as the process develops). This result is confirmd by transport data below (Fig. 4c) As seen in Fig.S1d: very low or no Raman D peak was observed after etching for multi-layer graphene on two different locations indicated by the green dots on the optical image. Note that the etching time is adapted to the thickness of the crystalline Si transferred. The SiO 2 "mask" was removed by a short buffered oxide etching (BOE) at room temperature (see methods section below for details).
Figure 6a-b show an example of the proposed integration. Windows (20µm side) were etched in the top Si/SiO 2 /Al 2 O 3 layer by a combination of standard dry and wet etching to partially expose a 4 m wide and about 30 m long EG area grown on the C-face. The EG area, shown by the white dashed contour in Figure 6a, lies partly underneath a 1 µm thick monocrystalline silicon layer. Eight evaporated metal strips (Ti/Pd/Au : 0.5 nm/20 nm /50 nm) are prepared by conventional lithography and lift-off techniques and connect the bottom EG to the top Si wafer die where the pads extend for electrical measurements. The resistance measurements below confirm the Raman data after ALD deposition and window etching that the characteristics of graphene are not affected by the process. From the resistance measurements several conclusions can be drawn. (i) The metal leads are continuous from EG to the top Si surface, as is also observed from the tilted view on Figure 6a. (ii)
( 9 )
9Windows in the Si/SiO 2 /Al 2 O 3 stack were opened with dry and wet etching after patterning a 1µm thick photoresist layer (Microposit SC1813) used as the dry etch mask: SiO 2 and Si were respectively dry etched in a CHF 3 /Ar RIE, and in SF 6 /O 2 plasma. Al 2 O 3 was removed in a solution of H 3 PO 4 : H 2 O (1:3) at 60˚C. For the sample of Fig. 4d, the following etching recipe was used. Si was etched in SF 6 /O 2 plasma and SiO 2 was etched in a CHF 3 /Ar RIE chamber. A shorter plasma etching recipe was used so that about 100 nm SiO 2 can be preserved and used as a "mask" to avoid plasma damage to the graphene underneath. The sample was further etched in a solution of H 3 PO 4 : H 2 O (1:3) to remove the Al 2 O 3 residues at 60˚C.
Figure
Captions
Figure 1 .
1Illustration of a silicon-on-EG/SiC monolithic wafer integration, showing CMOS technology on a Si thin wafer on top (grey layer) and graphene devices below (blue layer).The two electronic platforms are interconnected vertically by metal vias. There is no limitation a priori on the integration design on either platform.
Figure 2 .
2Process flow of silicon and EG/SiC integration: bonding;(8-10) smart-cut and metal vias fabrication to connect the top CMOS ready Si layer to the buried graphene.
Figure 3 .
3Demonstration of Si on EG/SiC wafer die bonding. In this case graphene was partially grown on the C-face of SiC. (a-b): Optical images of three 3.5mm x 4.5mm wafer die Si-on-EG/SiC; golden/purple color corresponds to the bonding areas. (a) after bonding; (b) after smart cut; (left) Si on EG/SiC substrate and (right) Si wafer die showing the trace of the removed Si layer. (c) SEM images of Si-on-EG/SiC sample. The image shows a cross sectional view of the sharp and clean interface between transferred Si-SiO 2 and the flat SiC substrate that is partially covered by Al 2 O 3 . (d) Depth profile on both wafer dies in (b)
Figure 4 .
4(a) AFM images of a partially graphitized epitaxial graphene on the C-face, after ALD Al 2 O 3 deposition. (Scale bar, 5µm). The dark area is bare graphene that drapes over the SiC steps (b): AFM height profile along the dotted line in (a), showing a increased roughness on the Al 2 O 3 coating compared to graphene. (c) Raman spectra of the graphene area in (a) before and after Al 2 O 3 coating, showing high graphene quality (no D peak). The SiC Raman peak contribution is subtracted. (d) Raman spectra of the two graphene area in the window opening after bonding and etching. The spectrum were taken at the green dots in the optical image in the inset; Very small or no D peak is observed. The noisy spectrum between 1500 and 200 cm -1 is due to the imperfect subtraction of the SiC Raman contribution.
Figure 5 .
5Si to structured EG/SiC wafer die bonding. Arrays of 200 parallel graphene ribbons (100nm x 100 µm) are grown on the sidewalls of trenches patterned in SiC-Si face before wafer die bonding. (a) Optical image of a 3.5 mm x 4.5 mm Si-on-structured EG/SiC wafer die; The purple color indicates bonding (b) Optical image of the graphitized array seen through the SiC substrate after bonding, indicating that the bonding doesn't damage the patterned structure. (c) AFM topographic image of the array of trenches patterned in SiC, after graphitization and prior to wafer die bonding, and AFM height trace (white trace-full amplitude is 50 nm). (d) Electrostatic Force Microscopy image of a similarly prepared sample showing the contrast between SiC (dark) and the 40nm wide graphene nanoribbons (light).
Figure 6 .Figure 7 .
67Scanning Electron Microscopy images of Si on-EG/SiC substrate. Openings are provided in the Si top layer to expose buried graphene and metal pads that connect the top Si wafer die to graphene. (a) top view. The graphene area is outlined with the dotted line (green), the pas are outlined in yellow, and the monocristalline Si in red. (b) tilted view with multiple windows opened in Si to expose epitaxial graphene. Scale bars: 5 m (c) room temperature resistance between any two pads in (a), showing that the same resistivity (proportional to the local slope R vs distance) is measured for exposed and buried (under the central pads) graphene Effect of annealing at 400°C for 30 minutes in air. Raman spectroscopy of a multilayer epitaxial graphene (MEG) sample before and after annealing in air at 400°C,
Figure 1
Figure 2 Height
2Figure 2
Figure 5
5Figure 5
Figure 6
AcknowledgementsThis material is based on research sponsored by DARPA/Defense Microelectronics Activity Government is authorized to reproduce and distribute reprints for Government purposes, notwithstanding any copyright notation thereon.
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| The effect of reaction temperature on the synthesis of graphitic thin film on nickel substrate was investigated in the range of 400°C to 1,000°C. Amorphous carbon (a-C) film was obtained at 400°C on nickel foils by chemical vapor deposition; hybrid films of multilayer graphene (MLG) and a-C were synthesized at a temperature of 600°C, while MLG was obtained at temperatures in excess of 800°C. Schottky-junction solar cell devices prepared using films produced at 400°C, 600°C, 800°C, and 1,000°C coupled with n-type Si demonstrate power conversion efficiencies of 0.003%, 0.256%, 0.391%, and 0.586%, respectively. A HNO3 treatment has further improved the efficiencies of the corresponding devices to 0.004%, 1.080%, 0.800%, and 0.820%, respectively. These films are promising materials for application in low-cost and simple carbon-based solar cells. | When epitaxial graphene layers are formed on SiC(0001), the first carbon layer (known as the ``buffer layer''), while relatively easy to synthesize, does not have the desirable electrical properties of graphene. The conductivity is poor due to a disruption of the graphene $\ensuremath{\pi}$ bands by covalent bonding to the SiC substrate. Here we show that it is possible to restore the graphene $\ensuremath{\pi}$ bands by inserting a thin oxide layer between the buffer layer and SiC substrate using a low temperature, complementary metal-oxide semiconductor-compatible process that does not damage the graphene layer. | Wafer-scale graphene devices processed entirely in a standard 200 mm silicon fab are demonstrated for the first time. New embedded gate structures enable full saturation of the drain current in graphene FETs with sub-μm channels, resulting in high intrinsic voltage gain. In addition, passive devices were monolithically integrated with graphene transistors to form the first GHz-range graphene IC using large-scale CVD graphene. The demonstration of high performance graphene FETs and IC fabricated using a 200 mm platform is a major step in transitioning this promising material from a scientific curiosity into a real technology. |
Introduction
A key requirement for the realization of the variety of envisioned graphene applications [1] is the availability of production methods delivering material with quality tailored to the specific needs of the particular application [2]. Microelectronics will likely require the highest quality graphene deposited inexpensively on large areas. Furthermore, graphene electronics with its anticipated unique features will most probably not be a stand-alone technology but will complement the existing technologies with new functionality. The ideal graphene deposition method should thus be compatible with the mainstream Si technology requirements and allow to grow high quality graphene directly on CMOS compatible dielectric and semiconducting substrates [3].
Currently, large area graphene [4] and even heterostructures of 2D materials including graphene, MoS 2 and BN [5,6] can be grown by CVD with high quality on metals such as Cu or Ni. Fabrication of electronic devices requires subsequent transfer to the target substrate. A variety of graphene devices can be then produced, as planar field-effect transistors [7], vertical transistors [8], and Schottky diodes [9], to name a few. Although transfer of graphene may be a viable option in some applications, it is not a generally preferred solution in microelectronic manufacturing where direct deposition would be ideal [2].
Direct growth on Si has been studied with not necessarily encouraging results [10,11] due to the high reactivity of Si against C, resulting in the formation of SiC [12]. Germanium does not form a stable carbide; the Ge-C and Cu-C systems [13,14] are similar. Ge is a semiconductor compatible with the Si technology and graphene grown on it can be directly used in such devices as the graphene base transistor [8,15,16]. Demonstration of catalyst-mediated [17] and catalyst-free [18] growth of graphene on Ge nanowires, and notably from CH 4 on Ge [19] make Ge a promising substrate. Yet, the growth of good graphene from CH 4 requires much higher temperatures on Ge [19] than on hexagonal BN [20], indicating that the Ge-C interaction plays a major role. This can be directly addressed by using atomic C instead of CH 4 .
We report on the first such study for this system. We show that graphene can be grown from atomic beam on Ge(001)/Si(001) and we use ab initio theory to analyze the C-Ge interaction with and without the presence of hydrogen. In accordance with the results of the CVD study [19], we find that the quality of graphene visibly improves if the Ge layer begins to melt during the deposition [21]; also this observation highlights the importance of the C-Ge interaction for the growth process. The unwanted side effect of the melting is however longrange roughening of the substrate. Given that the anticipated use of graphene grown on Ge(001) is in vertical transistor structures, in which the carriers travel across the interface between graphene and the germanium layer, such roughening is awaited to be at least problematic. Furthermore, heating the Ge layer up to temperatures close to the melting point is nearly certain to destroy any dopant profile in the layer. Studies of C-Ge interaction, as the study that constitutes a part of this work, may advance the knowledge needed to lower the growth temperature into the regime of safely low temperatures.
Results and Discussion
We apply the molecular beam epitaxy (MBE) process used for the growth of graphene on van der Waals substrates [22,23] to deposit carbon atoms onto Ge(001)/Si(001) templates. Ge layers are grown on Si(100) in tailored multistep processes compatible with standard Si technology [24]. High uniformity, low threading dislocation density, and low surface roughness of Ge can be achieved, providing a high-quality substrate for graphene deposition (see Methods). An oxide-free Ge surface is prepared using a combination of wet-etching and UHV annealing and exposed to a beam of thermally evaporated carbon atoms at various substrate temperatures. Analysis of the surface chemical composition and direct comparison with HF-last Si substrates reveals that in contrast to Si, Ge does not form a stable carbide phase (cf. Supporting Information for XPS spectra). Instead, as it is proved by Raman spectroscopy, at elevated temperatures the C deposit on Ge takes a form of sp 2 -hybridized carbon layer.
Experiments performed on SiO 2 -patterned Ge substrates ( Fig. 1) demonstrate that graphene is produced (as visualized by the 2D/G intensity ratio) and that there is marked difference in the quality of graphene produced at the same conditions on both materials. As we explain in the course of the discussion, albeit the graphene film still contains numerous defects and/or the domain size is clearly smaller than that achievable by growth using chemical vapor deposition (CVD) from CH 4 and H 2 mixture at atmospheric pressure [19], the electrical properties of graphene obtained in the current study by MBE are good enough to qualify it for the use in a terahertz graphene base transistor. We also analyze the reason for the observed differences in the quality of graphene grown on Ge(0010) substrates by CVD and by MBE. On Ge(001), the highest crystalline quality and lowest sheet resistance of the graphene layers is obtained at temperature approaching the melting point of the substrate. The drawback of high-temperature growth is an increased surface roughness, revealed by atomic force microscopy (AFM) images, cf. Fig. 2. Clearly, the surface topography changes significantly with increasing substrate temperature. While at low temperatures (below 550 • C) the surface roughness remains comparable with roughness of the initial Ge surface (0.12-0.16 nm), higher substrate temperature during growth results in strongly increased root mean square (rms) roughness exceeding 1 nm for growth temperature above 700 • C; furthermore, at 930 • C high-frequency wrinkles and low-frequency hills appear. The mechanisms responsible for this roughening are discussed by the end of this section on the basis of the measured activation energy and of the characteristic features of C-Ge interaction revealed by ab initio calculations. Figure 3a shows Raman spectra acquired from samples prepared at various substrate temperatures and at the same growth rate (estimated to be about 1.4 monolayers per minute) and time (200 s). The 2D Raman peak typical for Figure 3: (a) Raman spectra of films grown on Ge at various substrate temperatures by deposition of about 5 carbon monolayers. The peaks 1555 cm −1 and 2350 cm −1 are attributed to atmospheric oxygen and nitrogen that appear due to long integration times. (b) High resolution synchrotron radiation XPS. C 1s peak shape (T growth = 850 • C), compared to typical shapes for graphene grown on other substrates. The information depth is about 2 monolayers. The green component comes from graphene, the blue one may be due to O contamination. See the Supporting Information for more discussion of XPS and Raman spectra. sp 2 carbon [25] can be resolved only in films grown above about 750 • C, but the 2D/G peak area ratio is then close to 1 already at 800 • C and approaches 2 above 900 • C, when the substrate begins to melt (Fig. 3a). Such a high 2D/G ratio indicates that already at 800 • C sp 2 -hybridized carbon, i.e. graphene, covers most of the surface. In the C 1s core level peak in the XPS spectrum ( Fig. 3b), no other bonds as C-C sp 2 can be clearly resolved. The FWHM reflects the degree of crystallinity and strain; it is only slightly larger than that for CVD graphene grown on copper. The parameter α reflects asymmetry; the value of α = 0.07 implies graphene structure and metallic conductivity (cf. the Supporting Information on XPS).
The 2D Raman mode stems from an inter-valley double-resonant scattering process involving two TO phonons close to the K point (the Dirac point in single-layer graphene) on the Brillouin zone boundary. Since the doubleresonance process depends on both the electronic band structure and the phonon dispersion [26], the 2D-mode line shape gives information about both properties [27]. From the line shape (Fig 4a) and relative intensity of the 2D peak we conclude that the graphene consists of decoupled layers and is not singlelayer graphene. The 2D mode of the MBE-grown graphene is symmetric with a single-Lorentzian shape, but strongly broadened and up-shifted in comparison to that of graphene exfoliated on the same substrate; the same is true for the G peak (see the Supporting Information). Such behavior indicates the presence of nanocrystalline graphene. [28] The peak positions and widths (FWHM) of the G and 2D peaks may in principle be used to evaluate the doping level or the strain in graphene, but in this particular case they are more likely to be dominated by the nanocrystallinity of graphene. Figure 4: (a) Raman spectra of the MBE graphene sample grown at 930 • C (black) and exfoliated single-layer graphene (blue) on germanium in the range of the 2D mode. (b) Analysis of the 2D/G-mode intensity ratio r 2D/G in the investigated area (10×30 µm 2 ) of the same MBE sample. The intensity of the G and 2D mode is evaluated from the peak area. The data was fitted assuming a Gaussian distribution (red, dashed lines).
The statistical analysis of the G and 2D peak positions and the 2D/G intensity ratio ( Fig. 4b and Supporting Information) shows very low standard deviations (0.04 cm −1 and 0.12 cm −1 , respectively), demonstrating the very homogeneous growth of graphene on Ge.
The average 2D/G intensity ratio of 1.9±0.12 is significantly lower than the typical value of four typical for exfoliated single-layer graphene [27], which may again indicate that the film has multiple layers. However, graphene that is doped, interacts with the environment or is imperfect, may also have the ratio below four, even around one. [29,30] In contrast to the 2D Raman peak, the D mode scattering process involves a TO phonon and a defect. The D peak is absent in not sp 2 -hybridized carbon and symmetry-forbidden in perfect graphene or graphite. Therefore, it is a measure of disorder in the sp 2 carbon network. Its relative intensity reflects thus the density of defects, in particular, the presence of boundaries. The behavior of the D peak as revealed by Fig. 3a shows that sp 2 -bonded carbon is produced already at low substrate temperatures and that disorder is considerable also in samples grown at high temperatures. The D/G intensity ratio can be used to estimate the grain size of nanocrystalline graphene [25,31,32]; applying this method we deduce the average crystalline grain size to be about 10 nm. AFM topology is consistent with this estimate at least to the order of magnitude (Fig. 5). The AFM amplitude indicates that each of the grains has a sub-structure, which may explain the difference in these two estimates.
Electrical measurements performed with 4 colinearly arranged STM tips (see Supporting Information for detailed discussion) reveal ohmic IV characteristics of the MBE graphene. The metallic character is retained at temperatures below which the Ge(001) surface is semiconducting, i.e., below 200 K [33]; this excludes any contribution from substrate to the measured currents. However, the film contains high density of defects causing disorder in the electrostatic potential. This follows from the results of temperature dependent measurements ( Fig. 6a). With decreasing sample temperature the sheet resistance exponentially increases from 2 kΩ/2 up to 20 kΩ/2. Such a behavior is well known for disordered systems, including disordered graphene. It can be understood in terms of Anderson localization and variable range hopping (VRH) transport [34,35,36,37,38]. The fit in Fig. 6a proves that the dependence of log(R s ) on T −1/3 is linear, which is a signature of two-dimensional VRH. Indeed, as obvious from Fig. 6b) the resistivity is independent of the probe spacing clearly indicating 2D transport. As the substrate temperature during growth is increased from 900 • C to 930 • C, the sheet resistivity R s (Fig. 6b) drops fast to 2 kΩ/2, a value comparable to R s of CVD graphene [39,40,41,42] or to typical R s of base layer in a SiGe HBT transistor. On the other hand, there is little dependence of R s on the amount of deposited C. This may indicate that only the topmost carbon layers contribute significantly to the electrical conductivity of the film, or that the carbon that is deposited above a certain critical amount agglomerates in grains.
AFM statistics provides more support to the second of these hypotheses. Strong height variations appear in AFM topography on sub-micrometer length scale when the temperature exceeds about 600 • C (Fig. 7a): the film consists of grains. The surface roughness rms monotonously increases with the substrate temperature, suggesting that the roughening occurs by Ostwald ripening. [43] Ostwald ripening of grains is a phenomenon natural to expect during growth and for the purpose of further discussion we assume that this the mechanism that controls the grain evolution. When the substrate begins to melt, the surface topology changes: instead of randomly distributed grains, wrinkles forming a network of lines appear ( Fig. 2d). At the same time, the resistivity of graphene drops and the 2D/G Raman mode ratio sharply increases (Fig. 7b). The activation energy E RMS of the surface roughness rms, as obtained from Arrhenius plot, remains however the same in the whole range of temperatures (Fig. 7a). It is the same not only below 750 • C, where no 2D peak can be resolved ( Fig. 3a and 7b), and between 750 • C and 900 • C, where the 2D/G ratio r 2D/G remains close to 1, but also in the melting regime, where r 2D/G rises to 2. It seems that around 750 • C a nanocrystalline graphene layer is formed on top of the film, and that this layer improves as the substrate begins to melt.
The growth of grains supporting the graphene layer is limited by a process with low barrier height of E RMS = 0.66 eV, comparable to the barrier for migration of C ad-atom on graphene [44]. This implies that C atoms are easily liberated from one grain and then easily diffuse to another grain. Such easy detachment is hard to understand if the grain boundaries consisted of pure C, but can be rationalized if they are contaminated with Ge: as will be shown from DFT calculations, the barrier for a process in which Ge and C atoms exchange places may be as low as 0.65 eV, at least when the Ge atom belongs to Ge(001). Also the very presence of Ge at the boundaries can be understood on the basis of ab initio calculations (Fig. 8, cf. also the Supporting Information). The interaction of C atoms with the reconstructed Ge(001) surface leads to ejection of Ge atoms. As illustrated in Fig 8a-b, a C atom readily substitutes a surface Ge atom, which is kicked out into a mobile on-surface state. The energy barrier for ejection is only about 0.65 eV (Fig. 9a), low enough for the process to take place rapidly at any deposition temperature used in this experimental study.
The ejected Ge atoms become ad-atoms and are highly mobile. Their total amount is expected to be comparable to a monolayer. This is suggested by DFT molecular dynamics, according to which the probability of ejection before the adsorbed C atom thermalizes is of the order of 50%. The thermalized C atom resides for a while (microseconds at 850 • C, milliseconds at 450 • C) directly under the surface, as interstitials. Being strongly repelled from the bulk by elastic forces (Fig. 9b), it remains close to the surface and eventually ejects a Ge atom (Fig. 8b) or diffuses under the surface and then attaches itself to a C cluster ( Fig. 8c-d). The probability that a non-surface Ge atom will be substituted is negligible. As for the ejected Ge atoms, also they are trapped by the clusters (Fig. 8d), unless they find their way to a surface step or liberate a C atom from a C-Ge dimer by a kick-in process (reverse to Ge kick-out shown in Fig. 9a).
Indeed, the ejected Ge atoms are preferably attached between graphene edge and Ge(001). Depending on the adsorption geometry, the energy of the attached atom is by 0.6 eV to 2.3 eV lower than in the bulk. Furthermore, when a piece Fig. 8a and 8b, "X" is the lowest-energy subsurface interstitial, and "S" is the lowest-energy subsurface substitutional.
of graphene is placed on Ge(001), its edge atoms tend to make bonds with the substrate. Figure 8c-d illustrates both types of the graphene-Ge interaction for the case of a C 29 molecule. The molecule attached itself to Ge(001) with five of its edge atoms (Fig. 8c); graphene-substrate bond dissociation energy was 1.5 eV per C-Ge bond. In its stable state the molecule is bonded along the whole edge, as predicted for graphene nanoribbons on Si(001) [45], and the bond dissociation energy is 1.0 eV per C-Ge bond. But this optimum is difficult to reach for all nucleated graphene pieces (see the discussion in Supporting Information) and many of the molecules are expected to be trapped in a metastable state similar to that depicted in Fig. 8c. When a Ge ad-atom attaches itself between this molecule and the substrate (Fig. 8d) the energy is lowered by 2.8 eV and the molecule tilts back towards the horizontal orientation. Both tendencies (to stand up and to trap Ge) are pronounced, hence they both should have noticeable influence on the growth mode. Yet this influence is likely to be smaller when C is delivered from a molecular beam (as in this study) than when it is delivered from a mixture of CH 4 and H 2 (as in the CVD study reported in Ref. [19]), because hydrogen should preferentially etch graphene at sites where the edge is not protected by bonds formed with Ge(001), thus reducing the need to "glue" the standing molecules back to the substrate. Indeed, the CVD films are markedly better (in terms of D Raman mode intensity) than the MBE films grown at the same substrate temperature.
We suppose that the deterioration of the graphene growth mode by the formation of graphene-substrate bonds is reduced in MBE by the supply of C from subsurface "X" interstitial sites. These atoms have easier access to the graphene edge that is bonded to the substrate than to free parts of the edge.
The dominating mechanism by which carbon is supplied to the growing graphene is affected by the presence of hydrogen. Albeit -according to DFT results -when CH 4 molecule comes into a chemical contact with the surface, it adsorbs dissociatively, losing the first two hydrogen atoms one by one with barrier low enough to play no decisive role at the optimal deposition temperatures, the CH 2 produced by this reaction sequence is a relatively stable species on Ge(001). Carbon deposited from CH 4 is estimated to diffuse on the surface in the form of CH 2 , with the barrier of about 1.5 eV. The CH 2 molecule diffuses for a longer while (from hundreds miliseconds at 900 • C to about a second at 800 • C) before it decays into a subsurface "X" carbon and hydrogen atoms terminating the surface dimer atoms. The distance covered during this time at these temperatures is of the order of 200 nm and the life time of CH 2 is long enough (by the CH 4 flow rates used for deposition) for the molecule to encounter another molecule before decay takes place. In the CVD process, most of C atoms are therefore expected to be delivered to graphene from the top of the surface, while in the MBE process they should arrive from under the surface. For the same reason, the amount of ejected Ge should be considerably smaller during CVD than during MBE.
It follows that by varying the carbon flux (carbon source temperature in MBE, CH 4 flow in CVD) and the availability of H 2 , one tunes the balance between all these carbon delivery processes and influences the chemistry of at the edge of the growing graphene.
The surface roughening observed in the MBE process (and not reported for the CVD process) may be associated with the tendency of small graphene molecules to stand up. Since the boundaries of grains forming the roughened film are expected to be decorated with Ge atoms, it is plausible that on the grain boundaries there exist edge sites, from which atoms or dimers detach with energy barrier compatible to that observed in the experiment (Fig. 7a). One may speculate that the reaction that forms the bottleneck in the Ostwald ripening of the grains and is characterized by the measured barrier height E RMS = 0.66 eV is associated with Ge-mediated migration of grain boundary planes. The grains would then consist of graphene stacks with edge partially glued to Ge(001) by ejected Ge atoms, and partially glued to other such stacks, again by Ge atoms.
Finally we note that the observed defected character of the MBE film (relatively strong D Raman mode) is not associated with defects in the virgin Ge layers, because the defect density (dislocation density) in the Ge layer [24] corresponds to the average defect-defect distance of several µm, which is well below the average domain size deduced from the Raman spectra of the MBE graphene. The defects giving rise to the D Raman mode are not associated also with diffusion of Si from the wafer to the Ge(001) surface, because the amount of such Si should increase with the substrate temperature, while in contrary to this the quality of the film improves with the substrate temperature, being clearly the best when the Ge layer begins to melt. However, one cannot fully exclude the possibility that the observed roughening of the graphene film is connected with contamination of the surface with Si atoms segregated from the wafer. Yet if the Si contamination during the growth would be responsible for the roughen-ing, one would expect an activation energy reflecting the activation energy of Si diffusion in Ge. The rms surface roughness should be thus activated with nearly 3 eV [46]. The measured activation energy of 0.66 eV is too low to account for this process. We therefore tend to associate the roughening of the graphene film with the supposed vertical orientation of some of the graphene nuclei.
Conclusions
Germanium does not mix with carbon and as such it is a suitable substrate to grow graphene. We have demonstrated that molecular beam growth can be used to uniformly cover with decoupled few-layer graphene a Ge(001) film grown on a Si(001) wafer. The graphene sheets are free of carbon nanotubes and consist predominantly of grains with diameter in the range of tens of nanometers. This can be achieved at temperatures between 800 • C and the melting temperature of Ge. Films deposited at lower substrate temperatures are uniform as well and consist of sp 2 -bonded carbon (G Raman mode), but have no Raman signature of ordered sixfold rings (2D mode). On the basis of Raman and XPS spectra, AFM measurements, electrical measurements, and ab initio DFT calculations we argued that films deposited above 750 • C consist of grains built of stacked graphene layers (Fig. 10b). The measured low activation energy of the surface roughness rms (0.66 eV) suggests that the grain facets are contaminated with Ge. This is compatible with the DFT prediction that interaction of atomic C with the clean Ge(001) leads to ejection of surface Ge atoms to mobile ad-atom (monomer) state and that these Ge monomers are preferentially adsorbed on graphene edges. The grains of graphene seem to have nucleated in the initial stage of growth, when small graphene molecules have a tendency to stand up vertically because their edges make chemical bonds with the substrate. We argue that this tendency is reduced by Ge atoms released from the substrate by C atoms (Fig. 10b). Since the sheet resistance of the film hardly depends on film thickness and since the 2D/G Raman mode intensity ratio r 2D/G depends step-wise on deposition temperature (no 2D below 750 • C, r 2D/G 1 between 750 • C and 900 • C, and r 2D/G 2 above 930 • C), we suppose that the grains are covered by a graphene film of higher quality (cf. the cap layer in Fig. 10) that significantly improves when the attachment of graphene edges to Ge(001) weakens as the substrate begins to melt.
Indeed, AFM images show that as the substrate temperature approaches the melting point of germanium (T Ge melt = 937 • C), the grains of similar height become arranged into interwoven lines, but the grain growth mechanism (as characterized by surface roughness RMS activation energy) remains the same as at lower substrate temperatures. The process of line network formation may be driven by a stress relaxation mechanism. This relaxation may lead to electrical and vibrational improvement of the graphene cap layer.
Electrical measurements with 4-point probe STM prove ohmic behavior and 2D conductivity of the film. Independently, metallic behavior follows also from the asymmetry of C 1s core level XPS peak. Temperature dependence of the sheet resistance reveals the Anderson localization phenomenon, known to occur in disordered graphene.
The sheet resistivity drops significantly, down to 2 kΩ/2 for samples grown at 930 • C. Such a low value is comparable to (albeit higher than) that achievable in standard CVD graphene grown on copper substrates [39,40,41,42] and sufficient for application of the MBE graphene/Ge(001) film in a highfrequency graphene-base transistor. Transistors of this kind can be produced with graphene grown directly on the Ge(001) layer: there is no need to remove or further process the germanium.
These results indicate that germanium has some potential as a substrate for growth of graphene. The most advantageous property of germanium is here that, albeit it is a semiconductor, it does not form carbides and carbon hardly dissolves in it. The disadvantage is that carbon atoms at graphene molecule edges make chemical bonds with surface atoms of Ge(001). Nevertheless, Ge atoms ejected from the substrate act as glue that reduces the detrimental tendency of graphene molecules to stand up vertically on the surface and that works even at relatively low deposition temperatures. This leaves room for improvement of the film quality by properly tailored sequence of growth steps.
The implications of this study are strengthened by the very recent report that atmospheric pressure CVD can be used to grow high-quality graphene on germanium wafers [19]. Still, even the CVD process requires the substrate temperature to be above 900 • C, i.e., close to the the melting point of germanium. This is problematic, given that the direct growth of graphene on germanium is needed for devices such as the graphene base transistor, in which the transport of electrons takes place along the surface normal, that is, also from the graphene to the germanium substrate. When the substrate melts, the surface roughens (cf. the hills visible in Fig. 5d and forming a low-frequency pattern, independent of the high-frequency wrinkles), which may deteriorate the graphene-substrate interface by producing regions where the graphene hangs over above the substrate (i.e., the graphene-substrate distance is there significantly larger than when graphene is placed on top of a flat germanium surface). Other problems, such as strong broadening of the dopant profile by diffusion of dopant atoms or thickness inhomogeneity of the undoped germanium that should separate the graphene from doped germanium in such devices are anticipated as well. Lowering of the growth temperature seems therefore desirable.
From the comparison of the MBE and CVD results and from the accompa-nying DFT calculations one can conclude that in both cases the temperature around the surface melting point T sm of germanium is needed as a consequence of carbon-germanium interaction at the graphene edge. It follows that in order to lower this temperature to under T sm one should focus further studies on the control of this chemistry. According to the analysis presented in this report, hydrogen affects the balance between the major mechanisms by which C and Ge atoms are delivered to the graphene edge and thus allows one to use its availability as a means to tune the growth process. The exact method to lower the growth temperature remains however to be found.
Methods
Deposition. High quality Ge (001) layers used as substrates for graphene growth were deposited on non-patterned and patterned 200 mm Si(001) wafers using reduced pressure chemical vapor deposition in a two-step process described in detail elsewhere [47,24]. The thickness of Ge layer on non-patterned and patterned substrates was 1.1 µm and 200 nm, respectively. Clean Ge surfaces were prepared by dipping in HF:H 2 O solution followed by a flash anneal at 760 • C for 60 s [48].
The deposition of carbon was carried out in a DCA molecular beam ultra high vacuum (UHV) system on 1 µm Ge(001) CVD films grown on Si(001) wafers. The growth rate was about 1.4 graphene monolayer per minute, as estimated from X-ray reflectivity (XRR) measurements on a thick (15 nm) film grown at low temperature (200 • C) to suppress surface roughening (the sticking coefficient of C on graphite is close to 1 and its temperature dependence is weak). [49] The source (high-purity pyrolytic carbon) was placed 35 cm away from the sample and emitted mostly carbon atoms. The substrate temperature was varied between 350 • C and 930 • C. The growth time was typically 200 s, with some attempts performed for 100 s, 500 s, and 1000 s. The residual pressure during growth was in the range of 10 −7 mbar.
Characterization. The quality of the graphene film was studied ex-situ by µ-Raman spectroscopy using Renishaw In-viaFlex spectrometer and the green laser light (λ = 514 nm). Spatial resolution was 0.4 µm and spectral resolution was better than 2 cm −1 . Further Raman experiments and maps were done using a LabRamHR800 (JobinYvon Horiba) with 532 nm excitation wavelength. The chemical composition was monitored in situ by X-ray photoelectron spectroscopy (XPS, hν= 1486 eV). Ex-situ high resolution synchrotron radiation XPS measurements were performed in the SOLEIL synchrotron facility, Saint-Aubin, France, using photons with the energy of 350 eV and 600 eV. Topography of the film was assessed by atomic force microscopy (AFM). Air AFM images were taken with Digital Instruments NanoScope III device. Local transport experiments were performed by means of a four-tip scanning tunneling microscope (4-tip STM) in combination with a high resolution scanning electron microscope (SEM).
Theory. Ab initio density functional theory (DFT) calculations for total energies, atomic structures, energy barriers, and molecular dynamics (in the range of picoseconds) have been performed using Quantum Espresso. [50] Generalized Gradient Approximation (GGA) in the Perdew, Burke and Ernzerhof formulation [51] was used for the exchange and correlation energy. Ultrasoft potentials were used to lower the energy cutoff down to 30 Ry. The reciprocal space was sampled in two special points of the Brillouin zone of 4×4 Ge(001) surface area. Activation energies were obtained by the Nudged Elastic Band algorithm [52] and refined with the Climbing Image approach. [53] Ge slabs consisting of 8 Ge(001) layers, terminated on one side with H atoms, and separated with up to 3 nm of vacuum were used; dipole correction was applied to decouple the slabs.
Conflict of interest. The bibliography section is located after the Supporting Information section.
Supporting Information
The following Supporting Information is available: XPS measurements: comparison of C 1s, Ge 3d, and Si 2p spectra obtained at various stages of C deposition on Ge(001) and Si(00), and detailed description of the SR-XPS study. Raman spectroscopy: analysis of Raman peaks, statistical data. Electrical measurements: 4-tip STM setup, temperature dependence and Anderson localization, IV curves, SEM images. Ab initio calculations: expected growth modes, calculated energy barriers, kinetics of energy dissipation after C adsorption, comparison of C in the bulk of Ge and on the surface of Ge.
SI 1. X-ray Photoemission Spectroscopy
Chemical composition of the substrate surfaces and the deposited layers was investigated in-situ by x-ray photoelectron spectroscopy (XPS). Figure 1 shows XPS measurements performed during preparation of the Ge and Si substrates and after deposition of few monolayers of carbon at 850 • C. Native oxide layers in both cases is removed by HF dip. This standard cleaning procedure results in a clean oxygen-free Si surface (Fig. 11b), however, a residual signal from a substoichiometric oxide on germanium [54,55] is still detected (Fig. 11a). This Ge suboxide is effectively removed by a short annealing at 750 • C providing a clean Ge surface. Deposition of carbon on the Si surface results in a chemical shift of the main Si 2p photoemission line (∆E = 1 ±0.1 eV) which is attributed to the formation of SiC [10,56]. In contrast, the Ge 3d peak (29.6 eV) only decreases in intensity upon C deposition but no chemical shift is observed. In particular, no change in the position of the Ge 3d line proves that carbide formation does not take place which is in line with the Ge-C binary phase diagram [13]. Figure 3c compares the C 1s spectra on both samples measured after C deposition. Based on the peak positions (282.8 eV and 284.7 eV for Si and Ge substrates, respectively) and shapes (symmetric on Si and asymmetric on Ge) we conclude that the C deposit on Si substrate is converted into silicon carbide, while on Ge it takes the form of graphitic carbon [57].
SR-XPS C 1s core level spectra (cf. Fig. 3b in the main part) were obtained by measurements performed in the SOLEIL synchrotron facility. The spectra were taken for graphene films growth on different substrates: Cu foil, SiC(0001) (C face), SiC(0001) (Si face), and on Ge(001). The spectra were fitted accordingly with several peaks that describe various chemical carbon functionalities by taking into account the combined instrumental resolution of the experimental setup. The graphene component (green) has been fitted with a Doniach-Sunjic function which best reproduces the asymmetry on the higher binding energy side. The asymmetry on the higher binding energy side implies graphene structure and metallic conductivity. This can be measured by the singularity asymmetry factor α, which is related to the delocalization of the valence states. The green (graphene) component has an asymmetry factor a = 0.07. The spectra corresponding to SiC(0001) substrate consist of a SiC bulk component (in red), the graphene component (green) and two well-known interface contributions (blue).
The information depth was about two monolayers. The important energy to take into account for determined the mean free path (MFP) of the photoemitted electrons is the kinetic energy (KE) of the electrons. The photon energy used in the experiments was 350 eV for all substrates in exception of graphene on C face of SiC, where 600 eV photon energy was used. In the case of 350 eV photon energy, the KE of the C 1s core level is 61 eV. For 600 eV photon energy, the KE of the C1s is 326 eV. For electrons of 60 eV the MFP is close to 0.4-0.5 nm (1-2 monolayers) For electrons of 320 eV the MFP is 0.6-0.7 nm (near 2 monolayers).
Graphene on Cu: graphene was grown by Chemical Vapor Deposition of graphene on copper foils purchased from Alfa Aesar (50 mm thickness, 99.9995% purity). The details of the growth process are described elsewhere [? ].
Graphene on C-face of SiC: graphene was grown on nominally on-axis SiC substrates with the aim of obtaining graphene samples with a thickness from 1 or 2 to about 10 monolayers. The substrate was production grade n-type 6H from SiCrystal and was cleaned using the standard RCA cleaning procedure before introduction into the sublimation furnace. One sample was prepared on an on-axis 4H substrate. High temperature sublimation with a buffer inert gas was used. The temperature range was 1800-2000 • C, the pressure range was 500-850 mbar, and the average growth time was 15 min.
Graphene on Si-face of SiC: the sample was prepared by standard graphitization at IEMN, Université Lille 1. The graphitization of the SiC substrate at 1220 degC for six minutes produced a high quality bilayer graphene on the Si-terminated SiC(0001) surface. A more detailed description of the growth is described elsewhere. [58]
SI 2. Raman Spectroscopy
Ex-situ Raman spectroscopy measurements on a series of samples grown at various temperatures and having nominal thickness of 5 monolayers of carbon are summarized in Fig. 12. Figure 12a shows the Raman spectra. The sample prepared at 400 • C exhibits a broad peak over the 1000-1700 cm −1 range, which is an indicative of amorphous C. Increasing the growth temperature to 700 • C results in the transformation of this broad feature into two distinct peaks centered at around 1345 and 1600 cm −1 (D and G, respectively). Furthermore, a very weak local maximum can be recognized at 2679 cm −1 . It develops to a well defined peak (2D) as the growth temperature is raised to 800 • C. Presence of the characteristic D, G, and 2D bands proves the growth of sp 2 -hybridized carbon on Ge substrates already in the 700 − 800 • C temperature range. Narrowing of all peaks is observed when growth temperature is further increased (Fig. 12b), indicating the improving crystalline order. In addition, the 2D band gains intensity with respect to the D and G bands and at 900 • C the 2D peak becomes dominant in the spectrum.
Due to the versatility of Raman spectroscopy for studying all kind of graphitic systems [59], we also performed Raman measurements with a high spectral resolution on single spots, as well as large-area Raman mappings on the MBE-grown graphene samples on germanium. For comparison, we exfoliated graphene from natural graphite on Ge substrates. The substrates used in the exfoliation process and MBE growth are the same, however, the germanium for the exfoliation process was not heated in contrast to the germanium for the growth process. The typical Raman spectrum of graphene consists of the first-order Γ-point E−2g phonon around 1580 cm −1 (G mode) and several double-resonant Raman modes, namely the D, D , and 2D mode around 1350 cm −1 , 1620 cm −1 , and 2670 cm −1 , respectively (for the laser excitation energy of 2.33 eV). These Raman peaks result from a second-order double-resonsance process and can be activated either by a defect (D and D mode) or a second phonon (2D mode). Thus, the intensities of the defect-related Raman modes can be used to investigate the structural and crystalline quality of the grown layers. The 2D mode stems from an intervalley double-resonant scattering process involving two TO phonons close to the K point [26]. Since the double-resonance process depends on both the electronic band structure and the phonon dispersion, the 2D-mode lineshape gives information about both properties [27]. Figure 13: Raman spectra of the MBE-grown graphene (black, upper spectra) and exfoliated single-layer graphene (blue, lower spectra) on germanium in the range of the D and G mode (a) and in the range of the 2D mode (b). Spectra are normalized to the same G and 2D mode intensity, respectively, and vertically offset for clarity. The asterisk marks the Raman peak of atmospheric oxygen (O 2 ) that appears due to long integration times. Figure 13 shows Raman spectra of a MBE-grown graphene sample together with spectra from exfoliated single-layer graphene on germanium. The spectrum of the grown sample shows all prominent Raman peaks of graphene. Since the D and 2D modes possess a breathing-like vibrational pattern of a single hexagonal carbon ring, the appearance of these Raman modes proves the growth of sp 2hybridized carbon [25]. However, all Raman peaks are strongly broadened and up-shifted compared to exfoliated single-layer graphene on Ge. For instance, the G and 2D modes exhibit a FWHM of γ G = 40.8±1.0 cm −1 and γ 2D = 62.6±0.9 cm −1 , respectively, and a peak position of ν G = 1587.5±0.4 cm −1 and ν 2D = 2683.8±0.3 cm −1 (see Fig. 14). In contrast, the mechanically exfoliated single-layer graphene on Ge exhibits values of γ G = 1578 cm −1 and ν G = 13 cm −1 for the G mode and γ 2D = 2666 cm −1 and ν 2D = 26 cm −1 for the 2D mode. The positions and FWHMs of the Raman modes for exfoliated single-layer graphene on germanium resemble the typical values found for free-standing (undoped, unstrained) single-layer graphene [28]. The up-shift and broadening of the Raman modes in the MBE-grown sample indicate the presence of nanocrystalline graphene [60]. In general, Raman spectroscopy is used to evaluate the doping level or the amount of strain in graphene by the G-and 2D-mode position and their FWHM. In the present case this is rather difficult, since the influence of the defects superimposes possible effects from strain or doping. Instead, the up-shift of the Raman modes is most likely related to the nanocrystallinity of the graphene (see below).
The 2D mode of the MBE-grown graphene is symmetric with a broad single-Lorentzian shape. However, the symmetric line shape does not indicate singlelayer graphene in the grown samples but rather few-layer graphene. It was shown for CVD-grown graphene [61] and recently for MBE-grown graphene on sapphire [62] that in such samples the 2D-mode line shape remains symmetric up to approximately three layers but up-shifts and broadens. Since the 2D-mode line shape reflects to a certain extent the evolution of the electronic band structure of few-layer graphene around the K points, this single-Lorentzian shape in thicker graphene samples indicates that the grown graphene layers do not have a defined stacking order and thus are decoupled [63]. Since the grown graphene layers are decoupled, we cannot reliably determine the exact number of layers just from the 2D-mode line shape or by the appearance of other layernumber dependent Raman modes [27,64,65]. However, we can conclude that the MBE-grown graphene samples are not single-layer graphene. Furthermore, we can exclude the growth of carbon nanotubes, since no radial-breathing modes (Raman spectra not shown) nor a splitting of the G mode in G − and G + were observed (see Fig. 13) [66]. To evaluate the quality of the grown graphene samples, we performed Raman mappings covering an area of approximately 10×30 µm 2 with a total of 200 spectra. The statistical analysis of the peak position of the G and 2D mode is shown in Fig.14a and 14b. The data was fitted assuming a Gaussian distribution. The low standard deviation (σ=0.4 cm −1 ) of the experimental data from the peak positions γ G = 1587.5 cm −1 and γ 2D = 2683.8 cm −1 verifies the very high homogeneity of the grown graphene layers. Figure 14c shows an analysis of the 2D/G-mode intensity (integrated area) ratio in the investigated region, which has an average value of 1.90±0.12. This ratio is considerably lower than the values found for single-layer graphene, which exhibits typically a ratio of approximately four at excitation wavelengths of 532 nm and using SiO 2 substrates [27]. Again, this indicates that the MBE-grown graphene on germanium is not a single layer. The D/G-mode intensity ratio in the investigated area was determined to 2.19±0.07. It is well known that the D/G-mode ratio is indicative for the grain size L a of nanocrystalline graphene and graphite [25,31,32], where L a is defined as the average crystal diameter and given by L a (nm) = (2.4 × 10 10 )λ 4 Laser (
I D I G ) −1(1)
Using the D/G-mode intensity ratio from our samples, the laser wavelength of 532 nm used in our experiments, and applying the above equation, we deduce an average crystalline grain size of L a = 8.8±0.3 nm.
In summary, the Raman analysis proves the growth of sp 2 -hybridized carbon. The MBE samples consist of decoupled few-layer graphene. From the intensity ratio of the D and G mode we deduced a grain size of the nanocrystalline graphene of approximately 10 nm. Local transport experiments were performed by means of a four-tip scanning tunneling microscope (4-tip STM, Fig 15) in combination with a high resolution scanning electron microscope (SEM). The SEM gives a quick access to the local morphology of the samples and allows to choose a specific area for further transport experiments. After the first SEM analysis, all four tips of the 4-tip STM are brought into tunneling contact by a feedback control loop and are then navigated to the desired positions. Operating in tunneling mode ensures that the tips do not touch the surface while they are moved over the sample. Once the preselected area for transport experiments is reached, the feedback is turned off and all tips are lowered one by one until they make direct contact with the sample surface. During this process, contact resistances between the individual tips are checked simultaneously to ensure that all tips are in contact with the sample. All IV curves are recorded in a 4-point probe geometry. Hence, a current is driven through the outer probes and the voltage drop between the inner probes is recorded. This allows us to measure the resistance of the sample without parasitic influences of contact resistances. Four samples with different preparation conditions (growth time from 200 s to 1000 s, growth temperatures of 900 • C and 930 • C) were investigated. Two SEM pictures on different scalings ( Fig. 16a-b) of the sample prepared at 900 • C with a growth time of 500 s are shown exemplarily. The local morphology seen in the SEM images is consistent with AFM studies for all investigated samples.
SI 3. Electrical measurements
Local transport experiments were realized with tip spacings ranging from 500 nm to 3 µm. All transport data were recorded in an equidistant linear arrangement of the probes. A typical arrangement of the tips with the electrical wiring shown schematically can be found in Fig. 15. The recorded IV curves for a fixed probe spacing of 2 um are shown in Fig. 17a for all four investigated samples. Most striking is the linearity of the data indicating clearly a metallic behavior of the surface. Already visible is also a strong difference between the samples processed at 930 • C and those processed at 900 • C, which show a much higher resistance. To get more information about the nature of the electronic transport, probe spacing dependent measurements were carried out (Fig. 17b). All investigated samples exhibit a probe spacing independent resistance which is a typical property of two dimensional transport [67]. No contributions of the bulk Ge were seen in any of the transport experiments. A possible influence of the Ge surface state will be discussed below together with the temperature dependence. The probe spacing independence of resistance allows us to calculate the two dimensional resistivity (sheet resistance R S ) of the graphene crystallites in a very simple way with the expression [67] R S = π ln(2)
U 23 I 14 .(2)
The sheet resistances calculated with this formula are plotted in fig. 1(e). The samples processed at the surface melting point at 930 • C exhibit sheet resistances about 2 kΩ/2 which are 10 times smaller than those measured on the samples prepared below the surface melting point. This reflects the better electronic quality of the graphene nanocrystallites prepared at the surface melting point. However, no significant difference between samples processed at the same temperature but with different growth times could be found. This might be an indication that either only a fixed number of graphene layers participates in electronic transport, or that the additional amount of carbon is fully implemented into the carbon islands rather than into the graphene nanocrystallites in between.
To explore the influence of the strong inhomogeneity of the sample on the local transport properties, temperature dependent measurements were carried out. For this purpose, the sheet resistance of the samples which showed the lowest resistivity (those processed at the surface melting point) were measured within a temperature range from room temperature down to 36 K (obtained by cooling with liquid He). The results are shown in Fig. 6a in the main article. With decreasing sample temperature the sheet resistance is exponentially increasing from 2 kΩ/2 up to 20 kΩ/2. This behavior is well known for disordered systems and especially also for disordered graphene and can be understood in terms of Anderson localization [34,35,36,37]. To verify this assumption we fit the data according to the model of variable range hopping (VRH) which is the basic transport mechanism in Anderson localized systems [38]. For VRH the resistance is given by
ln(R) ∼ ( T T 0 ) d+1 ,(3)
where d denotes the dimension. From the inset in Fig. 6a (main article) it is already obvious that the data are best described with d = 2, which is consistent with the observation of two-dimensional transport behavior. The full fit is also shown in Fig. 6a (main article) and describes the data very well on the complete temperature range. A possible contribution of the Ge surface state [68] is also excluded by the temperature dependent transport data. The measured IV curves maintain their metallic behavior even at the low temperature. On the contrary, the Ge(001) surface was reported to be either only metallic at temperatures above 200 K and semiconducting below 200 K [69] or to be semiconducting even at room temperature [33]. In all cases, the temperature dependence of the surface state does not match our transport data, hence, a parasitic contribution of the Ge surface state can be excluded. Therefore we conclude that we probe only the electronic properties of the graphene and not of the underlying Ge substrate.
In summary, the local transport experiments have shown that the graphene nanocrystallites behave as a two-dimensional conductor. No contribution of the underlying substrate could be detected. The temperature dependent measurements reflect the inhomogeneity of the investigated samples showing strong localization effects. The data agree very well with the theoretical model of variable range hopping in two dimensions.
SI 4. Ab initio calculations
Results of ab initio calculations for the behavior of C atoms on Ge(001) p(2×2) surface point to three mechanisms as potentially responsible for the observed surface roughness. They are sketched in the next paragraph and discussed in more detail later on.
The first plausible reason for the roughness is ejection of Ge atoms from surface dimers by C atoms. This implies that during the first stage of deposition many carbon atoms are immobilized in surface substitutional sites, while the Ge atoms ejected from these sites agglomerate into islands, possibly with some of the deposited carbon atoms. During the second stage of growth, graphene nucleates and grows on top of such a pre-roughened surface (Fig. 18a). On the other hand, when one of the other two mechanisms dominates, the substrate surface retains its flatness. With the mechanism number two, the graphene growth mode is strongly influenced by the formation of chemical bonds between the edge of the first layer of graphene flakes and the topmost atoms of germanium (Fig. 18b). If this is the case, the aspect ratio of multilayer graphene islands is likely to depend on the crystallographic direction, along which the carbon atoms from the first graphene layer formed a stable chemical connection to the substrate. Finally, the mechanism number three works by combining the ejection of Ge and Ge-graphene interaction. This combination may lead to Ge contamination of graphene edges and to formation of Ge-rich facets on edges of multilayer graphene (Fig. 18c). Ge ejection (Fig. 19) is expected to take place because the energy barrier for this process is only about 0.65 eV. This is low enough to allow for ejection of Ge at any practicable growth temperature. For example, assuming the attempt frequency of 10 13 s −1 , the expected time needed to overcome this barrier is less than 0.1 ns at 850 • C and less than 5 ns at 450 • C. Furthermore, the barrier height is much smaller than the 4.6 eV released by C adsorption from vacuum to the top of the surface, meaning that from the energy point of view the Ge ejection process may take place athermally or may be athermally assisted. Namely, the energy needed to overcome the barrier, or part of this energy, may be taken from the adsorption energy and not from thermal vibrations, meaning that the ejection probability does not depend on substrate temperature or that the effective barrier showing up in the Arrhenius plot is smaller than the physical barrier.
Ge monomers are highly mobile. Thus, if the ejection process is indeed efficient, it is plausible that they agglomerate into islands. Such islands will grow provided that the monomer diffusion length is significantly smaller than on the clean surface. Otherwise, the surface will evolve as during molecular beam epitaxy of Ge on Ge(001): few islands will nucleate and flat Ge will regrow by step flow. One may speculate that the diffusion length of Ge monomers is reduced by interaction between the monomers and C atoms. In spite of high adsorption energy of C from vacuum to Ge(001), athermal emission of Ge seems to be a rare event. Figure 20 illustrates a typical evolution of the kinetic energy of a C atom during the first picosecond after adsorption. The average kinetic energy of nearby Ge atoms is plotted as well. We performed about a dozen simulation runs; in all cases the C atom did not diffuse away from the impact site further than a few Ångstroms before it finally occupied the sub-dimer interstitial site and thermalized to the energy well below the kinetic barrier for Ge ejection. Within the first picosecond, the impact energy was distributed among the neighboring Ge atoms, but the kinetic energy acquired by a single Ge atom was always lower than the kinetic barrier and, within a fraction of a picosecond, it dropped below 0.2-0.1 eV. One can associate this behavior with large mass ratio between light C and heavy Ge atoms. The surface processes caused by C adsorption on Ge(001) may thus be to good accuracy described as happening at thermal equilibrium.
The ejection barrier is low as long as a C atom occupies the sub-dimer interstitial site (Fig 19b). The efficiency of the carbon-assisted ejection of Ge depends therefore (a) on the probability that the reaction of C with Ge proceeds through the path containing the sub-dimer interstitial, and (b) on the total time spent by the C atom in this interstitial site. Indeed, C i may in principle drift away from the sub-dimer interstitial geometry (Fig. 19b) before its Ge neighbor had a chance to escape to the geometry shown in Fig. 19c, or it may diffuse deeper into the bulk of germanium, omitting the sub-dimer interstitial site altogether. Figure 9b of the main part summarizes the energies of carbon adsorbed at and under Ge(001) p(2×2) surface. It is apparent that C dissolved in Ge is unstable with respect to phase separation into graphene and clean Ge. The energy difference of about 2 eV between C in graphene and substitutional C in bulk Ge is compatible with very low solubility of C in Ge (up to 2.5·10 14 cm −3 in CZ-grown crystals, [70] which corresponds to 2.0 eV at Ge melting temperature of 937 • ). It is also clear that subsurface sites are generally more favorable for carbon than sites in the bulk; this effect is much stronger for interstitial (C i ) than for substitutional (C Ge ) geometries. The presence of numerous energetically nonequivalent subsurface sites of the same class (interstitial or substitutional) is due to the strain field caused by dimerization of the surface.
Closer examination of Fig. 9b (from the main part) reveals that the subsurface interstitial site with the lowest energy is not the one from which Ge ejection may be initiated (label "B", cf. the atomic structure in Fig. 19b), but one that is burried deeper under the surface (label "X", (001)-split interstitial, i.e. an atom sharing a lattice site with a host atom). The energy barrier from "B" to "X" is 0.9 eV, that is, nearly the same as the barrier for Ge ejection from "B". This means that, entropy factors ignored, it is expected that in about 90% of cases Ge atom is thermally ejected before the C atom leaves the sub-dimer site "B".
The carbon atom that has reached the burried "X" site does not eject Ge so easily. In order to do so, it would either have to return to the "B" site, or kick out and replace a neighboring Ge atom. The former process is associated with a kinetic barrier of 1.7 eV, meaning one successful return attempt per τ xb = 4 µs at 850 • C and one per τ xb = 70 ms at 450 • C; hence, this process takes much longer than about nanosecond needed from Ge ejection from "B". Going from "X" to the most favorable subsurface substitutional site ("S" in Fig. 9b in the main part) is even more time consuming. The corresponding barrier is nearly 3.0 eV, which translates into one successful substitution attempt per second at 850 • and one per year at 450 • C.
Deposited C atoms are thus expected to accumulate in close vicinity to the Ge(001) surface, partially in form of C-Ge dimers substituting Ge-Ge dimers (as in the structure "c", shown in Fig. 19c), and partially in form of interstitials (structure "X"). The "X" interstitial is highly mobile and it may also convert to other states, with various probabilities. It may substitute a fourfold-coordinated Ge atom, but this happens very rarely and is irrelevant for our discussion. Migration of the interstitial into deeper regions of Ge seems to be a rare event as well: in spite of low migration barrier of C i in bulk Ge (our calculations indicate the barrier of 0.9 eV, which is practically the same as the barrier for C i migration in Si), [71] the energy of bulk C i is substantially (by nearly 2.4 eV) higher than the energy of subsurface C i . However, the conversion of "X" to a C-Ge dimer (with Ge ejection from the surface dimer) is of importance, because it can happen within times several orders of magnitude shorter than the time τ CC that elapses between another C atom from the beam hits within the intermediate neighborhood. The latter time is namely comparable to the reverse deposition rate measured in monolayers per time: to seconds or minutes, which is orders of magnitude longer than the life time τ x of the "X" state.
The mobile C i spends therefore most of its life at one of the "X" sites. It ceases however to exist within τ x of the same length scale as the characteristic time τ xb of the conversion from "X" to "B": microseconds at 850 • C and milliseconds at 450 • C. This is because the "B" state decays, with about 90% probability, to a Ge surface monomer and a C-Ge surface dimer ( (Fig.19c)). The highly mobile monomer diffuses then away. The interstitial at "X" might also end up by reacting with another C i , but since the time τ CC is much longer than τ x , all interstitials are expected to be already in the C-Ge state before encounter with another C takes place.
The C-Ge surface dimer is likely to live until another Ge monomer kicks C from it; the corresponding barrier amounts to about 0.5 eV, it is therefore very low. For this reason we also suppose that overgrowth of the C-Ge dimer by a progressing surface step is quite improbable; what happens is rather that C is kicked out and either goes interstitial (to an "X" site") or ejects another Ge atom from another Ge-Ge surface dimer.
It follows that when two C atoms meet on Ge(001) surface, one of them has already substituted Ge in a surface dimer and is in the C-Ge dimer state. It seems plausible that this second C atom makes a bond to the C atom in the C-Ge dimer and that the whole object remains in its place until one more atom arrives. This can be either C or Ge. If C arrives, a C 3 cluster consisting of C in C-Ge dimer and of C 2 attached to it is expected to form. If Ge arrives, we suppose that a kick-in process takes place: Ge substitutes the C atom in the C-Ge dimer and a surface C-C dimer appears on top of the otherwise (locally) perfect Ge(001). The substitution of C in the C-Ge dimer a Ge monomer is however plausible only when the C cluster is small enough so that it does not block the access to the C-Ge dimer site. If the Ge monomer cannot approach the C-Ge dimer, it sticks to the C atoms at the edge of the growing cluster. The same is expected to happen when a Ge monomer encounters a C cluster sitting on top of the perfect surface: it attaches itself to the cluster edge.
This analysis indicates that Ge contamination (Fig. 18c) is a more realistic reason for film roughness than substrate roughening (Fig. 18a), albeit at submonolayer C coverages the amount of ejected Ge atoms is comparable to the amount of deposited carbon. It also shows that the role played by Ge ejection is ambivalent. On the one hand, Ge monomers may contaminate the C clusters, possibly hindering the growth of crystalline graphene at lower temperatures, and possibly contributing to the formation of facets on the edges of growing graphitic islands. On the other hand, Ge monomers may heal the surface from the defects at which the graphene seeds are strongly attached to the substrate. Which one of the two opposite actions dominates, it may depend on the growth conditions: on the substrate temperature and on the deposition rate.
So far we did not account for the possibility depicted in Fig. 18b: that graphene edge is bonded to the (otherwise perfect) substrate surface and that this effect limits the grain size. But when a small graphene molecule is placed on the Ge(001) surface, its edge atoms tends to make bonds with the substrate. Figure 21 illustrates this for the case of a molecule consisting of 29 carbon atoms. The molecule attached itself to the substrate with five edge atoms: four C atoms in a sequence became bonded to Ge atoms from the same dimer row, and one more distant C atom became bonded to a Ge atom from a neighboring dimer row. The bond dissociation energy was in this case 1.5 eV per C-Ge bond. Due to directional character of the bonds, the molecule experienced a force lifting it towards the surface normal. The effect is substantial and should have noticeable influence on the growth mode. Even though in its ground state a bigger molecule prefers to be parallel to the surface so that the number of C-Ge bonds at its edge is possibly high, many tiny C clusters will initially stand up, because there are many competing paths by which carbon clusters are built and grow, and some of these paths are likely to end up with a cluster that is bonded to the substrate only on one side. This non-parallel orientation is sustained by further C atoms. Namely, they reach the cluster in the form of "X" interstitials, i.e., from under the surface. In the main part of the article we explain why this tendency to produce graphene seeds that stand on the surface instead of lying on it is likely to be reduced by Ge monomers diffusing on the surface.
In summary, we find in the initial stage of growth C atoms substitute Ge atoms in surface dimers. At small sub-monolayer coverages, the overwhelming majority of C atoms is expected to occupy this position. As the coverage increases towards a monolayer, more and more C clusters are formed, more and more C substitutional atoms are replaced again by Ge monomers, and more and more C atoms can join the C clusters without previously ejecting a Ge monomer. As a result, carbon agglomerates on top of the substrate surface. Unless a significant amount of Ge monomers becomes trapped in the amorphous network of carbon, the germanium surface should become only slightly roughened, the expected rms being of the order of monatomic step height on on Ge(001), i.e., 0.14 nm. Any graphene molecules produced in this stage are likely to have many of its edge atoms chemically bonded to the surface Ge atoms and are likely to have a marked tendency to produce 3D structures. This detrimental tendency appears to be opposed by the Ge monomers, which glue graphene edge back to the surface. The diameter of graphene nanocrystallites is expected to be strongly affected by the efficiency of carbon incorporation at sites between carbon edge atoms and germanium surface atoms, while the distribution of the nanocrystallites on the surface is expected to be strongly affected by their diffusion. The latter is likely to be significant only at temperatures above the surface melting point of germanium.
Figure 1 :
1The difference in quality of graphene produced on Ge and on SiO 2 . (a) Optical microscope image of Ge pillars embedded in SiO 2 matrix. (b) Raman spectra and 2D/G mode intensity map. In all cases investigated in this study, if the substrate temperature T sub exceeds about 750 • C, graphene deposited on Ge has much higher quality than deposited on SiO 2 at the same conditions (here, T sub = 900 • C).
Figure 2 :
2AFM images acquired after C deposition at various substrate temperatures.
Figure 5 :
5Graphene grains in the sample on which about 5 monolayers of C have been deposited at 800 • C. (a) AFM corrugation averaged along the axis of the box shown in the panel b. (b) AFM amplitude image illustrating the sub-structure of the grains in the length scale below the grain size of about 20-50 nm recognizable in the AFM height scan (panel a).
Figure 6 :
6(a) Sheet resistance measured by 4-point STM as a function of temperature for a fixed probe spacing L = 2 µm (cf. Supporting Information for technical details). The symbols are the same as in panel b. (b) Sheet resistance as a function of probe spacing.
Figure 7 :
7(a) Arrhenius plot of AFM surface roughness RMS of nominally 6-monolayer films. (b) D/G and 2D/G Raman peak area ratio dependence on the growth temperature.
Figure 8 :
8(a) Carbon atom (black) adsorbed on Ge(001) dimer (red and blue atoms). (b) C atom substitutes Ge in the dimer and ejects a Ge atom. (c) A piece of graphene chemically bonded to Ge surface. (d) The ejected Ge atom (red, cf. panel c) attaches itself to its edge.
Figure 9 :
9a) Energy barriers for the process of Ge ejection. Adsorption of atom from vacuum to the configuration "a" proceeds withe the energy gain of 4.6 eV and no barrier. The states "A" and "B" are shown in Fig. 8a-b. Carbon in the intermediate state sits directly under the Ge dimer; the dimer bond is broken. b) Energy of C atom in various configurations, measured with respect to the energy of the substitutional C in the bulk. The ordinate is the vertical distance to the center of Ge dimers; the positions of dimer atoms and atomic layers of the perfect surface are indicated by dotted lines. Labeled larger symbols correspond to the configurations discussed: "A" and "B" are the structures shown in
Figure 10 :
10Supposed structure of MBE graphene on Ge(001). a) Overview. b) Details.
Figure 11 :
11In situ XPS investigation of C deposition on Si and Ge. (a) Ge 3d spectra for the Ge substrate at various stages. (b) Si 2p spectra for the Si substrate at various stages. (c) comparison of C 1s spectra for Ge and Si substrates after carbon deposition at 850 • C. XPS chemical shifts are attributed to the formation of carbide on Si substrate and graphitic carbon on Ge substrate.
Figure 12 :
12(a) Raman spectra from samples grown at at various substrate temperatures. (b) Peak width dependence on the growth temperature. (c) D/G and 2D/G peak height ratio dependence on the growth temperature.
Figure 14 :
14Statistical analysis of (a) the G-mode, (b) 2D-mode position, and (c) of the 2D/Gmode intensity ratio. The intensity of the G and 2D mode is evaluated from the peak areas. The data was fitted assuming a Gaussian distribution (red, dashed lines). Data collected from the area of 10×30 µm 2 with 200 spectra.
Figure 15 :
15SEM image of the 4-point probe configuration.
Figure 16 :
16SEM images of sample was grown at the substrate temperature of 930 • C. The nominal coverage is 12 monolayers of carbon.
Figure 17 :
17Room temperature measurements. (a) IV curves, L = 2 um (cf.Fig. 15) on various samples. (b) Sheet resistance as a function of probe spacing.
Figure 18 :
18Expected result of graphene growth dominated by each of the three roughening mechanisms discussed in the text. (a) Germanium ejection might lead to graphene covering a roughened Ge substrate. (b) Chemical interaction between graphene edge and substrate dimers might lead to formation of multilayer graphene islands with bonded edges. (c) Migration of ejected Ge to graphene edges might lead to formation of Ge-rich facets terminating the multi-layer graphene islands.
Figure 19 :
19Adsorption of C on Ge(001) and Ge ejection. (a-c) Atomic configurations: (a) a C atom (black) is adsorbed in a Ge dimer bond, (b) it moves into the nearest interstitial site, elevating one of the Ge dimer atoms, and (c) the Ge atom is ejected to the top of a neighboring dimer; the Ge-Ge dimer transforms into a C-Ge dimer. (d) Energy barriers; the labels correspond to the atomic geometries above. Adsorption of atom from vacuum to the geometry "a" proceeds with no barrier, the energy gain is about 4.6 eV.
Figure 20 :
20Kinetic energy of a carbon atom and average kinetic energy of seven Ge atoms in its nearest neighborhood, as a function of time elapsed from the moment of its first collision with the surface. Momentary and time averaged values are shown. At t = 0 ps the slab was at T = 0 K; the thick line shows the evolution of the average kinetic energy of slab atoms.
Figure 21 :
21Formation of bonds between a small (C 29 graphene molecule on Ge(001)-2×2.
The authors declare no competing financial interest. Acknowledgement Atomistic calculations have been done at the Jülich Supercomputing Centre, Germany, NIC project hfo06. The authors thank Ion Costina, Oksana Fursenko, David Kaiser, Wolfgang Mehr, Hans Thieme, Dominique Vignaud, Damian Walczyk, and Andre Wolff for technical support and discussions. Support from the European Commission through a STREP project (GRADE, No. 317839) is gratefully acknowledged. F.H. and J. M. acknowledge support by the European Research Council (ERC), grant number 259286.
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Since its discovery, graphene has been a top-list novel material thanks to its remarkable properties. 1,2 Beside extensive experimental and theoretical basic studies, graphene has been recently investigated for many novel device applications, like 2D-electronics and graphenebased transistors. 3 Nowadays, a limiting factor in the development of consumer electronics is the large chipset heat generation; therefore, intense research 4-6 is being focused in minimizing the energy losses due to heat generation or reusing the thermal energy by means of thermoelectric devices. The important thermal parameters are the thermopower (or Seebeck coefficient) and the ZT figure of merit, which must be as large as possible in order to maximize the efficiency of the thermoelectric conversion. Nanomaterials and composites are promising candidates since it is possible to engineer the phonon-phonon scattering, decreasing the thermal conductivity and in turn increasing the ZT factor. [7][8][9] In the case of graphene, after the growth process the layer can be transferred onto an arbitrary substrate. 10 This substrate may be used as source of electrical carriers (electrons or holes) and may also transfer heat from the electrically functional layer. 11 In particular, due to the very good thermopower properties of silicon carbide (SiC), where the Seebeck coefficient exceeds −480 µV/K, it has been shown that this substrate can be used as a very good heat sink, performing in some cases even better than a copper plate. 11,12 Moreover, silicon carbide is especially convenient as a material for graphene deposition, since it is a growth substrate for graphene monolayers when the Si-terminated (0001) surface is used as substrate, 13,14 or for graphene multilayers when the C-terminated face is used. 15 Additionally, SiC doped with boron is a superconductor 16 and can be used for graphene-superconductor junctions, where the Seebeck coefficient can be strongly enhanced at specific temperatures in the Andreev reflection regime. 17 Moreover, doping SiC with boron might not only cause superconductivity but also increase the thermoelectric efficiency as it happens in many other boron-containing materials. 18 The experimental and theoretical studies of the graphene structure on SiC reveal interesting geometric phases: i) graphene monolayers grown onto the Si-face of SiC have a strong buckling, 19,20 i.e. variable graphene to Si interatomic distances, ii) graphene grown onto the C-face of SiC form flat multilayers which occur in different stackings, i.e. orders of atoms in subsequent layers. Multilayers can be classified in one of three families: 15 C atoms exactly on top of each other (AA stacking), the Bernal stacking (AB stacking) and the rhombohedral stacking (ABC stacking), as illustrated in Fig. 1. Graphene layers interact among each other mainly via van der Waals interactions, and different stackings exhibit distinct properties. For instance, the stacking order and the presence of defects in graphene can be visualized via a measure of the thermoelectric power. 21 In the literature, extensive studies have been focused on evaluating and measuring the thermopower and the thermoelectric figure of merit for pure graphene monoand bilayers, often at the Si/SiO 2 substrate; a comprehensive review collects these data. 22 However detailed knowledge of these parameters for various stackings and number of multilayers is missing, and the effect of the SiC substrate also has not been investigated yet, except in the case of a single graphene layer. 23 Therefore, we report here theoretical calculations of the Seebeck coefficient and of the electrical contribution to the ZT figure of merit for different mono-and multilayers, both free-standing and on top of a SiC substrate, at various temperatures and for the three dif- FIG. 1: (From left to right) Band structure, top-view geometry, and Seebeck coefficient (two columns) as a function of chemical potential for monolayer graphene (first row), graphite (second row), and the AA, AB and ABC stacked graphene (third, fourth and fifth rows, respectively). In the third column, the Seebeck coefficient is shown as a function of the chemical potential for different temperatures; in the fourth column, it is shown as a function of the chemical potential for different monolayer (ML) thicknesses.
ferent possible stackings. Calculations were performed within the density-functional theory (DFT) framework with the PW91 approximation for the exchangecorrelation functional, 24 as implemented in the Quantum ESPRESSO suite of codes 25 which adopts a planewave basis set and uses pseudopotentials to approximate the core region. We used the PW91 ultrasoft pseudopotentials freely available from the Quantum ESPRESSO website, with energy cutoff of 20 Ry for the wavefunc-tions and 200 Ry for the charge density. The experimental geometry was used for the interlayer distances: 3.2Å for the first graphene layer above the SiC substrate, 3.7Å between the first and the second graphene layer, and 3.4Å for each subsequent layer. 15 The SiC substrate (when present) was simulated using 10 monolayers, more than sufficient to properly approximate a semi-infinite bulk, with an in-plane atomic arrangement for the first graphene monolayer on the C face of the SiC surface that fits a √ 3 × √ 3R30 • supercell with respect to the SiC surface atoms. 41 The bottom-face of the substrate has been passivated with H atoms. The Brillouin zone has been sampled using a 20 × 20 × 1 Γ−centered Monkhorst-Pack uniform k-mesh. The vacuum separation between periodic slabs in the direction perpendicular to the surfaces has been kept to about 50Å in order to avoid interactions between periodic images. After the DFT calculations, we used the Wannier90 code 26 in order to obtain the maximally-localized Wannier functions (MLWFs) 27 . We minimized the spread of the MLWFs using atom-centered orbitals as starting guess: the p z orbital on each C atom and hybridized sp 2 orbitals on every other C atom of the graphene layer; and sp 3 orbitals on every Si atom of the SiC substrate. The frozen energy window was chosen from the bottom of the valence band to the Fermi energy, and the outer energy window 30 eV above the Fermi level. We verified that the obtained MLWFs were real-valued. We then used the MLWFs to interpolate the band structure on a very dense k−mesh (2400 × 2400 points in the graphene plane for free-standing cases and 1600 × 1600 points for the deposited cases, which are characterized by smaller Brillouin zones). Such a dense mesh is necessary for an accurate description of the band structure of the systems un-der investigation, as we also discuss later, and is possible thanks to the extremely accurate and fast interpolation of the electronic bands in a maximally-localized Wannier functions basis set. 28 Finally, we calculated the transport distribution function and the other transport properties presented here using the semiclassical Boltzmann equations in the constant relaxation time approximation using the BoltzWann post-processing code 29
distributed with Wannier90 v.2.0.
The band structures for bulk graphite, free-standing monolayer graphene and multilayer graphene in the AA, AB and ABC stackings are compared in Fig. 1, where we also show the atomic arrangement and the calculated Seebeck coefficients S for different temperatures and number of monolayers (ML). We emphasize here that, in the constant relaxation time approximation, S is independent of the value of the relaxation time τ . For all the free-standing systems, the Seebeck coefficient as a function of the chemical potential displays the electron-hole symmetry, at least in a range of about 1.5 eV around the Fermi energy. This symmetry is also revealed in experiments for deposited graphene with small doping rates, i.e. close to the Fermi level of the undoped system 30 , and is particularly useful since it allows to determine whether a graphene layer is p− or n−type doped by means of a thermopower measurement.
Our results show that the maximum of the Seebeck coefficient at T=300 K achieves 86 µV/K for graphene, 66 µV/K for graphite, 12 and 69 µV/K for 4-monolayers (4ML) in the AA-and AB-stackings respectively, and 71 µV/K for three monolayers (3ML) in the ABC-stacking. Inspecting the graphene band structure near the Dirac point, we notice that "V-shaped" bands give a negative contribution to the Seebeck coefficient, while the "A-shaped" bands give a positive contribution. This explains why the AA stacking shows suppressed Seebeck effect with respect to other types of stackings. The tiny details of complex "V-A-shaped" band structure in the ABC stacking are also reflected in the oscillating Seebeck coefficient at low temperatures. We emphasize that we verified by increasing the k−mesh that these oscillations are a real physical effect and not the result of numerical noise. We also note that a change in the number of monolayers (ML) in free-standing graphene significantly influences the thermoelectric response only for the AA stacking. This is due to the stronger interactions between carbon atoms stacked on top of each other with respect to interactions between monolayers in the AB and ABC stackings. On the other hand, the AB stacked graphene is thermoelectrically similar to the single graphene monolayer and, as expected due to the similar stacking, to graphite.
The values that we obtain are in good agreement with experiments for samples mechanically exfoliated on Si/SiO 2 . For instance, a thermopower (TEP) value of about 93 µV/K is reported in 31 for p-type graphene at 300 K under a gate voltage of V g = −5V. A maximum TEP value of around 95 µV/K at 280 K is re- ported in Ref. 33 . Other authors obtain 40 µV/K close to V g =0V at 255 K, with a maximum around 50 µV/K near V g =25V 32 . Our results also agree with previous model calculations for graphene, that report a TEP close to 80 µV/K at 300 K. 34 The efficiency of a thermoelectric material, however, does not depend uniquely on the Seebeck coefficient, but is instead described by the thermoelectric figure of merit ZT, defined as ZT = σS 2 T /κ, where S is the Seebeck coefficient, σ is the electrical conductivity, and κ = κ e + κ l is the thermal conductivity, having an electronic contri-bution κ e and a lattice contribution κ l . In many systems of interest at room temperature, κ l is the leading term. The evaluation of κ l requires however the evaluation of phonon-phonon scattering terms, that is beyond the scope of this work. We therefore show in the following figures the quantity σS 2 T /τ , that is independent of the value chosen for relaxation time τ , and is an indicator of the behavior of the electronic contribution to the ZT coefficient.
The calculated values for free-standing structures in all stackings are shown in Fig. 2 in the 200 − 400 K temper- ature range and for different number of monolayers. The maximal value of σS 2 T /τ increases monotonically with temperature for all stackings. We emphasize that this is not only due to the factor T , because the increase is more than linear (compare for instance the curves at 200 K and 400 K). Moreover, for the AB and ABC stackings, the dependence of σS 2 T /τ partially loses the electron-hole symmetry. As already discussed above, interlayer interactions in the AA stacking strongly suppress the Seebeck coefficient and in turn the σS 2 T /τ factor, especially for values of the chemical potential near the Fermi energy. On the other hand, both the AB and ABC stackings show an increase of σS 2 T /τ as the number of layers increases, with peaks located at both sides of the Fermi energy. This is particularly relevant for applications, showing that an increased thermoelectric efficiency could be achieved if multilayer systems (with the proper stacking) are considered, mainly thanks to the increased number of transport channels available and therefore to an increase of the electrical conductivity. In particular, at 300 K, the maximum value reached by σS 2 T /τ in the case of the AB stacking is 1.22, 2.67 and 4.07 times larger than the maximum of graphene for 2, 4, 6ML respectively. In case of the ABC stacking the corresponding numbers are 2.18, 4.66 and 6.85, for 3, 6 and 9ML respectively.
The effect of the deposition on the C-face of a SiC(0001) substrate on the properties of graphene and its multilayers is technologically extremely relevant. For this reason, we also investigate here the transport properties of the same systems after deposition onto such a substrate. In Fig. 3 the band structures, the Seebeck coefficient S and the σS 2 T /τ contribution to the thermoelectric efficiency are reported for all investigated stackings at different temperatures. The flat interface states are visible in the band structures and appear in all cases on the hole side. In order to better understand the origin of the different bands and features in the transport spectra, we also report in Fig. 4 the projected local density of states for all structures, where we show the projection onto the p−like states of each graphene layer and of the first two layers of the substrate. In all cases, a fully occupied high density peak is present, originating from the p z states of the topmost C-layer of the C-face SiC(0001) substrate, corresponding to the almost flat bands just below E = 0. A broader maximum, also of p z −type but on the unoccupied side, arises from the graphene layers. In order to understand whether the most relevant contributions to the Seebeck coeffiecient and to σS 2 T /τ originate mainly from the overlayers or from the substrate, we also show the total density of states decomposed in its contributions from the substrate and the overlayers.
The Löwdin analysis shows in all cases a similar charge distribution over the substrate and graphene multilayers: the substrate Si atoms have a net charge of −1.2 electrons per atom with respect to their valence charge of four; the substrate C atoms have net charge of +1.1, except for the topmost C layer where each C atom has a charge of +0.8. The C atoms in the graphene multilayers have a net charge in the range of [−0.06, −0.04], with the smallest net charge (in absolute value) reached for the central layers of the multilayer graphene. We emphasize here that the charge disproportion effect that we find is not very pronounced, at least for the thin multilayers (a few ML) that we investigated. In Ref. 35 , instead, three differ-ent types of electrical conductivities -electron, intrinsic, and hole -have been reported for multilayer graphene sheets, indicating that the position of the Fermi level is layer-dependent. In the experiments of Ref. 35 , however, the number of graphene MLs is expected to be larger than 10. It is therefore possible that, when increasing the number of ML, the charge disproportion effect becomes more important, giving rise to different spatial regions that are effectively neutral, n− or p−doped.
In Fig. 3, the Seebeck coefficients show two main oscillatory behaviors, one occurring near the Fermi energy where the localized states are present, and one at the energy at which the Dirac cone is localized. Indeed, as already discussed using the results of Fig. 4, the PDOS for energies above the Fermi level is almost completely due to the graphene overlayers, and indeed the Seebeck coefficient for positive µ resembles the corresponding spectrum of the free-standing case (after having taken into account a shift of the Fermi energy upon deposition). However, if we focus on the σS 2 T /τ contribution, our result show that a very strong enhancement is obtained for ABC stacking upon deposition on SiC. In fact, at 300 K, the maximum value (for µ > 0) reached by σS 2 T /τ in the case of deposited graphene is 0.95 of the maximum of the same layer in the free-standing case. The same ratio of the deposited with respect to the free standing AAAA (4ML) is 0.37, for ABAB (4ML) it is 0.97, and for ABC (3ML) it is 2.99.
The thermopower coefficient calculated in this work for graphene deposited on SiC achieves a maximum value of 86 µV/K for a temperature of 230 K, to be compared with the experimental value reported in Ref. 23 for a single hole-doped graphene layer on the C-face of the same substrate, and at the same temperature, which is 55 µV/K. The difference is large but within our theoretical range. For a comparison with the deposition on different substrates, the measured TEP for graphene deposited on SiO 2 substrate is about 20 µV/K for p−type or −50 µV/K for n−type samples. 36 For multilayered graphene deposited on Si/SiO 2 , there are several exper-imental reports showing values ranging from 40 µV/K up to even 700 µV/K for gapped graphene functionalized with molecules. 37,38 The upward shift of the Dirac cone in graphene deposited on a substrate is an usual phenomenon, which is also visible in thermoelectric experiments for graphene grown at SiO 2 and published by Novoselov et al. 39 (see Fig. 2 in that paper). Another important issue to take into account is the fact that we assumed a perfect in-plane periodic structure in the calculations, but in the experiments the buffer layers between the substrate and the perfect graphene layer are discontinuous, island-like shaped. 40 Summarizing, we calculated the DFT band structures of graphene mono-and multilayers both free-standing and deposited on the C face of 4H-SiC(0001). The corresponding thermoelectric properties (Seebeck coefficient and σS 2 T /τ ) were evaluated adopting the semiclassical Boltzmann equations in the constant relaxation time approximation, where the electronic bands were interpolated using a maximally-localized Wannier functions basis set. The Seebeck coefficient is similar for the AB and ABC stackings and monolayer graphene in the freestanding case. The AA stacking is instead thermoelectrically much less efficient. The effect of deposition on SiC strongly increases the σS 2 T /τ contribution to ZT in the case of the ABC stacking. For the AA stacking, instead, the interactions between the graphene layers and with the C-face of the SiC substrate reduce the parameters of interest. These results are optimistic for a design of graphene and SiC-based heterostructures as future electronic devices and provide an indication of which type of stacking can be the most suitable.
This work has been supported by the European Funds for Regional Development within the SICMAT Project (Contract No. UDA-POIG.01.03.01-14-155/09). Calculations have been performed in the Interdisciplinary Centre of Mathematical and Computer Modeling (ICM) of the University of Warsaw, within the grants G51-2 and G47-7, and partially supported by PL-Grid Infrastructure.
FIG. 2 :
2σS 2 T /τ for different number of free-standing graphene monolayers (ML) and types of stacking as a function of the chemical potential, for different temperatures. (a) Four monolayers (4ML) with AA stacking. (b) Four monolayers with AB stacking. (c) Three monolayers with ABC stacking. (d) AA, (e) AB and (f) ABC stacking at T = 300K for different number of monolayers. The Fermi energy is set to E = 0.
FIG. 3 :
3Band structure, Seebeck coefficient and σS 2 T /τ for graphene (a-c), 4ML graphene in the AA stacking (d-f), 4ML graphene in the AB stacking (g-i), and 3ML graphene in the ABC stacking (j-l). In all cases, the layers are deposited on the C-face of a SiC(0001) substrate.
FIG. 4 :
4Projected local density of states for graphene monoand multilayers deposited on the C-face of SiC(0001). Red solid lines denote the projection onto pz states, whereas black dashed lines denote the projection onto px and py states. The local DOS projected on each of the graphene layers, as well as on the first two layers of the substrate, is reported. Total DOS of substrate and overlayers is presented in the upper part of the panels.
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Grill and P. Avouris, Multicarrier transport in epitaxial multilayer graphene. Y.-M Lin, C Dimitrakopoulos, D B Farmer, S.-J Han, Y Wu, W Zhu, D Kurth, J L Gaskill, R L Tedesco, C R Myers-Ward, A EddyJr, Appl. Phys. Lett. 97112107Y.-M. Lin, C. Dimitrakopoulos, D. B. Farmer, S.-J. Han, Y. Wu, W. Zhu, D. Kurth Gaskill, J.L. Tedesco, R.L. Myers-Ward, C.R. Eddy Jr., A. Grill and P. Avouris, Mul- ticarrier transport in epitaxial multilayer graphene, Appl. Phys. Lett. 97 112107 (2010).
Thermoelectric power of graphene as surface charge doping indicator. A N Sidorov, A Sherehiy, R Jayasinghe, R Stallard, D K Benjamin, Q Yu, Z Liu, W Wu, H Cao, Y P Chen, Z Jiang, G U Sumanasekera, Appl. Phys. Lett. 9913115A.N. Sidorov, A. Sherehiy, R. Jayasinghe, R. Stallard, D.K. Benjamin, Q. Yu, Z. Liu, W. Wu, H. Cao, Y.P. Chen, Z. Jiang, G.U. Sumanasekera, Thermoelectric power of graphene as surface charge doping indicator, Appl. Phys. Lett. 99, 013115 (2011).
Power factor enhancement for few layered graphene films by molecular attachments. D Sim, D Liu, X Dong, N Xiao, S Li, Y Zhao, L Li, Q Yan, H H Hng, J. Phys. Chem. C. 115D. Sim, D. Liu, X. Dong, N. Xiao, S. Li, Y. Zhao, L. Li, Q. Yan, H.H. Hng, Power factor enhancement for few layered graphene films by molecular attachments, J. Phys. Chem. C 115, 1780-1785 (2011).
Enhanced thermopower of graphene films with oxygen plasma treatment. N Xiao, X Dong, L Song, D Liu, Y Y Tay, S Wu, L Li, Y Zhao, T Yu, H Zhang, W Huang, H H Hng, P M Ajayan, Q Yan, ACS Nano. 5N. Xiao, X. Dong, L. Song, D. Liu, Y.Y. Tay, S. Wu, L. Li, Y. Zhao, T. Yu, H. Zhang, W. Huang, H.H. Hng, P.M. Ajayan, Q. Yan, Enhanced thermopower of graphene films with oxygen plasma treatment, ACS Nano 5, 2749-2755 (2011).
Electric field effect in atomically thin carbon films. K S Novoselov, A K Geim, S V Morozov, D Jiang, Y Zhang, S V Dubonos, I V Grigorieva, A A Firsov, Science. 306K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, A. A. Firsov, Electric field effect in atomically thin carbon films, Science 306, 666-669 (2004).
. K Grodecki, private communicationK. Grodecki, private communication.
We did not perform calculations of graphene deposited on the Si-face of the SiC substrate because they would require a much larger supercell. We did not perform calculations of graphene deposited on the Si-face of the SiC substrate because they would require a much larger supercell.
| Abstract We report here on a new way to tailor the structure of large-scale graphene during its growth. Monolayer graphene formed on Pt substrate using a modified hot-filament-assisted chemical vapor deposition setup, which does not require any control of crystal nucleation and orientation. The underlying growth mechanism includes a stage of structural evolution from nanocrystalline to microcrystalline graphene film. An enhanced recrystallization process in the graphene film is assessed by means of Raman spectroscopy and atomic resolution transmission electron microscopy. Moreover we demonstrate that the rotational angle between neighboring graphene grains can be tuned to 30° or to small misalignment about 1–2° only. This process opens a new route to control the electrical properties of large-scale uniform graphene film. | Local electron–phonon coupling of a one-dimensionally nanorippled graphene is studied on a SiC(0001) vicinal substrate. We have characterized local atomic and electronic structures of a periodically nanorippled graphene (3.4 nm period) prepared on a macrofacet of the 6H-SiC crystal using scanning tunneling microscopy/spectroscopy (STM/STS) and angle-resolved photoelectron spectroscopy (ARPES). The rippled graphene on the macrofacets distributes homogeneously over the 6H-SiC substrate in a millimeter scale, and thus replica bands are detected by the macroscopic ARPES. The STM/STS results indicate the strength of electron–phonon coupling to the out-of-plane phonon at the K points of graphene is periodically modified in accordance with the ripple structure. We propose an interface carbon nanostructure with graphene nanoribbons between the surface rippled graphene and the substrate SiC that periodically modifies the electron–phonon coupling in the surface graphene. | 483 | We introduce a novel approach to the synthesis of high-quality and highly uniform few-layer graphene on silicon wafers, based on solid source growth from epitaxial 3C-SiC films. Using a Ni/Cu catalytic alloy, we obtain a transfer-free bilayer graphene directly on Si(100) wafers, at temperatures potentially compatible with conventional semiconductor processing. The graphene covers uniformly a 2″ silicon wafer, with a Raman I/I band ratio as low as 0.5, indicative of a low defectivity material. The sheet resistance of the graphene is as low as 25 Ω/square, and its adhesion energy to the underlying substrate is substantially higher than transferred graphene. This work opens the avenue for the true wafer-level fabrication of microdevices comprising graphene functional layers. Specifically, we suggest that exceptional conduction qualifies this graphene as a metal replacement for MEMS and advanced on-chip interconnects with ultimate scalability. | The development of graphene electronics[1,2]requires the integration of graphene devices with Si-CMOS technology. Most strategies involve the transfer of graphene sheets onto silicon, with the inherent difficulties of clean transfer[3][4][5]and subsequent graphene nano-patterning that degrades considerably the electronic mobility of nanopatterned graphene[6,7]. Epitaxial graphene (EG) by contrast is grown on an essentially perfect crystalline (semi-insulating) surface, and graphene nanostructures with exceptional properties[8][9][10][11]have been realized by a selective growth process on tailored SiC surface that requires no graphene patterning[9,12,13].However, the temperatures required in this structured growth process are too high for silicon technology. Here we demonstrate a new graphene to Si integration strategy, with a bonded and interconnected compact double-wafer structure. Using silicon-on-insulator technology (SOI)[14][15][16]a thin monocrystalline silicon layer ready for CMOS processing is applied on top of epitaxial graphene on SiC. The parallel Si and graphene platforms are interconnected by metal vias. This method inspired by the industrial development of 3d hyper-integration stacking thin-film electronic devices[17,18]preserves the advantages of epitaxial graphene and enables the full spectrum of CMOS processing. Figure 1 is an illustration of the monolithic integration of both Si and SiC devices onto the same double wafer, showing CMOS devices patterned on the thin crystalline Si wafer on top, | Among the many anticipated applications of graphene, some -such as transistors for Si microelectronics -would greatly benefit from the possibility to deposit graphene directly on a semiconductor grown on a Si wafer. We report that Ge(001) layers on Si(001) wafers can be uniformly covered with graphene at temperatures between 800 • C and the melting temperature of Ge. The graphene is closed, with sheet resistivity strongly decreasing with growth temperature, weakly decreasing with the amount of deposited C, and reaching down to 2 kΩ/2. Activation energy of surface roughness is low (about 0.66 eV) and constant throughout the range of temperatures in which graphene is formed. Density functional theory calculations indicate that the major physical processes affecting the growth are: (1) substitution of Ge in surface dimers by C, (2) interaction between C clusters and Ge monomers, and (3) formation of chemical bonds between graphene edge and Ge(001), and that the processes 1 and 2 are surpassed by CH 2 surface diffusion when the C atoms are delivered from CH 4 . The results of this study indicate that graphene can be produced directly at the active region of the transistor in a process compatible with the Si technology. | Direct graphene synthesis on a Si/SiO2 substrate by a simple annealing process | Graphene has attracted tremendous interest due to its unique physical and chemical properties. The atomic thickness, high carrier mobility and transparency make graphene an ideal electrode material which can be applied to various optoelectronic devices such as solar cells, light-emitting diodes and photodetectors. In recent years, there has been a growing interest in developing graphene/silicon Schottky junction solar cells and the power conversion efficiency has reached up to 15.8% with an incredible speed. In this review, we introduce the structure and mechanism of graphene/silicon solar cells briefly, and then summarize several key strategies to improve the performance of the cells. Finally, the challenges and prospects of graphene/silicon solar cells are discussed in the development of the devices in detail. | Single-Crystalline Monolayer Graphene Wafer on Dielectric Substrate of SiON without Metal Catalysts | Two limiting factors for a new technology of graphene-based electronic devices are the difficulty of growing large areas of defect-free material and the integration of graphene with an atomically flat and insulating substrate material. Chemical vapor deposition (CVD) on metal surfaces, in particular on copper, may offer a solution to the first problem, while hexagonal boron nitride (h-BN) has been identified as an ideal insulating substrate material. The bottom-up growth of graphene/h-BN stacks on copper surfaces appears therefore as a promising route for future device fabrication. As an important step, we demonstrate the consecutive growth of well-aligned graphene on h-BN, both as single layers, by low-pressure CVD on Cu(111) in an ultrahigh vacuum environment. The resulting films show a largely predominant orientation, defined by the substrate, where the graphene lattice aligns parallel to the h-BN lattice, while each layer maintains its own lattice constant. The lattice mismatch of 1.6% between h-BN an... | In this letter, we report the first experimental demonstration of wafer-scale ambipolar field-effect transistor (FET) on Si (111) substrates by synthesizing a graphene layer on top of 3C-SiC(111)/Si(111) substrates. With lateral scaling of the source-drain distance to 1 μm in a top-gated layout, the ON-state current of 225 μA/μm and peak transconductance of > 40 μS/μm were obtained at Vds = 2 V, which is the highest performance of graphene-on-Si FETs. The peak field-effect mobilities of 285 cm2 /Vs for holes and 175 cm2 /Vs for electrons were demonstrated, which is higher than that of ultra-thin-body SOI (n, p) MOSFETs. | III-V Nanowire-based Solar Cells on Si and Graphene |
484 | where did the first professional tennis tournament take place | Which country played tennis first? | U.S. Pro Tennis Championships The U.S. Pro Tennis Championships (also for a period known as the World Pro Championships) was the oldest professional tennis tournament played until its final year of 1999 and is considered as a part of the professional grand slam from 1927–1967 until the advent of Open Era. Pancho Gonzales holds the record for most wins with eight. The tournament only had a men's draw. American's first prominent professional player, Vinny Richards, arranged what became the first U.S. Pro by negotiating with Doc Kelton to have a tournament played on the Notlek courts, located at 119th Street | Grand Prix tennis circuit they remained amateurs – had become apparent to Herman David, the chairman of The Wimbledon Championships at that time. In 1967, David announced that a professional tournament would be held at Wimbledon after the Championships that year. The tournament was televised by the BBC and succeeded in gaining public support for professional tennis. In late 1967, the best of the remaining amateur players turned professional, paving the way for the first open tournament. Some professionals were independent at this time, such as Lew Hoad, Luis Ayala and Owen Davidson, but most of the best players came under contract to one | Major professional tennis tournaments before the Open Era Wembley Championship. Played between 1934 and 1990, at the Wembley Arena in England, it was unofficially usually considered the world's championship until 1967. The third professional major was the French Pro Championship, played between 1934 and 1968, on the clay-courts of Roland Garros, apart from 1963–1967, when it was played on the indoor wood courts of Stade Coubertin. The U.S. Pro Tennis Championship, also known as the US Pro, was an annual tournament, later known as MFS Pro Championships. It was first organized by player Vinnie Richards when promoter C. C. Pyle withdrew interest in the project. It was first | Colonial National Invitational (tennis) The Colonial National Invitation was a men's tennis tournament played at the Colonial Country Club in Fort Worth, Texas from 1962 to 1973. The club hired Tut Bartzen as a tennis pro and made him responsible for hosting the tournament. The inaugural edition in 1962 was an amateur-only event. In 1967 it became a professional tournament, which meant that amateurs could not compete. The total prize money for the tournament that year was $15,000 and the first-prize, won by Rod Laver, was $1,700. The following year, 1968, a women's professional event was added which was won | Tennis tour A tennis tour (or tennis circuit) is tennis played in tournament format at a series of venues – a tour – over during a set period of weeks or months. Professional tour tennis is played globally with one season consisting of one calendar year. Several tournaments are held each week as players win prize money and earn ranking points. A player's ranking determines her or his ability to enter a particular tournament, as tournaments vary in the amount money and points obtainable. Winning a tournament typically requires winning four to six matches in succession, generally a match a | French Pro Championship In 1930 the "Association Française des Professeurs de Tennis (AFPT)" held its first pro tournament, titled "Championnat International de France Professionnel" (French Pro Championships) June 18–22, 1930, and is considered as a part of the professional grand slam from 1927 to 1967 till the advent of Open Era. The tournament only had a men's draw. From 1930 the French Pro Championship were always played at Paris, on outdoor clay at Roland Garros except from 1963 to 1967 where it was held at Stade Pierre de Coubertin on indoor wood. Ken Rosewall holds the record for 8 wins | Team tennis Team tennis is a tennis tournament which consist of matches between different groups of players each competing to win the tournament for their team. It is played at the collegiate or national level in the United States. The United States Tennis Association promotes junior team tennis and USTA League Tennis. The National Collegiate Athletic Association organizes competitions such as the NCAA Division I Men's Tennis Championship and NCAA Division I Women's Tennis Championship. In the United Kingdom, team tennis is played through schools and clubs from local to national levels. The Lawn Tennis Association have an 'AEGON Team | 484 | History of tennis held in August 1967, the first tournament where professional tennis players were allowed to play at Wimbledon. The "Open Era" began in 1968 when major tournaments agreed to allow professional players to compete with amateurs. Before 1968, only amateurs were allowed to compete in Grand Slam tournaments and other events organized or sanctioned by the ILTF, including the Davis Cup. The move is made because the English are tired of the hypocrisy in the sport, the shamateurism that plagues high-class tennis. It is well known that amateurs bargain for – and receive – exorbitant expenses to compete at many tournaments. | Portugal Ladies Open (tennis) | when did the german open tennis tournament change to doubles | when was the last time the hall of fame tennis tournament was held | when was the first lawn tennis tournament played | in which historical event was tennis played a major role | how many professional levels were there in the first tennis tournament | 1972 First National Tennis Classic | 1941 U.S. National Championships (tennis) |
485 | Royal Purple Synthetic motor oil | How synthetic oil made? | As with all Royal Purple Synthetic Products, this is a high quality, top performance grease, I had all the front end parts of my 1994 GMC K-2500 replace with life time warranty Moog premium parts, the original factory parts were worn-out @ 224,000 miles, I immediately noticed a smoother ride and operating ease with this grease, much better than the old "Dino" type, it's a bit more costly, but I'm sure it will last and protect against wear much better, Thanks Amazon for the ability to find and receive such a high quality product in a quick manner, and to Royal Purple for your wonderful products | When I noticed that STI in Japan sold this product as their recommended oil, I looked up the specs from Motul. Unlike most companies, Motul is not afraid to share their additive information. After reading all day, I decided to switch! I now use Motul in the engine, trans and rear diff. | It's motor oil and it's in a convenient gallon jug. I use it in my garden tractor but for my car, I use only name brand oil. | There are different types of synthetic oil: synthetic blend oil and full synthetic oil. Here's the difference. Synthetic blend oil is a mix of conventional motor oils and synthetic base stocks. | Can you switch to regular motor oil after using synthetic? | Well constructed of solid materials. I use it in conjunction with Liqui Moly synthetic oil and am pleased with the results so far. | What do you do with used motor oil? | 485 | This is an area that I am very familiar with as I previously distributed synthetic oil. During this period I befriended one of the leading scientists who created and formulated synthetic oils. We spent a lot of time together and I often accompanied him to several industry seminars. I received quite an education about oil.
Briefly synthetic oil does everything your regular motor does, only it does it better. Plus Royal Purple uses a different base stock (ester) then regular and most other synthetic oils--it clings naturally to metal. thus reducing dry starts (a leading cause of engine wear), reduces friction; has a higher film strength; reaches operating temperature quicker; runs cooler and has a greater resistance to high temperature breakdown;has a high solvency action that will clean the inside of your engine; it has a lower freezing point point, ie. it will not thicken up in the coldest of climates, and finally it will increase your HP and mpg. In my opinion Royal Purple is the best synthetic oil on the market today.
Is there a downside? Yes it is expensive and hard on valve seals. If you have an older car with a lot of miles on it (about 60,000 or more) the seals and rings may be filled up with engine deposits and the oil may flush them out,leaving you with an oil burner. We had this problem on my friends Saturn. The oil made a big difference in performance and mpg, but it began to burn oil. So on the next change we dumped it and replaced it with Mobil ! and Lucas Synthetic Oil Stabilizer and the oil consumption ceased. It was a better performance then regular oil but not as good as the Royal Purple. Also suggest you use Royal Purple transmission oil, it does make a difference.
Don F Los Angeles | Synthetic oil | Is Goon Grease/Oil-based just not for me? | Excellent oil for 4-Stroke Racing and V-Twin Motorcycle Engines - tried in extreme conditions! | Should you use Synthetic oil to break in new engine? | I use it in conjunction with Liqui Moly synthetic oil and am pleased with the results so far | I try to take as much care for my car as possible and I feel like my car runs the best on this oil | Is it badd to switch to synthetic oil? | This is really good oil for high performance cars duh |
486 | who said that raymond rubicam's copywriters and art directors were the most | Robert Bresson views of two people: one is called Bresson and one called Bergman". In his book "Sculpting in Time", Tarkovsky describes Bresson as "perhaps the only artist in cinema, who achieved the perfect fusion of the finished work with a concept theoretically formulated beforehand." Bresson's book "Notes on the Cinematographer" (1975) is one of the most respected books on film theory and criticism. His theories about film greatly influenced other filmmakers, such as the French New Wave directors. Opposing the established pre-war French Cinema (Tradition de la Qualité) by offering his own personal responses to the question 'what is cinema?', and | Arthur Secunda (born November 12, 1927, in Jersey City, New Jersey) is an American painter, sculptor, and printmaker. Secunda had his first one-man show in 1950 at the Galerie Lucien Gout in Montpellier. His works hang in the permanent collections of institutions worldwide including the Museum of Modern Art in New York, the Library of Congress in Washington, DC, the Chicago Art Institute, the Honolulu Academy of Fine Arts, and the Detroit Art Institute.
References
American male painters
20th-century American printmakers
1927 births
Living people
Sculptors from New Jersey
20th-century American sculptors
20th-century male artists
Painters from New Jersey | elements, some—but rarely and perhaps never all—of which are found in each of the genre's films. Because of the diversity of noir (much greater than that of the screwball comedy), certain scholars in the field, such as film historian Thomas Schatz, treat it as not a genre but a "style". Alain Silver, the most widely published American critic specializing in film noir studies, refers to film noir as a "cycle" and a "phenomenon", even as he argues that it has—like certain genres—a consistent set of visual and thematic codes. Other critics treat film noir as a "mood", characterize it as | Mark Landis November 2010, "The Art Newspaper" published a comprehensive article on the matter, inspiring other publishers such as the "Financial Times" to follow suit. Despite these exposés, Landis has continued his forgeries intermittently, with attempted gifts in November 2010 to the Ackland Art Museum (as Father Arthur Scott); in September 2012 to William Carey University (as Martin Lynley); and in October 2012 to several southern museums (as Lynley and as John Grauman). It appears that in donating forgeries to art museums, Landis has not actually broken any laws, even though his activities were clearly deceptive. If he had sold the work | Gilles Larrain Gilles Larrain (Dec 5, 1938) is a French-American photographer who believes photography is a way to “capture the landscape of the soul of a person.” By taking a unique approach to photography, which includes creating his own lighting, managing the entire darkroom process, and always having subjects come to his personal studio space, Larrain has created acclaimed pieces of art since 1969. In 1973, Larrain published the highly successful photographic book, "Idols", which presented portraits of transvestites. Two generations later, the book inspired American photographer Ryan McGinley who wrote an April 2010 article in Vice Magazine, which identified | the field of computer graphics has expanded over time. An astonishing amount of the breakthroughs in the field in this decade - particularly many important early breakthroughs in the transformation of graphics from utilitarian to realistic - occurred at the University of Utah in the 1970s, which had hired Ivan Sutherland. Sutherland was paired with David C. Evans to teach an advanced computer graphics class, which contributed a great deal of founding research to the field and taught several students who would grow to found several of the industry's most important companies - namely Pixar, Silicon Graphics, and Adobe Systems. | Who the $&% Is Jackson Pollock? mémoire "Framed" to solicit his help in selling her painting. With Volpe's vision a business venture was formed, Legends Art Group, to manage and sell works of art authenticated by science; the formation of this venture is discussed in the documentary. Volpe also brought Horton's story to producer Steven Hewitt, who, along with his father, executive producer Don Hewitt (creator of "60 Minutes"), had formed the Hewitt Group to produce documentaries. Harry Moses, an Emmy, Peabody, and Directors Guild of America award-winner, and a recipient of a lifetime achievement award from the National Academy of Television Arts and Sciences, is | as to the exact size of Caravaggio's oeuvre, with counts as low as 40 and as high as 80. In his biography, Caravaggio scholar Alfred Moir writes "The forty-eight colorplates in this book include almost all of the surviving works accepted by every Caravaggio expert as autograph, and even the least demanding would add fewer than a dozen more". One, "The Calling of Saints Peter and Andrew", was recently authenticated and restored; it had been in storage in Hampton Court, mislabeled as a copy. Richard Francis Burton writes of a "picture of St. Rosario (in the museum of the Grand | 486 | advertising. David Ogilvy credited Rubicam with assembling "the best team of copywriters and art directors in the history of advertising," whose advertisements "were read by more people than any other agency's." The agency represented many of America's leading companies, such as Gulf Oil, General Electric, Johnson and Johnson, Fortune, Life and others. While Raymond Rubicam's emphasis on creativity was innovative in itself, his philosophy and copywriting approach revolutionized the industry. He believed that an advertisement should "mirror the reader." Knowledge and understanding of the customer was a critical component of effective advertising. With the help of Dr. George Gallup, Rubicam | gil oved is the co-founder of which south african advertising agency | [For Hire] Advertising Agency Producer | The Bastrop Advertiser | who was the founder of the american advertising museum | Article focused on advertising as one of the most important parts of marketing communication in one of the online shop, TokoBagus. Advertising communicated a message from a certain brand to the target audience through a particular medium. The aim of this research was making advertising with a powerful message, so it was able to become a captain of consciousness that could play an important role in economic and social systems of modern society. Because of its potential power, the creative advertising workers had a big responsibility in their hands. It was not only to explore the creativity visually or verbally to a creative worker, but also, they should understand the purpose of communication, the communication strategy, and the creative strategy. In this case, TokoBagus run this in making advertisement campaign to promote its brand. The method used in this research was the qualitative method and inductive model. Data were collected through an interview, literature, and visual data. Those collected data were analyzed using a qualitative-verificative strategy and case study method. The case study was Toko Bagus advertising campaign from the year 2011 to the year 2014 when finally its name changes into OLX. It finds that the advertisements only become beautiful works of art, but it does not solve the problem of the brand. Therefore, this research is important to document the communication strategy and the creative strategy of an advertising campaign so it can be a reference for a young designers or students. | The article analyzes the notions of persuasion, persuasive techniques, manipulation, its types, and their application in business advertising. Advertising has covered the way from informing a target audience to asking and convincing, from convincing to working out conventional reflexes, from working our traditional reflexes to the unconscious suggestion, and from the cold advice to the projection of a symbolic image. Advertisers have been consistent in making customers perceive a picture of a promoted product consciously and then make them buy it automatically.Advertising is so powerful that it can form and change the worldview and behaviour of people. That is why professionals in many spheres study and investigate the phenomenon of manipulative potentials of advertising. The term manipulation stands for the art of managing people's behaviour and thinking with a focused impact on the social consciousness, a type of psychological influence, a hidden inducement of people to perform specific actions, an invisible socio-psychological control of a target audience.A successful manipulation requires exploiting human beings' critical weaknesses, such as the limited capability of strategic reasoning, little awareness, susceptibility to cognitive biases, or potentially indirect social pressure.As to the to the persuasive techniques, the most effective ones are lexical (descriptive adjectives, clichés, coloured words, emotive and inclusive vocabulary, colourful words and descriptive language, loaded words; associations and connotations, subtexts, anecdotes), rhetorical and stylistic (rhetorical questions, argumentation, reasoning and logic, evidence: exaggeration, hyperboles, alliteration, metaphors, repetitions, similes, irony, pun) and the visual ones (iconic signs, graphs, tables etc.). Combined together they make advertisements eye-catching, bright, memorable, informative, thought-provoking, persuasive, and manipulative.Creating advertisements by borrowing methods from psychology has been quite successful. Psychology is an inseparable part of human being activity, including advertising and business. That is why knowledge of psychology, psycholinguistics, and NLP provides a better understanding of consumers' needs, desires, and preferences and positively influences a company's image and profits. At present, advertising is, on the one hand, an organic part of modern life. With its assistance, we find out about new products, goods, shops, and services. On the other hand, advertising is a means of massmedia communication that influences people by implementing modern psychology and psycholinguistics's practical methods and tactics. To achieve their set goals, advertisers use a unique language and select lexical units that create anxiety, fear of being late or missing a chance or a sale. | Development Report on the Advertising Industry (2011–2012) | along with walter d. scott, what psychologist contributed to the field of advertising |
487 | What is Kate Gosselin's book tour schedule 2010? | When is kate gosselin's new book coming out? | Kate gossilin coming back with a show? | Is kristen stewart going to be in eclipse? | Please enable Javascript to watch this video
Two local artists are celebrating the separate releases of their albums -- together!
Kathryn Rose Wood and Mikayla Braun both have new albums coming out. And they are throwing a double album release party Sunday (Oct. 8) at 8 p.m. at Gasa Gasa. Doors open at 7 p.m.
Look for Kathryn Rose Wood on Facebook for more information on her new album, In the Ashes.
And check out Mikayla Braun's website for more information on her new album, Synapse. | Jon and kate plus 8 websites? | How many movies has kristen stewart has made? | What is kellie picklers phone number? | What is kate walsh cadillac commercial? | 487 | What is kate gosselin book tour schedule? | What are the names of kate gosselins books? | What church does kate gosselin belong to? | Did kate gosselin spank leah gosselin? | How long has Kendall Schmidt and Katelyn Tarver been going out? | Is kate gossilin coming out with a new sereas? | When is Kate plus 8 coming back? | Can someone look up the contact & IRB info on Kate Kenski? | Where does mary-kate olsen live? |
488 | Technically faulty! - Dieses Ebook ist technisch fehlerhaft! | I'll start off by saying that I'm only 100 pages into it, but it's baaaaad. Some reviewers state that there's nothing here that can't be found on the web, but that's true of any tech book right? It's always on the web in my experience. What disturbs me about this book is the copious amounts of errors and other typos. I've never seen a book w/ so many! Was there no editor? Did the author not read their own work? Also, this book doesn't read well in English. I realize that English is not the author's native language, but I'd assume that it would be somebody at Packt's job to help w/ this. The lack of flow coupled w/ the rampant errors makes this book a tough read. Top that off w/ poorly conceived examples and you've got a real stinker of a book. Oh, and I might not win any points w/ this last one, but avoid Ext JS at all costs if you can. | The editorial content is good. Author is excellent and well researched. The problem is a physical one. The book is not bound properly. There are loose pages throughout and makes reading difficult Would not have purchased it if I had known of this defect. | I have recently purchased SEVEN Nora Roberts and JD Robb audio books which were manufactured by Brilliance Audio and ALL of them were defective. I have yet to receive any explanation other than "send it back and we'll send you another." I am reluctant to make another purchase. | There was no proof reading of this book. It is distracting to have to figure out what word to add or delete. I can't believe the copy of this book was not checked for errors before printing.
I am learning to tap and there are some pointers but he goes over the same routine for each issue which is a lot of the book. | This product is the result of OCR scanning technology and is very difficult to read. The only redeeming factor is that the OCR errors are consistent so that, once one understands the errors,the book, although annoying, becomes readable. | A german/english publication with 320 pages and 1000 images...The oeuvre of the Stuttgart based talented engineers Jorg Schlaich and Rudolf Bergmann, is presented in this publication.
Book covers extraordinary designs/examples of successful symbiosis of structure and architecure through presented projects of towers,roofs, facades,bridges, which are examples of unique engineering solutions of real elegance and beauty.
The reader will not like at all the fact that book is in two languages simultaneosly (one page in german ,next in english), what makes it unpleasant for reading..some drawings are only german,scale of well done graphics is often smaller that should be etc....these are the main 'spoilers' of this valuable publication. | This book as multiple errors throughout the entire book with the test questions and reductive reasoning. I bought this book to help me study for my upcoming test and it's left me nothing but frustrated. I notified the manufacture of this book and they informed me they know about the errors and there is nothing they can do since I purchased from Amazon. Save your money and don't purchase this book!!! | It is somewhat difficult to read, but that is not its fault. This is complicated material. I have no complaints but the reader should be prepared for some work in reading this. | 488 | I own several antiquarian issues of Ferdinant Avenarius' Hausbuch and was really excited to find it as an ebook as well. The book's content therefore deserves five stars. However, this ebook is technically faulty. The distributors obviously either scanned or collated it twisted 90 degrees counter clockwise, which makes it illegible. I hope that Amazon will approach the distributors to ensure mitigation. Amazon should have a proper control in place that ensures they only sell technically flawless ebooks.
Ich besitze mehrere antiquarische Ausgaben des Hausbuchs von Ferdinand Avenarius und war glcklich, dieses nun auch als Ebook erwerben zu knnen. Diese Lyrikanthologie verdient inhaltlich fnf Sterne, aber des Ebook ist technisch felherhaft. Die Seiten wurden entweder um 90 Grad verdreht gescannt oder digital gebunden. Es ist daher unlesbar. Ich hoffe, dass Amazon den Verlag auffordern wird, diesen Fehler zu beheben. Amazon sollte als Vertreiber ausserdem eine Endkontrolle auf technische Fehler durchfhren, um sicherzustellen, dass lediglich einwandfreie Ebooks verkauft werden. | What’s the best bang for buck Android tablet for simply reading giant PDF’s / scanned books? | PAPERBACK Publishing on Amazon problems [AUS] | when is the book of earthsea the complete illustrated edition released | Trying to get "O'Books" O'Reilly books downloader to work with no luck | eBook sources and conversion issues | Tor ebook club for September? | Terrible PDF support for students | ebook version does not meet Amazon format standards |
489 | Game is starting to lose its fun (assassin, level 23)... | Tried the sortie assassination six times today. Every single time we defeated the first phase the host would crash, we would migrate, and the game would reset with us unable to fight the next phase.
The least you could do DE is make sure the game is playable without game breaking bugs.
I don't care about how hard the mission is, that's half the fun of it - epic boss fights with coordinated team work. But having a game breaking bug like this which has been in the game for ages is absolutely ridiculous.
Fix host migration! New content is great, but ignoring massive issues and piling new stuff on top just makes the foundation weaker and eventually it's all going to come tumbling down. | Now I know for the most part, the people who comes to these forums are generally decent people who are looking to have a good time in game. We care enough about the franchise to discuss it outside of the game, and that's more than the average player is going to take it.
That being said, the community is what drives this game. With each release, there are issues. Yeah, you can blame the developers, but at a certain point you need to start blaming the players.
There's a lot of complaints about lag. Yeah. We're all having some issues. They're working on it. The game is a week old. Relax.
There's a lot of complaints about weapon balancing. Again, they'll sort it out. They always do. In the meantime, the guns are at everyone's disposal. If you're getting mowed down by the BAL and AK12 every game, equip it for yourself and fight fire with fire.
There are a lot of complaints about the spawn mechanics. I get it. You're dying because people are spawning behind you. But for every bullshit death you receive at the hands of a poor spawn, there is someone you're killing who is at the mercy of the game's spawn mechanics. Again, they'll tweak it if deemed necessary. In the meantime, learn how to avoid it. This game pushes a fast-paced gameplay. On the objective modes, the spawns are predictable. On TDM, it's a clusterfuck like it's always been. I don't know why you expected anything else.
The funny thing is I don't see many people complaining about the biggest problem that continues to plague this game; the community. Thankfully I'm on a next gen console and therefore don't have to listen to hundreds of angsty 16 year olds cuss me out every day. But for those of you who do, I feel for you. Now what about the people who think Domination is really just a mode to increase your K/D? Sitting behind home flag and getting 10 kills, 2 deaths every game sounds like fun, right? I just played an exciting game of DOM (TDM) on Riot where the enemy team failed to reach 30 points because they hung out back by A flag sniping or with thermal ARs. Was that the game's fault?
This game is fine. It's a whole hell of a lot more fun than Ghosts was, and we all played the hell out of that.
You can curse SHG for making a different type of CoD, but in the end, that's what we wanted. It's time for a little self reflection. We are the problem. | I played EQ2 way back at release, and have tinkered around a little bit recently on the free live servers. As I understand it, the TLP is a few expansions deep.. Is it still similar in style to the released version of the game? ie, Is there any challenge to the game? Is leveling a bit more of a process? Is grouping a core aspect of the game?
I can't stand playing the live server when everything is a cake walk and you just get chain fed kill quests. | Now this is the Diablo I wanted. When i first played Diablo 3 I quit after 20 levels because I hated the auction house, etc... With the new patch and this expansion pack Blizzard has fixed the issues gamers were complaining about. They earned my money on this one. | I'm giving this game a 4 because I love the game I love the story I love the characters I love the concept arts I love the unlockables I love everything about it except the difficulty in my opinion certain sections of this game are way way way too hard like this one level when your iron man and you have to fight a whole room of enemies and you cannot get hit and if you do you lose the level a lot of stuff like that in the game but otherwise amazing game | So. Can it please be fixed so I'm not forced to play with bad players and leavers for 30 levels?
Getting tired of games like these. | Can you please stop playing arty as it Ruins my enjoyment of the game | Ive been playing Killing Floor since it came out for PC, and it instantly became my favorite shoot 'em up game, so I was so fucking pumped to hear that they were making a sequel. Finally the game came out and I was instantly hooked again, and it felt so much better than the original. HOWEVER, I noticed that not a lot of people actually play for a long time and move past the game fast. I asked a couple of my friends why they stopped playing, and the answers were: "Stopped being fun.", "Same thing over and over." and the dumbest one "Doom is better." (I love Doom, but they are just two complete different games.) Sadly my old computer broke and I haven't gotten around to get a new one, so I've been playing my PS4 since. Its not hard to find a game, but it seems that not a lot of people still play Killing Floor, which is sad, especially when you see how much the developers put fine detail into this one. Maybe its because Im on console or haven't found the right people who play a lot like me, or its just because I haven't seen it covered all that much at all since its release. Do you think that Killing Floor is underappreciated or is it just me? | 489 | I can't exactly explain why, but this game is starting to lose its appeal for me somewhat. It seems like every new mission I go on (even one which was at my level and was "trivial") gets me killed and facing hoards of enemies I can barely stay alive against. One bad hit gets my shields down, a few more and my life is dead (and my shields and health have been priorities in terms of upgrading).
Yes, I'm playing solo, but I hope the fun factor of this game doesn't hinge entirely on that.
Any thoughts? Am I playing wrong? | I feel like my game is boring | How do you stop combat from becoming boring when you have 5+ players? | This game has the most infuriating enemy balance curve I have ever experienced. | Looking to play for the first time. How do people in the game feel about newbies? | Am I the only person who still really enjoys playing Clash Royale! | This game feels OVERWHELMING, help? | Riot, you are killing all the fun in this game | I'm new to MMORPGs. I have no idea what's going on or what I'm doing. I'm struggling to enjoy the game. Help me? |
490 | In vitro activity of cefodizime (HR-221). | A mixture of cerebrosides, called poke-weed cerebrosides, was purified from Phytolaccae Radix (Phytolaccaceae) and characterized as 1-O-β-D-glucopyranosides of phytosphingosine type ceramides comprised of a common long chain base (2S, 3S, 4R, 8Z)-2-amino-8-octadecene-1, 3, 4-triol and fatty acids. The fatty acyl chain of ceramide moieties was determined as (2R)-2-hydroxypentacosanoic acid, (2R)-2-hydroxylignoceric acid, (2R)-2-hydroxytricosanoic acid, (2R)-2-hydroxybehenic acid, (2R)-2-hydroxypalmitic acid, and palmitic acid. The poke-weed cerebroside inhibited the cyclooxygenase-2 dependent phase of prostaglandin D2 generation in bone marrow-derived mast cells in a concentration dependent manner with an IC50 of 6.2 μg/ml. | The immediate and delayed toxicity of Cerbera odollam leaf extract was studied in mice. Under the experimental conditions adopted, using macroscopic and microscopic examinations, Cerbera odollam leaves appeared to be relatively devoid of the marked toxicity found in seeds, a common source of poisoning. At doses smaller than the maximal dose never lethal (14.5 gkg i.p.), the leaf extract decreased mice spontaneous motor activity significantly, increased the reaction time to a thermal stimulus, reduced the duration of pentylenetetrazole-induced tonic seizures and mortality, and potentiated sodium pentobarbital-generated hypnotic effects. | Cefradine Cefradine (INN) (formerly cephradine BAN) is a first generation cephalosporin antibiotic. Cefradine has similar spectrum of activity to cefalexin. It is used in the following instances: Cefradine is distributed in the form of capsules containing 250 mg or 500 mg, as a syrup containing 250 mg/5 ml, or in vials for injection containing 500 mg or 1 g. The antibiotic is produced under many brand names across the world. Cefradine is known as Cefradina in Portuguese and Spanish and is produced by the following companies under this name: AC Farma, Peru; Andromaco, Chile; Anglopharma, Colombia; AZ Pharma, Colombia; Biogalenic, | Pharmacokinetics of ceftizoxime | Cefapirin (INN, also spelled cephapirin) is an injectable, first-generation cephalosporin antibiotic. It is marketed under the trade name Cefadyl. Production for use in humans has been discontinued in the United States.
It also has a role in veterinary medicine as Metricure, an intrauterine preparation, and combined with prednisolone in Mastiplan, an intramammary preparation. Both are licensed in cattle.
Synthesis
In one of the syntheses, 7-aminocephalosporanic acid (7-ACA) is reacted with bromoacetyl chloride to give the amide. The halo group is then displaced by 4-thiopyridine.
References
Cephalosporin antibiotics
Enantiopure drugs
Pyridines
Acetate esters | Two Cecropia species (Cecropia obtusifolia and C. peltata), known as guarumbo, are employed in Mexican traditional medicine to treat diabetes mellitus; the leaves of both species contain phenolic bioactive compounds such as chlorogenic acid (CA) and isoorientine (ISO), which have been attributed with hypoglycemic, hypolipidemic, and antioxidant properties. An in vitro propagation protocol was developed from existing apical bud meristem from C. obtusifolia seedlings; the shoot generation was induced on Murashige and Skoog (MS) medium supplemented with varying concentrations of 6-benzylaminopurine and kinetin (Kn) combined with either α-naphtalene acetic acid (NAA) or indole-3-acetic acid (IAA) auxins. Best morphogenetic response was developed with Kn 26.64 μM combined with either NAA or IAA 0.57 μM, respectively; likewise, C. peltata-seedling apical buds were subjected to these best selected treatments. Cecropia obtusifolia and C. peltata shoots were rooted in growth regulator-free half-strength MS medium, and regenerated whole plants were adapted successfully under greenhouse conditions and field. Leaves from both Cecropia-micropropagated plants produced the phenolic compounds CA and ISO, with highest concentrations in leaves from 18-month C. obtusifolia (12.28 ± 7.06 mg g−1 dry leaves of CA and 8.30 ± 2.70 mg g−1 dry leaves of ISO) growth in the field. Our results offer a protocol of apical-bud use for multiplication and curative-property conservation of the two previously mentioned important Mexican medicinal plants. | SUMMARY Mitochondrial respiration can be impaired in vitro by a toxic complex, termed succinic oxidase factor (s.o.f.), produced by 70 yo of coagulasepositive staphylococci. The oxidation of succinate is most sensitive, cytochrome oxidase less so while succinic dehydrogenase is resistant. The complex consists of at least two components, differing in degree of heat sensitivity. The more heat-resistant component impairs electron transfer in the region of ubiquinone (ubiquinone can reverse the impairment), while the other component impairs electron transfer in the region of cytochrome c (cytochrome c can reverse the impairment). It is thought that the components of s.0.f. may be of enzymic nature, acting on the phospholipids responsible for the integrity of the electron transport chain. | The mechanism of action of cerumenolytics vary by the classification. Water-based cerumenolytics, including water itself, work by hydrating ear wax, fragmenting corneocytes (a type of skin cell) within the ear wax itself. Cerumenolytics with peroxides release oxygen upon contact with the skin, inducing effervescence (bubbling) that mechanically fragments ear wax. Oil-based cerumenolytics provide lubrication to the ear wax, softening the surface without fragmenting the ear wax. The mechanism of action of non-water- and non-oil-based cerumenolytics is unknown. Using carbamide peroxide as an example, the pharmacokinetics of cerumenolytics are not well studied. Cerumenolytics are used to treat cerumen impaction in cats | 490 | The in vitro activity of cefodizime (HR-221), a new cephalosporin antibiotic, was compared with the activities of selected antimicrobial agents against a broad spectrum of aerobic bacteria. Cefodizime concentrations of 2 micrograms/ml inhibited about 90% of Enterobacteriaceae studied. Serratia marcescens required 8 micrograms/ml to inhibit 90% of strains. Among gram-positive cocci, 50% of strains were inhibited by 2 micrograms/ml of cefodizime (including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, and penicillin-resistant Streptococcus pneumoniae). Pseudomonas aeruginosa was less susceptible to cefodizime. Cefotaxime, an antibiotic closely related to cefodizime structurally, was about fourfold more active. | The in vitro activity of a new parenteral cephalosporin cefepime (BMY 28142) was compared with that of ceftazidime, cefotaxime, piperacillin, imipenem, gentamicin, amikacin and ciprofloxacin against 173 recent multiresistantPseudomonas aeruginosa isolates of nosocomial origin using an agar dilution technique with an inoculum of 104 CFU per spot. The activity of cefepime was comparable to that of ceftazidime, superior to that of cefotaxime, piperacillin, gentamicin and amikacin, but inferior to that of imipenem and ciprofloxacin. Cross-resistance ofPseudomonas aeruginosa to ceftazidime and cefepime occurred in nearly 50% of the cefepime resistant strains and 61.5% of the ceftazidime resistant strains respectively. | In vitro activity of cefoperazone-sulbactam combinations against cefoperazone-resistant clinical bacterial isolates | The treatment of systemic infections, especially meningitis, caused by Streptococcus pneumoniae nonsusceptible to third-generation cephalosporins, is extremely difficult due to the paucity of therapeutic options. The main objective of this study was to characterize isolates of S. pneumoniae with reduced susceptibility to cefotaxime (MICs, ≥1 μg/ml) by different typing methods and to evaluate whether clonal dissemination of this pathogen had occurred among Latin American medical centers. A total of 46 isolates collected from respiratory tract specimens, blood cultures, cerebrospinal fluid, eye, and other sources were analyzed. The isolates were collected from Latin American medical centers located in Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay through two multicenter surveillance programs, in 1997 and 1998. Isolates were serotyped and molecular typed by pulsed-field gel electrophoresis (PFGE) and automated ribotyping. Antimicrobial susceptibilities were determined to 19 drugs by reference broth... | Background: Serratia marcescens is an important nosocomial pathogen and the characteristic property of resistance conferred by extended-spectrum beta-lactamase or a novel AmpC cephalosporinase was not unusual in Taiwan. This study investigated the trends in antimicrobial resistance in S. marcescens from a nationwide surveillance in Taiwan. Materials and methods: S. marcescens isolates were collected biennially between 2002 and 2010 from medical centers and regional hospitals throughout Taiwan, as part of the Taiwan Surveillance of Antimicrobial Resistance program. Minimal inhibitory concentrations were determined by the Clinical and Laboratory Standards Institute reference broth microdilution method. Results: A total of 403 nonduplicate S. marcescens isolates were collected, mostly from respiratory samples (157, 39.0%), followed by the urinary tract samples (90, 22.3%). Overall, 99.3% isolates were susceptible to imipenem, 93.8% to ceftazidime, 89.2% to minocycline, 87.8% to amikacin, 86.8% to cefepime, 82.9% to aztreonam, 73.2% to ceftriaxone, 72.7% to levofloxacin, 63.8% to ciprofloxacin, 60.8% to trimethoprim/sulfamethoxazole (TMP/SMX), and 59.6% to gentamicin. A significantly increased susceptibility rate after 2004 was observed for the following antibiotics: amikacin (73.8% vs. 97.1%), gentamicin (40.0% vs. 72.4%), ciprofloxacin Journal of Microbiology, Immunology and Infection (2014) 47, 387e393 (53.8% vs. 70.4%), ceftriaxone (53.8% vs. 86.0%), cefepime (74.4% vs. 95.1%), aztreonam (72.5% vs. 89.7%), and TMP/SMX (41.3% vs. 73.7%). Conclusion: In this 8-year study, the susceptibility of S. marcescens to ceftazidime and imipenem remained consistently high in Taiwan. S. marcescens isolates demonstrated relatively higher resistance to ciprofloxacin and levofloxacin, and therefore continued surveillance of antimicrobial resistance, especially for fluoroquinolone, is warranted. | Inhibitory and bactericidal activity of cefpirome and cefotaxime against blood culture isolates. | Transfer, by conjugation and transduction, of resistance to ceftazidime and cefotaxime from the same clinical isolate of Pseudomonas aeruginosa. | Abstract ::: HR810 (Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.) is a new, cyclical-pyridinium cephalosporin that appeared superior to numerous comparison drugs against 658 strains of aerobic and facultative anaerobic bacteria. Seventeen Enterobacteriaceae spp. were tested by broth microdilution methods, and the 50% MICs (MIC50S) and 90% MICs (MIC90s) were 0.03 to 0.12 and 0.03 to 2.0 micrograms/ml, respectively. Only one strain had an MIC greater than 8.0 micrograms/ml (99.6% is considered susceptible). HR810 inhibited 98% of Pseudomonas aeruginosa isolates at less than or equal to 16 micrograms/ml, and the MIC90 for Acinetobacter spp. was 4.0 micrograms/ml. It was also very active against Pseudomonas spp. and Staphylococcus aureus (MIC90, 0.5 micrograms/ml) but marginally active against methicillin-resistant staphylococcal strains (MIC90, 16 micrograms/ml) and enterococcus (MIC90, 32 micrograms/ml). Non-enterococcal streptococci had MIC50s ranging from 0.008 micrograms/ml for Streptococcus pyogenes to 0.12 micrograms/ml for pneumococci. All MICs of HR810 against Haemophilus and Neisseria spp. were less than or equal to 0.03 micrograms/ml (MIC50, 0.002 to 0.008 micrograms/ml). HR810 poorly inhibited beta-lactamases and was very stable against 11 tested beta-lactamases of plasmid (TEM, OXA, SHV-1, and PSE) and chromosomal (K1, K14, P99) types. | The comparative activity of cefepime and other current antibiotics against microorganisms isolated from patients in pediatric intensive therapy units |
491 | Daily Giveaway #833 - 10x Random Steam Keys | Hi GOG'ers. I've got a spare Fortune-499 key lying around, which is probably much better off with one of you than it is laying in my library unredeemed. Want to be a part of the giveaway? Post a comment including the Steam ID. To give all commenters an even chance, I'll use a random comment picker. Comment will be picked on the 19th of December, 11 PM CET. Good luck! :)
&#x200B;
EDIT: Winner is Naum21! PM Incoming, congratulations! ( | What you have to do to enter the giveaway:
* Choose a number between 1 and 1000
* Tell me which prize do you want
* Link your Steam ID
Prizes:
* 1 20% discount on OlliOlli
* 2 25% discounts on Pixel Piracy
* ~~1 Counter Strike Source Guest Pass~~ **Gone**
* ~~1 Dead Island Epidemic~~ **Gone**
* 1 Dell code for $15 off a purchase of $50 or more on electronics or accessories
Winners will be chosen with random.org an announced in 24 hours from the time of posting.
Good Luck everybody!
EDIT: Formatting
EDIT 2: Giveaway is over! But some prizes are left to be claimed, just PM me if you want any. | Hi again!
Got another two lucky random steam keys to giveaway to anyone that might want them. Post a number between 1-500 and state which game you want, first number that comes up wins the game stated, second number that comes up wins the other game. I'll use an RNG for the draw. Winners will find out tomorrow evening (+1 GMT) if they've won.
Can only enter for one game.
Both winnings are, as mentioned in the title, in the form of steam keys from the excellent Lucky Random Steam Keys site. Just not something I'm interested in :)
Games are:
1) Two Digits by CleverWeek
2) Fireflies by Bugendai
Good Luck!
Winners have been notified, Thanks. Another in a month or so! | So a friend was kind enough to give me a key, but unfortunately, I already own Saints Row 3..so I decided I would giveaway the key here! :) All you have to do is put your Steam ID and the reason why Saints Row 3 is a game you'd like to play. I'll choose a random winner 12-24-ish hours from now. :)
Good luck people! :D
**Edit:** Seems like this giveaway will end earlier than expected. I'm having internet problems and won't be around for awhile. Hopefully not. :(
And the random number generator (which took a few minutes coz fml) has chosen /u/ChimBlade as the lucky winner! Will send the PM in a minute.
**Edit 2:** Sent! Thanks to all who participated in my first giveaway! (Since the last one had no participants, I guess it doesn't count) | Welcome to 60 Days, 60 Games. I have 60 Steam keys for games mostly leftover from various Humble Bundles, and it's time to get rid of them. I'm giving away one game per day for 60 games total. The games are roughly ordered by their regular price on Steam, so the game values will get more expensive as time goes on.
Since some of these keys are quite old, I make no claims that the code I'm giving you will actually work. I'll be pressing the "Click to redeem on Steam" button right before sending the resulting code to the winner, but I've had problems with that in the past, people have reported back that their code didn't work. If a code doesn't work, well, there's always tomorrow.
Today's game is:
#Super Meat Boy
Post your favorite meme, tweet, or video about meat to win. | Welcome to 60 Days, 60 Games. I have 60 Steam keys for games mostly leftover from various Humble Bundles, and it's time to get rid of them. I'm giving away one game per day for 60 games total. The games are roughly ordered by their regular price on Steam, so the game values will get more expensive as time goes on.
Since some of these keys are quite old, I make no claims that the code I'm giving you will actually work. I'll be pressing the "Click to redeem on Steam" button right before sending the resulting code to the winner, but I've had problems with that in the past, people have reported back that their code didn't work. If a code doesn't work, well, there's always tomorrow.
Today's game is:
#Turmoil
Post your favorite meme, tweet, or video about dirt or oil to win. | So I got 2 steam keys and while redeeming it i found that i own the game, most probably both of the games are indie but will be having cards (if you are into them)
For entering just guess my favorite number between 1-250. There will be 2 winners,so good luck. I'll pm the winners and edit here for everyone to know.
Edit1: Now there is 1 more key!
Edit 2 : F me 1 more now!
Edit 3 : If this post gets 1k i'll tell everyone how to get the steam keys for free easily with guaranteed trading cards , DON'T message me begging for keys or for the exploit!
Still giving keys sent a little bit more and now having even more XD.
edit again : Lost count of how many sent XD!
Number bigger than 250-500 are allowed now!
I am bored now! so will make another post so that people can apply again! also let me grab some keys first. | Hi guys,
I purchased two Steam keys for Rust by accident, I only actually needed one. As such, I have one spare so thought I'd give it away.
I don't really know how people usually go about giveaways so I'll just ask that if you're interested, just comment below and I'll at random scroll and stop with my finger on someone 24 hours from now to allow time for people to get involved.
I'll edit and update the post with the winner's name (if this is the done thing?) and PM the code in 24 hours.
EDIT:
It's been 24 hours. Thanks to everyone who posted, I was surprised to see so many people interested and based on that, I'll be doing some future giveaways with more this month in the spirit of Christmas and giving to others.
Anyway, without further ado, the winner is... Dawnbreaker107 | 491 | This giveaway is over! Here are the 10 lucky winners:
/u/ravencroft135
/u/ashwin1927
/u/-AJDJ-
/u/hakeem1279
/u/yaromirro
/u/willie012328
/u/TrueGamer7
/u/WildZeebra
/u/NachoDraws
/u/Dani3BR
The next giveaway will start soon, stay tuned! | I won a giveaway but received nothing. | Only one day left. Join the contest of Ugotvape First Giveaway! Kanger Aerotank & Vision Spinner 2 | just started out, missed the 10k giveaway, any more comming? | Last chance to join our giveaway! | Giveaway for the active kind redditors here! (Stars, Bells and furniture!) | My First Giveaway! Shiny Diancie!! (Additional Wonder Trades!!) | Hi Hi! (1000 member giveaway) | Holy shit I won an Exchange sweepstakes! |
492 | Current source LCC resonant converter for an EDM power supply | The paper presents and validates a straightforward design methodology for realising LCC current-output resonant converters, with the aim of reducing tank currents, and hence, electrical stresses on resonant components. The scheme is ideally suited for inclusion in a rapid iterative design environment e.g. part of a graphical user interface | LCLL resonant step-down/up converters | Characteristics and Design of an Asymmetrical Duty-Cycle-Controlled LCL-T Resonant Converter | Operation and characteristics of resonant converters on the utility line are presented. Series-parallel (LCC-type) resonant converter operating with discontinuous current mode and continuous current mode (variable frequency control as well as fixed-frequency) are considered. Design examples are presented. SPICE simulation and experimental results obtained for the designed converters (rated at 150 W) are presented to verify the theory. It is shown that high line power factor (>0.95) and line current total harmonic distortion (THD) of <25% are obtained for the LCC-type converter for a wide load range (from full load to 10% rated load) without any active control, and the switch peak current decreases with the load current. With active line current control, low distortion and zero voltage switching for the entire cycle are realized. | Quasi-Resonant Converters-Topologies and Characteristics | A method for improving output current of series-resonant AC link cycloconverter | The transformer winding capacitance, which is significant in high-voltage power supplies, is not gainfully utilized in an LCL-T resonant converter (RC). A simplified analysis presented in this paper predicts the severe degradation of output current regulation of an LCL-T RC due to transformer winding capacitance. The presence of winding capacitance, in fact, changes the third-order LCL-T resonant tank into fourth-order LC-LC topology. Using an AC analysis, it is shown that, under the derived design conditions, LC-LC RC also exhibits constant output current and in-phase source voltage and current, simultaneously at all loading conditions. Thus, the transformer leakage inductance and winding capacitance are gainfully utilized as a part of a resonant network, resulting in improved output characteristics. Closed-form expressions for the converter gain and component stresses are derived. The condition for converter design optimized for the minimum size of the resonant network is obtained. Experimental results on a prototype 100-mA 2-kV DC power supply confirm the observations of analysis. | High Efficiency Resonant DC/DC Converter Utilizing a Resistance Compression Network | 492 | In this paper, the design of a low size power supply prototype for electrical discharge machining is presented. The system is a DC to DC LCC resonant converter whose switching frequency is tuned at the natural resonant frequency where the converter tends to act as a current source. In this way, two effects are achieved: (1) the necessary over-voltage, first to ionize the dielectric and then to establish the electric arc is generated, and (2) a constant current is supplied during the erosion of the workpiece, providing the circuit with inherent protection under short circuit conditions. The output voltage is intended to be adjusted by an external system that controls the arc distance. | The micro-discharge tool setting method is to apply a certain voltage between the tool and the tool set. When the distance between the tool and the tool set is less than the discharge gap, the medium between the tool and the tool set discharges and the voltage decreases, monitoring the voltage signal and recording the machine coordinates when the voltage changes times to complete the tool setting process. In this paper, the influence of the times and duration of the discharge on the tool clearance and the shape of the tool nose during the micro-discharge tool setting process is studied. The research shows that the tool clearance is related to the discharge duration. When the duration of the discharge is less than 4s, the tool clearance becomes smaller with the increase of the times of tool setting process, and the governing tool nose damage is breakage by the discharge current impact. When the discharge duration is greater than or equal to 4s, the tool clearance becomes larger as the number of times of tool setting process increases. The burr that grows at the nose of the tool changes the nose shape. The above research provides support for the subsequent micro-discharge tool setting tool to achieve high-precision non-destructive tool setting process and optimize tool setting parameters. | Electrical discharge machining with ultralow discharge energy | THE NEW DEVLOPMENT OF ELECTRIC DISCHARGE MACHINING TECHNIQUE | We present here a new design of ion blower based on bipolar corona discharge of needle-dielectric-needle configuration. With the help of the dielectric plate, the new ion blower can endure higher voltage before spark than the traditional bipolar corona discharge, with the similar discharge characteristics and current-voltage curve. The high operating voltage provides a much greater ionic wind velocity compared with the traditional needle-to-needle corona. The neutral ionic wind can reach as far as 0.45m with a velocity of 0.2m/s. This feature is very helpful for eliminating mechanical rotating components in the design of ion blowers, so that the size of ion blower can be greatly compressed and the noise significantly reduced, which is more suitable for electrostatic elimination in small areas and some specific scenarios. The design of self-balancing regulation unit can effectively monitor the charged characteristics of mixed ion wind and make corresponding adjustments to ensure the neutrality of positive and negative discharge. This new ion blower has an excellent performance, including an offset voltage of ±5V, a short discharge time of 5s or less at distance of 20cm. | Vacuum circuit breakers are considered an attractive solution to climate change concerns regarding SF 6 gas. From the beginning of vacuum circuit breaker, it was known that vacuum has the high arc extinguishing ability and insulation performances. At the time of the electrode, spiral electrode on contrate electrode is used, The contact materials were Bi on CuCr. There was a problem that was not able to be intercepted when the breaking current became 40 kA or more as the development of vacuum circuit breakers advanced. In the 1970s, vacuum circuit breaker was successfully interrupting from 12kV–200kA by the development of axial magnetic field (AMF) electrode. Research on AFM electrodes was carried out in laboratory. | Application of permanent magnet vacuum switch in low-voltage reactive power compensation installation | A low cost partial discharge measuring system for a high voltage laboratory | Considerations for installing and applying arc resistant low and medium voltage control equipment in forest products industries |
493 | Where do I find eSports VODs? | As a new guy to Starcraft where is there a site for starcraft vods. particularly so I can watch Proleague. For league of legends there is /r/loleventvods. Is there a starcraft equivalent? | I know AfreecaTV has VODs for GSL on Youtube, and there are some old WCS Challenger videos on Youtube, but is there any place to watch VODs for the current WCS Challenger? | Hey guys!
Someone suggested in another thread that we should have a youtube channel that has vods from tournaments so that it would be easy for people to search and find gameplay in one place. I have created a youtube channel called Rivals of Aether Vods that will have some games from the tournaments that have been streamed or sent in. I ask the streamer before using their content also.
If you guys have any vods that you would like to add from a tournament or have any suggestions for the channel, then you can link me to a stream's past broadcasts through steam, reddit message, or the email for the channel at [email protected].
The only vods I have currently are from Kenneth's NCS run last night but will be looking for more to expand the content. | I just bought a twitch sub to dropoutlive so i could watch fantasy high season 2 but i cant find the vods and i dont know if im just stupid and dont know how it works please help | Is there a VOD of the Korean team participating in CoD Champs 2013? I want to laugh. | there was a game where fnatic played Carry Alchemist and i wanted to ask you whether you got the link to the vod. | Hey all, been trying to review VODs as a team exercise the last couple weeks. Originally I was planning to just go over a VOD and record it myself, but would prefer to do in real time with my team right after scrims are over.
We've tried doing it via stream on twitch and talking about it in discord, but it got awkward when people would hear themselves on stream. Or muting the stream, but then they cant hear the in-game sounds. (There might be something here I'm not thinking of to avoid all that).
We tried rabb.it, but I think overwatch has a lot of video data with the colours and fast paced action as I can only stream that on 360p via youtube or twitch vod.
Any other recommendations for programs or anything? | Already subscribed to one provider but looking at a second must have a good range of sports channels and a good range of vod
pm me with details | 493 | Every time I've really tried watching HOTS pros it has been because some kind soul took the time to post here with convenient links to the VODs of all the games.
How can I find these videos on my own? Is there a convenient source that has them all in one place or do I need to search through the various different twitch/youtube channels regularly? | Having trouble finding videos of the LCS matches (not live) | Anyone know where I can find game vods without getting spoilers? | Where can I find all videos from VimConf 2020 | Find clips from your favorite channels | Where can I find videos featuring Ashens that are not on his own channel? | I want to make motiavtional videos on youtube, but where do I find videoclips? | Tracking Audios & Videos for Free :) | Where can I find dota meme videos? |
494 | who directed the closing ceremony of the 2014 winter olympics | 2014 Winter Olympics closing ceremony blown out by the Polar Bear, who shed a tear after the flame went out – an homage to Misha and the 1980 Summer Olympics closing ceremony. A remix of Aleksandra Pakhmutova's "Goodbye, Moscow" played and the Polar Bear extinguished the Olympic flame. The 1,000 members of the Pan-Russian Children's Choir assembled with the mascots carrying small flames in their hands and Abkazian-Russian soprano Hibla Gerzmava sung "Goodbye, Sochi!" with the children's choir to close the games. The ceremony ended with a fireworks display set to the music of Tchaikovsky. Russian DJ Kto hosted the afterparty. 2014 Winter Olympics closing | 1968 Winter Olympics display team, flew over the stadium and marked out the colours of the Olympic rings with their vapour trails in the sky. The Winter Olympics ended on 18 February, on a Sunday evening, with the closing ceremony in the "Stade de glace". The first highlight showed the figure skaters putting on an exhibition skating session. It also included ice dancing, an event that was first introduced into the main programme in 1976. The best ten partners from the last World championship took part in the event and there was no scores. After that the last award ceremonies then took place. | United States at the 2014 Winter Olympics President Barack Obama and Vice President Joe Biden did not attend the 2014 Winter Olympics. American nordic combined skier Todd Lodwick was the flag bearer of Team USA for the Parade of Nations during the opening ceremony. Four-time ice hockey Olympian Julie Chu was the flag bearer during the closing ceremonies. The results may be amended due to the Russian doping scandal. The United States has qualified a total quota of twenty athletes in alpine skiing. The full list of the U.S. alpine skiing team was officially announced on January 27, 2014. Based on their performance at the 2012 and | 2014 Commonwealth Games opening ceremony The opening ceremony for the 2014 Commonwealth Games was held at Celtic Park in Glasgow, Scotland, between 21:00 and 23:40 BST, on 23 July 2014. The ceremony was directed by David Zolkwer and included the 2014 Commonwealth Games Parade of Nations where 71 athletes, bearing the flags of their respective nations and territories, led their national delegations as they paraded into the stadium. The games were formally opened by Her Majesty Queen Elizabeth II. She referred to the Commonwealth's "shared ideals and ambitions" and the "bonds that unite" its members. The programme, which included about | 2014 Winter Paralympics opening ceremony song based on the poetry of Mikhail Lermontov while riding an ice-breaking ship named "Mir" (Peace). During his speech at the opening ceremony International Paralympic Committee president Philip Craven noted that Russia had declined to host the 1980 Paralympics in conjunction with the 1980 Summer Olympics, making this the first time Russia has hosted the games. He called on spectators to have "barrier-free mind" just as "the city of Sochi has built a barrier-free environment for athletes and officials to enjoy". Russian President Vladimir Putin declared the games open, drawing loud cheers. Russian Paralympians Olesya Vladykina and Sergey Shilov shared | Bermuda at the 2014 Winter Olympics the 1980 Summer Olympics in Moscow. The only medal the territory has won so far is a bronze in the sport of boxing at the 1976 Summer Olympics. The Bermudian delegation consisted of one cross-country skier, Tucker Murphy. This was Murphy's second consecutive Olympics as Bermuda's only representative. Despite the terrorist threats on the Sochi Olympics, there were no plans to withdraw unless Murphy felt unsafe, and the delegation ultimately competed as scheduled. The delegation marched in the opening ceremony with their traditional Bermuda shorts. Murphy was selected as the flag bearer for the opening ceremony, while a ceremony volunteer | When are the 2008 olympic closing ceremonies? | of Beijing, Chen Jining. 2018 Winter Paralympics closing ceremony The 2018 Winter Paralympics closing ceremony was held at Pyeongchang Olympic Stadium in Pyeongchang, South Korea, on March 18, 2018. The flag bearers from each participating country entered the stadium informally in single file, ordered by "ganada" order of the Korean alphabet, and behind them marched all the athletes, without any distinction or grouping by nationality. The IPC President Andrew Parsons paid tribute to the late Stephen Hawking in his closing speech. The flag was passed by the mayor of Pyeongchang, Shim Jae-kook, to IPC President, Andrew Parsons, who then handed | 494 | 2014 Winter Olympics closing ceremony The closing ceremony of the 2014 Winter Olympics was held on 23 February 2014 from 20:14 to 22:25 MSK () at the Fisht Olympic Stadium in Sochi, Russia. It was designed to show Russian culture, through a European perspective, and featured performances by Yuri Bashmet, Valery Gergiev, Denis Matsuev, Hibla Gerzmava, and Tatiana Samouil, among others. The closing program presented "Reflections of Russia"; that is, highlights of Russian culture, presented through a European perspective. It was directed by Daniele Finzi Pasca. Konstantin Ernst served as creative director and Andrei Nasonovskiy was the executive producer. Throughout | what animal lit the flame at the closing ceremony of the 2014 winter olympics | how many people watched the 2014 winter olympic games | where did the closing ceremony of the 1968 winter olympics take place | what tv network aired the 2014 winter olympic games | who did n 't finish slalom in 2014 olympics | what is the official slogan of the 2014 winter olympics | 2010 olympics when is the closing ceremony? | how many athletes did croatia have in 2014 winter olympics |
495 | Hot take: What are everyone’s opinions on Christopher Bell? | JOHN W. TUKEY: HIS LIFE AND PROFESSIONAL CONTRIBUTIONS 1 | I love the wikis and YouTube videos dedicated to him and solely him, are there others as documented and interesting as Chris? | Bryan Cranston once again is amazing. Another sad blight on the face of America and our rush to judgement of others. While the focus is about name calling him a communist it fits right in with the current political atmosphere, anger and finger pointing. The desire to black ball anyone who may think differently than we do, including among our own peers. Amazing piece of work about a pretty fascinating man that I had never heard of and whose movies I grew up loving! | So recently, the Youtuber Shaun has made a video disputing The Bell Curve (1994) - which is odd since most HBD people don't cite TBC too much:
I wrote an article a while back that responded to some critiques Shaun brought up indirectly, but not every argument was responded to since Shaun was mostly focusing on TBC and I wasn't. My article is here:
I'm surprised no mention was made of the works of Jensen, Levin, Carrol, Gottfredson, Sesardic etc, especially since they contributed a lot when it comes to factor analysis, intelligence testing, the genetics of intelligence, within-group heritability and between-group heritability and responses to the environmentalist case. (Thanks to u/TrannyPornO and u/BasementInhabitant for their thread on heritability as I relied on it a lot.) | Great Rob Thomas, again. What can I say except he is at his best, again. | When the "tough guy" character was first introduced I was like -Eye roll...here we go again. Yawn.
However, he does such an amazing job of acting and his story is so rich that I can see why he is the way he is. It's deep.
So refreshing to not have the standard cookie cutter tropes!! | Gives a little background to the T.V. personality but nothing indepth. I bought this because I was curious.
Didn't satisfy me too much as a book. But it did give a look into Derek's background and ability.
I would recommend this only to the true Derek Acorah fan. | Who don't love this man ?every man and a woman in this planet admired him,this book make me to understand him,show us he is very smart ,he make his way to USA and start making movies with very famous people,and he ended to be a big star and to be loved from everyone,he keeps his life privacy with no scandals,that is a man ,I wish the young generation who wants to be famous read this book,he can be you teacher,they think more scandals they have people have something to talk about them,that is sad , Sean is one of the kind. Thelma | 495 | It seemed as if the handful of fans last night were pretty silent during the S/F line interview. Do people not like Christopher Bell? Is it because he wins too much? On the flip side, because of Christopher Bell’s success, how far can he go in his career? Will he be the next Kyle Busch, or better? Any C-Bell fans out there? | Ted Bell is consistent. | Any worry about James Connor’s role in Pittsburgh? | Fun for Saved by the Bell fan | who signed bell and james to a full album deal | We should be talking more about Connor's MVP like numbers instead of an absentee Bell. | where did chris bell record his first album | how much did the city of bell workers get | should I switch back to bell? |
496 | [F4A] Overprotective older sibling | Soooo I know for sure that I put a brother and sister in a living quarter and then the sister came out pregnant and when the baby was born the brother was the dad... there was no one else in the room with them and I even double checked to make sure they shared the same parents or mom or dad and they do so I’m just very confused on how this even unfolded? I thought this couldn’t happen?? LOL. Yikes.
Has this happened to anyone else before?? I’m like 1000% sure these were siblings because I documented their birth for this exact reason. And if they aren’t siblings then they’re still family in some way and I didn’t want inbreeding to happen and this is where I wish we had a family tree feature in the game that could prevent this from happening and save me a headache lol but oh well.
I’m just trying to figure out if this is a mishap on my part or those were in fact siblings and I’m wondering if this has happened to anyone else. | Sister of My Heart turned out as she expected. Anju and Sudha exchange regular letters and short phone calls, but their old intimacy is missing. The friends discover they are pregnant at the same time and both seem finally happy. Sudha's mother-in-law finds out that Sudha's child is a girl. She demands Sudha abort the baby, believing the first child should be a son. Sudha has nowhere to turn, leaving her husband would be grounds to talk to each other again as true sisters. Refusing to tie her life to another man and realizing Anju needs her, Sudha and her daughter decide to go | My mom (F/45) and younger brother (M/16) are extremely co-dependent on to each other. To summarize things, their relationship is eerily similar to that of Norman and Norma Bates. He's an extremely intelligent child which only makes things that much worse. He's angry, manipulative, aggressive, and has my mom wrapped around his finger. He's verbally and mentally abusive towards my mother. She understands that something isn't right but continues to defend him and make excuses to everything he does because "he's her baby". This is harmful to the both of them and needs to stop, what can I do to help them? | My brother's daughter is my what? | Hi guys. My mom [43F] has been dating this guy [40sM] for about 5 months now, and I recently found out that my sister [14F] is dating his son [17M]. I'm not sure how long they have been dating, but it has to have been at least 2 months. Besides the age difference (which I think is a bit too large) I don't have any problem with the relationship itself. He seems to make my little sister happy, and I never hear about them arguing or anything. The fact that they could be brother and sister and some point, though, is really creeping me out. However, I'm the only one who feels this way. Neither of the parents think its creepy, nor does my sister's best friend or my other sister. However, from what I can tell, they haven't told anyone else (friends, family, etc), and the only other person who knows about it is my aunt. Am I just overreacting to the whole thing?
tl;dr: My little sister [14F] is dating our potential step-brother [17M]. I find it creepy. Am I overreacting?
Edit: The general consensus seems to be that I have nothing to worry about, so I'll just leave it be for now. Thanks guys, I appreciate it. | Six cases of familial fibrocystic pulmonary dysplasia are described involving five siblings and their father. The clinical findings and radiological features were similar in all six patients although there was some variation in the period of survival following the onset of the disease. In three the diagnosis was confirmed pathologically; the two brothers, who did not have lung biopsies, had disturbances in respiratory function which are considered typical of the impaired diffusion produced by interstitial fibrosis. One hundred and five members of the family were surveyed for evidence of this disease, but no further cases were discovered. Four of the patients had some elevation of their gamma globulin. Immunoelectrophoretic analysis, which was performed on three of the patients, the two healthy siblings, and 16 of their offspring, showed elevated immunoglobulin patterns. This evidence suggests the possibility of an inherited aberration in the immune response in this family. | Older sister here. I don't get along with my younger sister and never have. Not for any particular reason other than clashing personalities. But both she and my parents have the ridiculous idea that siblings are automatically like "built in bffs" and I literally base my preference for having only one kid off of not wanting to make a kid deal with a sibling that won't leave them alone. Though I guess I'd be SOL if they turn out super extroverted or would actually fancy the idea of being a mentor to a young child during their "monkey see monkey do" phase (yes I resent having to be responsible for a toddler wanting to copy everything I did as a kid and not being able to get away with normal kid stuff because of it) | I'm SAHP with 2 kids to take care. The elder one is 8 yr who has goes to school already. The younger one is 17 months old. I spend more time taking care the younger one, and we allowed the old brother to have a phone in case he needs to contact us for help. Here comes the problem. The elder one now has his own WhatsApp account and often chats on WhatsApp. When I ask what he is chatting, he just smiles and says nothing. I may have been separated too long from the outside world. Is any suggestion for this? Should I try some WhatsApp spy app to protect my boy? | 496 | *I am an adult looking for another adult for a fantasy roleplay.*
Greetings, APP!
I'd like to play a beautiful younger girl who is growing up far too quickly for her older sibling's liking. Perhaps I'm getting attention from a selfish older sister's male friends (or boyfriend) or and older brother's best friends keep calling me *hot.* You don't like all the positive attention I'm getting and decide to do something about it.
There are more extreme and more relaxed ways to do this, either of which I'm okay with:
Relaxed: You decide to teach me about my body and its development with a hands-on lesson. You train me to be the perfect sexual subject for you.
Extreme: You hate all the attention I'm getting and rape me. You could put a chastity belt on me afterward, too, so there's no way I feel any pleasure without you allowing it.
My age is flexible and we can discuss our favorite ages to play.
I'm into the more depraved side of D/s and I consider myself completely submissive. My only limits are scat, extreme ageplay, and extreme pain. | 27 [F4F] Scandinavia/online - I am bi and I feel like I want to be a gentle dominant with a girl but I am inexperienced and shy | I'm impotent and young. What should I do? | 26 [F4F] Scandinavia/Online - I am bi and I feel like I want to be a gentle dominant with a girl but I am inexperienced and shy | I'm ashamed of being submissive, please help? | 31(m) questioning sexuality and looking to experiment: Need advice (some nsfw language) | [Sexual Insensitivity] My girlfriend [18F] doesn't get turned on by foreplay and I want to know what I can do to change that. | Need advice/tips for sexting (22F/21M) | Looking for guidance for sexually inexperienced thirtysomethings |
497 | Progresses of Researches on Numerical Weather Prediction in China： 1999-2002 | Sea surface wind field is a basic parameter of marine dynamic process, Catastrophic Sea-state such as marine tropical storm and storm surge are all driven by marine wind, wave monitoring and prediction also need sea surface wind field. By combining the new generation of weather research and prediction model (WRF) and the third generation wave model (WAVEWATCHⅢ), establish an Atmospheric-Wave numerical prediction system. Developing sea surface wind field numerical forecast in East China Sea, it makes up the shortage of wind field data’s absence. By using the numerical prediction results, the wave model will provide accurate and reliable wave forecast products for Chinese shipping, ocean activities and military affairs. | A possible abrupt change in summer precipitation over eastern China around 2009 | History of numerical weather prediction The history of numerical weather prediction considers how current weather conditions as input into mathematical models of the atmosphere and oceans to predict the weather and future sea state (the process of numerical weather prediction) has changed over the years. Though first attempted manually in the 1920s, it was not until the advent of the computer and computer simulation that computation time was reduced to less than the forecast period itself. ENIAC was used to create the first forecasts via computer in 1950, and over the years more powerful computers have been used to increase | The statistical scheme using the results of the best foreign global schemes and the COSMO-RU7 regional scheme is proposed for forecasting surface temperature for five days and the amount of precipitation for three days. Presented are the estimates of prognostic values of the surface air temperature and amount of precipitation for the period of July 2010–June 2013. The joint statistical taking account of different types of systematic errors in the complex forecasting scheme enables excelling all initial schemes in quality. A scheme of the complex forecast is operationally used and its results are daily updated on the website of the Hydrometcenter of Russia at 09:15. The forecasts of extreme temperature, dew-point temperature, and wind speed near the surface are also presented on the website. | A statistical approach is developed to forecast rainfall with a lead time of 3 days on the basis of factors from 500-hpa, 700-hpa and 850hpa isobaric surfaces. The procedure to set up the forecasting model includes the steps of partitioning storm-type and constructing the forecasting equations for each storm-type. Application of the proposed method is presented for Panjiakou Reservoir basin in the north of China. Results indicate that the approach is easy to use with fewer data requirements. It is shown that the method is applicable to the prediction of medium-term rainfall for practical use. | East Asian winter monsoon forecasting schemes based on the NCEP’s climate forecast system | Weather data is the most important information for the farmers, Agricultural products, their sales and marketing people, Tourist Travel agencies, Meteorological department scientists & Analysts, Agriculture university people and Environmental department people etc. It is very difficult to predict or analyze Temperatures effectively across various regions to help the farmers and others to safeguard their properties and lives. An attempt has been made in this direction with the help of data science techniques, Data Mining Techniques and Machine learning algorithms to analyze the Chennai temperature data effectively and to bring useful conclusions in this direction. | History of numerical weather prediction 1971. Efforts to involve sea surface temperature in model initialization began in 1972 due to its role in modulating weather in higher latitudes of the Pacific. A global forecast model is a weather forecasting model which initializes and forecasts the weather throughout the Earth's troposphere. It is a computer program that produces meteorological information for future times at given locations and altitudes. Within any modern model is a set of equations, known as the primitive equations, used to predict the future state of the atmosphere. These equations—along with the ideal gas law—are used to evolve the density, pressure, and potential | 497 | Recent Progress in Numerical Atmospheric Modeling in China | Wind-Wave Numerical Prediction Model in East China Sea | Mesoscale modelling in China: Risø DTU numerical wind atlas calculation for NE China (Dongbei) | Mathematical Modeling of Atmosphere; Predictability and Feasibility | Impact of different cumulus convective parameterization schemes on the simulation of precipitation over China | The Impact of Convective Transport and Wet Deposition of Airborne Dust Particles on the Numerical Simulation of Northeast Asian Storms | Modelling air-pollution in Beijing: Emission reduction vs. meteorological influence | where was the first atmospheric model used | The Regional Integrated Environmental Model System(RIEMS 2.0) coupled with a chemistry-aerosol model and the Princeton Ocean Model(POM) is employed to simulate regional oceanic impact on atmospheric circulation and the direct radiative effect(DRE) of aerosol over East Asia.The aerosols considered in this study include both major anthropogenic aerosols(e.g.,sulfate,black carbon,and organic carbon) and natural aerosols(e.g.,soil dust and sea salt) .The RIEMS 2.0 is driven by NCEP/NCAR reanalysis II,and the simulated period is from 1 January to 31 December 2006.The results show the following:(1) The simulated annual mean sea-level pressure by RIEMS 2.0 with POM is lower than without POM over the mainland and higher without POM over the ocean.(2) In summer,the subtropical high simulated by RIEMS 2.0 with POM is stronger and extends further westward,and the continental low is stronger than without POM in summer.(3) The aerosol optical depth(AOD) simulated by RIEMS 2.0 with POM is larger in the middle and lower reaches of the Yangtze River than without POM.(4) The direct radiative effect with POM is stronger than that without POM in the middle and lower reaches of the Yangtze River and parts of southern China. Therefore,the authors should take account of the impact of the regional ocean model on studying the direct climate effect of aerosols in long term simulation. |
498 | Estimating the Quantitative Demand of NOAC Antidote Doses on Stroke Units | BACKGROUND AND PURPOSE ::: Intravenous tissue plasminogen activator (tPA) is an economically worthwhile but underused treatment option for acute ischemic stroke. We sought to identify the extent of tPA use in Canadian medical centers and the potential savings associated with increased use nationally and by province. ::: ::: ::: METHODS ::: We determined the nationwide annual incidence of ischemic stroke from the Canadian Institute of Health Information. The proportion of all ischemic stroke patients who received tPA was derived from published data. Economic analyses that report the expected annual cost savings of tPA were consulted. The analysis was conducted from the perspective of a universal health care system during 1 year. We estimated cost-savings with incrementally (eg, 2%, 4%, 6%, 8%, 10%, 15%, and 20%) increased use of tPA for acute ischemic stroke nationally and provincially. ::: ::: ::: RESULTS ::: The current average national tPA utilization is 1.4%. For every increase of 2 percentage points in utilization, $757,204 (Canadian) could possibly be saved annually (95% CI maximum loss of $3,823,992 to a maximum savings of $2,201,252). With a 20% rate, >$7.5 million (Canadian) could be saved nationwide the first year. ::: ::: ::: CONCLUSIONS ::: We estimate that even small increases in the proportion of all Canadian ischemic stroke patients receiving tPA could result in substantial realized savings for Canada's health care system. |
Introduction
Randomized clinical trials (RCTs) and real-world data (RWD) in patients with atrial fibrillation have shown that-compared to vitamin K antagonists (VKAs)-non-VKA oral anticoagulants (NOACs) are at least as effective in the prevention of ischaemic stroke, while decreasing the risk of bleeding.
Objective
We aim to evaluate the cost-effectiveness of the NOAC apixaban versus other NOACs (dabigatran, edoxaban and rivaroxaban) and VKA, for stroke prevention in patients with atrial fibrillation by including the available data both from RCT and real-world analyses of all NOACs into one integrative previously published model.
Methods
The model was updated to the current Dutch healthcare situation. The incremental costeffectiveness ratio was calculated using either efficacy/effectiveness and safety data derived from a network meta-analysis (NMA) synthesizing NOAC RCTs or RWD. We conducted a systematic literature search to identify eligible publication to best inform the RWDbased analysis. Additional sensitivity and scenario analyses were conducted to test the robustness of the outcomes.
Results
In the NMA-based analysis, apixaban appeared to be cost-effective compared to VKA (€3,506 per quality adjusted life-year) and dominant (cost-saving and more effective) over PLOS
Introduction
Atrial fibrillation (AF) is the most common cardiac arrhythmia and is a major cause of ischemic stroke, heart failure and cardiovascular morbidity [1]. In the Netherlands, the prevalence in the total population is 2-3% [2] and in people of 75 years and older the prevalence of AF increases to more than 10% [3]. Furthermore, also due to the aging population, the number of patients with AF is predicted to rise steeply in the coming years [1]. AF constitutes a significant public health problem, and estimates suggest that this condition accounts for 1.3% of the national healthcare budget in the Netherlands with a total estimated cost of €583 million in 2009 [4]. In patients with AF, the associated increased risks of ischaemic stroke and systemic embolism (SE) present a major challenge. Approximately 20-30% of the patients experiencing ischaemic stroke have been diagnosed with AF before, during or after the stroke event [1]. Embolic events, such as ischaemic stroke, lead to high hospitalization costs and long-term maintenance costs which contributes to the high economic burden to the Dutch health care system [5].
In patients with AF, anticoagulant (AC) therapy with vitamin K antagonists (VKAs) has been the most effective treatment for the prevention of stroke over the last decades. However, on the basis of randomized clinical trial (RCT) results, the European guidelines have adopted the non-vitamin K antagonist oral anticoagulants (NOACs) as the preferred treatment for the prevention of stroke in patients with AF since 2012 [1,6]. A network meta-analysis (NMA) on pivotal RCTs in treatment groups with NOACs versus the VKA warfarin included data on almost 72,000 patients [7], showed that NOACs were at least as effective in the prevention of ischaemic stroke compared to VKA while there was a 50% decrease in the incidence of haemorrhagic stroke. Over the last years, real-world data (RWD) on the effectiveness and safety of NOACs have been published, confirming the results of the RCTs.
In terms of effectiveness, safety and costs it is relevant to investigate whether the results of the cost-effectiveness using RWD support results of analyses based on RCTs (external validation). Multiple cost-effectiveness analyses have been published on the different NOACs versus VKAs on the basis of the respective RCTs results, using different models and cost assumptions. Here, we aim to evaluate the cost-effectiveness of the NOAC apixaban versus other NOACs and VKAs for stroke prevention in patients with AF by including the available data both from NMA and real-world analyses of all available NOACs in one integrative previously published model [8].
Methods
We aim to evaluate the cost-effectiveness of apixaban compared to VKA, dabigatran, rivaroxaban and edoxaban, by capturing all costs and health outcomes related to the disease, treatment and complications, during the lifetime of a patient with AF. The primary outcome of our analysis is the incremental cost-effectiveness ratio (ICER), calculated by dividing the incremental costs by the incremental health outcomes, presented in life-years LY and quality adjusted lifeyears (QALY). The ICER is compared to a willingness-to-pay (WTP) threshold of €20,000/ QALY. This threshold is based on the disease burden calculation (proportional shortfall estimate of 0.14) in the calculation tool as recommended by the Dutch guidelines for pharmacoeconomic research [9]. The ICER will be calculated using either efficacy/effectiveness and safety data derived from an NMA synthesizing RCTs on NOACs [10] (NMA-based analysis) or RWD (RWD-based analysis).
We conducted a systematic literature search to identify eligible publication to best inform the RWD-based analysis. The systematic literature search was conducted with help of the 'Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement', to identify RWD studies eligible for the RWD-based analysis [11]. Details on the method as well as the results of the search strategy are described in S1. Following the pre-defined eligibility criteria, the real-world study of Lip et al. [12] was considered the most appropriate for use in the RWD-based analysis. From this real-world study we obtained patient characteristics and real-world event rates to populate the RWD-based model. The model structure and all other input parameters were equal for both analyses.
Model structure
Both the NMA-based and the RWD-based analyses were based on a previously published Markov model [8]. The model was validated and updated with data based on the most recent literature to evaluate the lifetime costs and health effects of treatment with apixaban compared to VKA and other NOACs in AF patients in the Netherlands. Since this model was designed with apixaban as the reference drug, indirect comparison between other NOACs (dabigatran, edoxaban and rivaroxaban) and comparison of other NOACs to VKA, was not possible [8]. To model the disease course of AF, different health states were included in the model. During a lifetime, a hypothetical cohort of 1,000 AF patients could remain in or move through these different health states. The risk to move to another health state, hereafter mentioned as the transition probability, was calculated per six weeks cycle. This cycle length was chosen to capture all events related to AF within such a short time frame [8]. As shown in Fig 1, the model included the following health states: 'AF', 'ischaemic stroke' (including unspecified strokes), 'SE', 'bleeding', myocardial infarction ('MI'), 'other deaths' (all deaths unrelated to the aforementioned events) and 'event unrelated AC discontinuation' (AC discontinuation unrelated to the events explicitly included). Bleeding events were divided into intracranial haemorrhage (referred to as 'ICH', which was assumed to be the composite of 'haemorrhagic stroke' and 'other ICH'), other major bleeding (referred to as 'other MB', defined as all non-ICH MBs) and clinically relevant non-major bleeding ('CRNMB'). Additionally, a distinction was made between different levels of ischaemic and haemorrhagic stroke severity (i.e. mild, moderate and severe).
All patients entered the model in the 'AF' state. Upon the occurrence of a clinical event the patient remained in the corresponding health state for one cycle and then moved to one of the transient or absorbing states. 'AF' and 'AF without AC therapy' (red squares) reflect transient states, meaning that patients were able to remain in them or move to other states in the model. In absorbing states (e.g. 'death' [blue squares]) patients remained until death. Upon the occurrence of 'event unrelated AC discontinuation' patients were assumed to discontinue AC therapy permanently.
Patient characteristics
In the NMA-based and RWD-based analyses, all patients entered the model with baseline characteristics as presented in S1 Table. In the NMA-based analysis, we used a Dutch study [13] to simulate specific Dutch patient characteristics, adapting the NMA to the specific Dutch context. The patients entered the model aged 72, 64.7% were male and the average CHADS 2 score and CHA 2 DS 2 -VASc score were 1.7 and 3.1, respectively. In the RWD-based analysis, the patient characteristics were based on the selected real-world study itself. The patients were on average 74.3 years old, 54.1% were male and the average CHA 2 DS 2 -VASc score was 3.7.
Transition probabilities
Event rates. In the NMA-based analysis, transition probabilities were based on the clinical event risks in patients receiving apixaban and a VKA (warfarin) in the ARISTOTLE trial [14]. Hazard ratios compared to apixaban for the comparators dabigatran (110 mg and 150 mg), rivaroxaban and edoxaban were obtained from an NMA by Lip et al [10], who made pairwise indirect comparisons between the ARISTOTLE [14], RE-LY [15], ROCKET-AF [16] and ENGAGE-AF [17] trials, using propensity score adjustments. S2 Table shows the event rates per 100 patient-years (PY) for apixaban and VKA and hazard ratios for dabigatran 110 mg, dabigatran 150 mg, rivaroxaban and edoxaban. Lip et al. also included edoxaban 30 mg in his analysis, however this low-dose was not approved by the European Medicines Agency and therefore not included in our analysis. Average stroke and bleeding (ICH and other MB) risks were weighted by multiplying the CHADS 2 score distribution in Dutch patients (S1 Table) with the corresponding event risks (S2 Table). Subsequently, the event rates were calculated per six weeks (42 days) cycle [18]. Event rates for the other NOACs (dabigatran 110 mg, dabigatran 150 mg, rivaroxaban and edoxaban) were based on the event rate of apixaban per cycle multiplied by the hazard ratios (S2 Appendix) [18]. 'Ischaemic stroke' and 'haemorrhagic stroke' health states were specified per severity, by the modified Rankin Scale (mRS), specifying mild (mRS 0-2), moderate (mRS 3-4), severe (mRS 5) and fatal, based on RCT data [14][15][16][17]. The model allowed for one recurring ischaemic or haemorrhagic stroke, with an event rate of 2.72 per 100 PY, which was assumed to be equal across all treatments [19].
The transition probabilities used in the RWD-based analysis are summarized in S3 Table. Based on the real-world study by Lip et al. [12] we included RWD-based event rates of apixaban and VKA and hazard ratios of dabigatran and rivaroxaban for ischaemic stroke, ICH, other MB and SE, and distributions of haemorrhagic stroke among ICH and GI bleeding among other MB. If transition probabilities were unavailable from RWD, we used the same transition probabilities as used in the NMA-based analysis.
In sensitivity analysis, transition probabilities, hazard ratios and distributions were varied with a beta, log normal and Dirichlet distributions, respectively.
Mortality. Mortality rates are summarized in S4 Table. Background mortality was calculated by fitting the Gompertz distribution to 2017 Dutch life tables to obtain a proper representation of the mortality risk for each six weeks cycle [20]. For each health state, background mortality risks were updated by case fatality and mortality risk adjustment factors by using data from the ARISTOTLE trial [14] within the trial period and data from a Danish study by Brønnum et al. [21] for long term outcomes.
Treatment switch or discontinuation. Treatment discontinuation could be related or unrelated to an event. Upon the occurrence of non-fatal other ICH, patients were assumed to discontinue AC therapy for six weeks after which they resumed their original AC [22]. Patients who experienced a non-fatal haemorrhagic stroke or non-fatal MI were assumed to discontinue their AC therapy permanently and were assigned acute and long-term maintenance costs. All patients experiencing any other event such as ischaemic stroke or SE were assumed to continue their current AC therapy.
In case of 'event unrelated AC discontinuation', all event risks (e.g. event risk, case fatality, severity distributions) were updated to no treatment. These event rates for patients receiving no treatment, summarized in S5 Table, were obtained from a meta-analysis of placebo, aspirin and warfarin controlled studies in AF [23]. After AC discontinuation we assumed no further bleeding risk, and a constant risk of ischaemic stroke, SE, MI, and other CV hospitalisation, independent of time, prior AC therapy or patient characteristics.
Utilities
In absence of health-state-specific EQ-5D Dutch utilities, health-state-specific utility scores were obtained from a UK EQ-5D catalogue by Sullivan et al. [24] (Table 1), similar to the previously published UK model [8]. A specific score for AF patients was used and updated upon the occurrence of a clinical event. Stroke (ischaemic and haemorrhagic) utility scores were subdivided per severity. Utility decrements for other ICH, other MBs, CRNMB, other CV hospitalization and use of AC therapy were taken into account [25,26]. Health outcomes were discounted at a rate of 1.5% per year [27]. Utility (decrement) parameters were varied using beta distributions for sensitivity analyses.
Costs
Direct and indirect costs in-and outside the healthcare sector were considered, since the ICERs were calculated from a societal perspective, as recommended by the Dutch cost manual for pharmacoeconomic evaluation [27]. All costs with their corresponding ranges are summarized in Table 2. Ranges were based on a 95% confidence interval (CI). If 95% CI was unavailable, ranges were calculated with a standard error of 25% of the mean. Although this approach has limitations, as discussed in ISPOR-SMDM modelling good research practices report [28], the exclusion of parameters from a sensitivity analysis based on the fact that there is no data available to estimate probabilistic parameters would be less appropriate. Drug costs were derived from the offxicial Dutch price list (Z-index) from July 2018 [29]. The drug cost of VKA was based on the weighted average cost of phenprocoumon (21,1%) and acenocoumarol (78,9%) as reported in the annual medical report of the Dutch Federation of Thrombosis Service (FNT) [2]. For VKA patients the cost of an INR monitoring visit was calculated as the average cost of INR measurement at thrombotic service (45.6%), at home (34.0%) and self- Treatment with VKA 0.0130 (0.00-0.08) While receiving treatment [26] Treatment with apixaban, dabigatran, rivaroxaban or edoxaban a 0.0020 (0.00-0.04) While receiving treatment [26] a Utility decrement related to anticoagulation with NOACs is assumed to be equal to aspirin. [27,30]. Based on the FNT medical report an average frequency of INR monitoring visits was set at 22.3 per year. Routine care for NOACs consisted of an annual general practitioner (GP) visit, since the European Society for Cardiology guidelines mention that chronic care management can be handled by GP [1]. AC management consisted of an additional GP visit for AC related dyspepsia and renal function monitoring, which should for NOACs be done at least once per year according to international guidelines [31]. Event costs per health state were divided into acute and long-term maintenance costs. Acute costs were related to hospital and rehabilitation facility costs and maintenance costs reflected medical costs during the patient's lifetime [18]. Event costs were derived from our previous Dutch economic study [5]. Costs for other CV hospitalizations were based on Dutch hospital tariffs for hospitalization related to heart failure, high blood pressure and heart infections [32].
Indirect costs included travel expenses and event related informal care costs. Travel costs were included for 45% of the INR monitoring visits (patients who visited thrombosis service themselves) and for each routine care or dyspepsia related GP visits [33]. These costs were based on an average distance to the GP of 1.1 km, with a cost of €0.19 per km [27]. Informal care costs were added to other long-term maintenance costs following events with absorbing states. Informal care costs for stroke (ischaemic and haemorrhagic) were based on the Dutch costing study by van den Berg et al. (based on a mean of 29.4 hours of informal care per week) [34]. MI and SE were assumed to require less intensive informal care compared to stroke, and were therefore based on a Dutch report that defined non-intensive informal care as eight hours per week. The price per hour (€14) was derived from the Dutch cost manual for pharmacoeconomic evaluation [27].
All costs were inflated to 2018 using the consumer price index from Dutch Statistics (CBS Statline) [35]. Cost outcomes were discounted at a rate of 4% per year [27]. For sensitivity analyses the costs parameters were varied over a gamma distribution.
Sensitivity and scenario analyses
Next to base case analyses based on the NMA and RWD, sensitivity analyses were conducted to evaluate the influence of uncertainty in input parameters on the ICER. In the probabilistic sensitivity analysis (PSA) costs, utilities, transition probabilities were varied simultaneously over their 95% CIs and ICERs were calculated during 2,000 simulations. Outcomes were presented in cost-effectiveness planes and cost-effectiveness acceptability curves. In the univariate sensitivity analysis input parameters were varied one by one over their 95% CI, to examine the influence on the ICER for each parameter separately. The input parameters with the highest impact on the ICER were presented in a Tornado diagram. Three additional scenario analyses were conducted to capture the effect of healthcare payer's perspective (scenario 1), equal drug prices for all NOACs (scenario 2) and equal event unrelated AC discontinuation rates for all NOACs (scenario 3). Table 3 summarizes the costs outcomes per category. Event costs are the largest contributor to the total costs (45-49% and 47-53% in the NMA-based and RWD-based analyses, respectively). Indirect costs also have high impact on the total costs: in both analyses 39-45% of the total costs are related to indirect costs. In VKA treated patients, the impact of drug costs is negligible compared to NOACs (<1%% vs. 8-10% of total costs). Overall relatively small VKA (3 mg daily) €0.05 a [29] Dabigatran (110 mg BID) €2.44 [29] Dabigatran (150 mg BID) €2.44 [29] Rivaroxaban (20 mg daily) €2.29 [29] Edoxaban (60 mg daily) €2.39 [29] Monitoring and routine care costs Table 4. In NMA-based analysis, apixaban increases costs with €902 and QALYs with 0.262 over lifetime compared to VKA, resulting in an ICER of €3,506/QALY. In both analyses, all other ICERs were dominant, meaning that apixaban treatment is cost-saving while increasing patient's health.
Results
Deterministic results
Sensitivity analyses
The results of the PSA are plotted in cost-effectiveness planes, shown in S1 File. Figs 2 and 3 show the probability of being the most cost-effective treatment alternative per WTP threshold for the NMA-based and RWD-based analyses, respectively. In NMA-based analysis, apixaban is the most cost-effective treatment option at the €20,000/QALY WTP threshold with 50%. At the same threshold, the probabilities of VKA, dabigatran 110 mg, dabigatran 150 mg, rivaroxaban and edoxaban being the most cost-effective treatment option are: 8%, 1%, 11%, 9% and 21%, respectively. In RWD-based analysis, similar results were found: apixaban is the most cost-effective treatment with 90%, and apixaban was-compared to VKA, dabigatran and rivaroxaban respectively-cost-effective in 0%, 0% and 9% of the iterations. Nevertheless, apixaban was only significantly dominant compared to VKA in the RWD-based analysis, as in more than 95% of the PSA simulations apixaban was cost-saving and more effective compared to VKA. In all other PSAs the dominancy of apixaban was non-significant.
Additionally, we conducted a univariate sensitivity analysis. Fig 4 represents the tornado diagram for apixaban versus VKA (NMA-based analysis). Univariate sensitivity analyses comparing apixaban with other NOACs identified similar parameters to be the most influential on ICER. The parameters with the most influence are the ischaemic stroke and ICH hazard ratios for VKA compared to apixaban, INR monitoring costs, daily costs of apixaban and stroke (ischaemic or haemorrhagic) case fatalities.
Scenario analyses
We conducted three additional scenarios to assess the influence of the healthcare payer's perspective (scenario 1), equal drug costs among NOACs (scenario 2) and equal rates of event unrelated AC discontinuation among NOACs and VKAs (scenario 3). Scenario 1 showed that compared to societal perspective the incremental costs of apixaban versus VKA and edoxaban in the NMA-based analysis increase, resulting in an ICER of €5,787/QALY and €206/QALY.
In RWD-based analysis, apixaban is cost-effective compared to VKA (€292/QALY), and cost-saving (dominant) compared to dabigatran and rivaroxaban. In scenario 2, equal drug costs for NOACs resulted in an ICER of €2,884/QALY for apixaban versus edoxaban in the NMA-based analysis. All other ICERs are dominant.
In the third scenario of the NMA-based analysis, equal event unrelated AC discontinuation rates for NOACs and VKAs resulted in an ICER of €244,079/QALY for apixaban versus dabigatran 150 mg, which is not considered cost-effective at the WTP threshold of €20,000/QALY. In NMA-based analysis, apixaban appeared to be dominant over dabigatran 110 mg and rivaroxaban and cost-effective compared to VKA (€5,648/QALY) and edoxaban (€10,243/QALY). In RWD-based analysis apixaban was still dominant over VKA, dabigatran and rivaroxaban. Detailed results of the three scenarios are presented in Table 5.
Discussion
To our knowledge, this is the first cost-effectiveness study comparing different NOACs and VKA with RCT data as well as RWD in patients with AF. The RWD-based analysis was specifically implemented to externally validate conclusions found in the NMA-based analysis based on RCT data. In the NMA-based analysis, apixaban appeared to be cost-effective compared to VKA and cost-saving compared to dabigatran 110 mg, dabigatran 150 mg, rivaroxaban and edoxaban. The RWD-based analysis showed that apixaban is cost-saving compared to all comparators. Apixaban was shown, in both analyses, to be the most cost-effective treatment option at a WTP threshold of €20,000/QALY (50% and 90%, respectively). In the scenario analysis apixaban appeared to be not cost-effective compared to dabigatran 150 mg, when using equal event-unrelated treatment discontinuation rates for each drug. In all other scenarios apixaban is cost-effective or cost-saving compared to VKA and other NOACs. A previously published cost-effectiveness analysis, which was based on the same model as our analysis, was conducted in the UK setting [8]. They found that apixaban was cost-effective compared to dabigatran 110 mg, dabigatran 150 mg and rivaroxaban with ICERs of £4,497/ QALY, £9,611/QALY and £5,305/QALY, respectively. In our analysis, we found apixaban to be dominant (cost-saving while increasing QALYs) over the other NOACs. This difference can be explained by the drug costs (apixaban is relatively lower priced in the Netherlands) as well as the difference in perspective, with UK consistently using the health-care perspective, i.e. that of the National Health Service (NHS). In our scenario analysis conducted from a healthcare payer's perspective, the incremental costs did decrease compared to the societal perspective, supporting this hypothesis, although the ICERs remained dominant. Moreover, in Table 5. Results of the scenario analyses: NMA-based and RWD-based analyses calculated from healthcare payer's perspective (scenario 1), equal drugs costs for NOACs (scenario 2) and equal event unrelated AC discontinuation rates for NOACs and VKAs (scenario 3). Real-world and trial based cost-effectiveness of oral anticoagulants in atrial fibrillation our scenario analysis assuming equal AC costs apixaban was still dominant over all other NOACs except edoxaban in the NMA-based analysis, with an ICER of €2,884/QALY. This suggests that these differences in methodology are not solely the cause of the difference in ICERs.
In a follow-up publication [18], it was concluded that, apixaban was dominant over 30 mg and 60 mg edoxaban in the UK setting (with edoxaban not being included in the previous publication [8]). Two other studies in the UK calculated the cost-effectiveness based on alternative NMAs [36,37]. Both studies showed that, compared to the other NOACs, apixaban is the most cost-effective treatment option at a WTP threshold of £20,000/QALY.
A Dutch study that compared the cost-effectiveness of apixaban, rivaroxaban and dabigatran to VKA [38] found that from a healthcare payer's perspective apixaban was the most costeffective option compared to VKA with an ICER of €14,626/QALY. The difference in outcome compared to our study can be explained by differences in input parameters (e.g. apixaban drug costs were higher compared to our analysis) and model structure. For example, they did not include haemorrhagic stroke costs and QALYs, which in our univariate analysis had the highest influence on the ICER. Also, they used the healthcare payer's perspective and US tariffs for the quality of life, which might also contribute to different outcomes.
Event unrelated AC discontinuation rates differ among the anticoagulants [10]. To show the impact of these differences in event unrelated AC discontinuation rates on cost-effectiveness, we included a scenario assuming equal rates for all anticoagulants. This scenario shows to have a high influence on the ICER of apixaban compared to dabigatran 150 mg, which changed from dominant to €244,079/QALY. Apixaban remained cost-effective or cost-saving compared to the other NOACs and VKA. Firstly, dabigatran has the highest event unrelated AC discontinuation rate compared to apixaban (hazard ratio versus apixaban = 1.500). Secondly, dabigatran 150 mg has a better protective effect (hazard ratio versus apixaban = 0.790) compared to apixaban, while bleedings are a bit more frequent (hazard ratio versus apixaban ICH = 1.020 and other MB = 1.340). When assuming equal event unrelated AC discontinuation, the incremental QALYs of apixaban versus dabigatran 150 mg decreases from 0.131 in the base-case analysis to 0.008, resulting in a high ICER.
Notably, costs and QALYs are highly driven by the patients who discontinue AC therapy. Treatment discontinuation in patients with AF is very undesirable, because from that moment on they are not protected for stroke or other thromboembolic events. In practice, a certain percentage of the patients who discontinue their initial AC therapy, will be encouraged to switch to another AC. However, it is hard to get supportive data on the percentage of patients fully discontinuing AC therapy, as well as reliable data on what that exactly means for stroke and bleeding risks. By emphasizing the importance of AC therapy and discussing the reason for the AC discontinuation the patient can be encouraged to restart or switch AC therapy.
The major advantage of this study is that both an NMA and RWD were used for cost-effectiveness. For the RWD-based analysis we used the publication of Lip et al. that best met the inclusion criteria for the systematic literature search underlying the NMA [12]. Differences in estimated hazard ratios in NMA and RWD led to differences in results, although the overall outcomes of the RWD-based analysis support the conclusions of the NMA-based analysis.
Unfortunately, we were unable to include RWD on Dutch patients. Throughout the next years, a recently launched project "DUTCH-AF registry" aims to include 6,000 Dutch patients with AF to collect and assess data on effectiveness, adherence and bleeding risk of NOACs and VKAs [39]. It is interesting to have this data specific on the Dutch patients, since the patient characteristics, such as CHADS 2 /CHA 2 DS 2 -VASc scores and age, can obviously differ among populations. Obviously due to the fact that RWD studies include heterogeneous populations from different countries, differences in clinical outcomes can be expected, which potentially also leads to differences in economic outcomes. Therefore, the use of another RWD study might lead to different outcomes. Also, outcomes of NMAs can vary when making use of different combinations of clinical trials, differences in methodology and the differences in definitions of the outcomes [40], again potentially leading to other results than those based on our selected NMA and RWD.
Notably, we included low-dose dabigatran (110 mg) in our analysis. We included this dose since it was officially approved by European Medicines Agency [41], although it was approved with the condition that this low-dose should be reserved for patients older than 80 years and/ or with a decreased renal function (eGFR<50 ml/min). Since this was not the population that was exclusively followed in the dabigatran (RE-LY) trial included in the NMA [15], the risks used in the model might not adequately represent the real-world situation, and possibly this leads to a undervaluation of the (cost-) effectiveness of dabigatran 110 mg.
Conclusion
Based on RCT-data as well as RWD, we conclude that generally apixaban is cost-effective or even cost-saving (less costly and more effective) compared to VKA and other NOACs in the overall population of AF-patients. It should be mentioned that, especially compared to other NOACs, only marginal differences in costs and health effects were found and the use of other effectiveness and safety data sources or assumptions, considerations of specific patient populations, costs might lead to different outcomes. Table. Patient baseline characteristics model inputs used in the NMA-based and RWDbased analyses. Abbreviations: CHADS2 score, congestive heart failure, hypertension, age �75 years, diabetes mellitus, prior stroke or transient ischemic attack or thromboembolism; CHA2DS2-VASc score, congestive heart failure, hypertension, age �75 years (2), diabetes mellitus, prior stroke or transient ischemic attack or thromboembolism (2) and vascular disease (peripheral arterial disease, previous MI, aortic atheroma). (DOCX) S2 Table. Event rates for apixaban and VKA and dabigatran 110 mg, dabigatran 150 mg, rivaroxaban, and edoxaban and distributions of patients across different levels of ischaemic and haemorrhagic stroke severity. a The event rates of ischaemic stroke, ICH and other MB were adjusted by CHADS2-scores with the distribution presented in S1 Table. Abbreviations: AC, anticoagulant; CHADS2, congestive heart failure, hypertension, age �75 years, diabetes mellitus, prior stroke or transient ischemic attack or thromboembolism; CI, confidence interval; CRNMB, clinically relevant non-major bleeding; CV, cardiovascular; GI, gastro-intestinal; HR, hazard ratio; ICH, intracranial haemorrhage; MB, major bleed; MI, myocardial infarction; NOAC, non-vitamin K antagonist oral anticoagulant; PY, patient-years; SE, systemic embolism; VKA, vitamin K antagonist. (DOCX) S3 Table. Input parameters for the RWD-based analysis obtained from real-world study comparing apixaban with VKA and other NOACs by Lip et al. [3]. Abbreviations: CI, confidence interval; HR, hazard ratio; ICH, intracranial haemorrhage; MB, major bleeding, PY, patient-years; SE, systemic embolism; VKA, vitamin K antagonist. a Hazard ratio apixaban versus comparator = (1/HR comparator versus apixaban). (DOCX) S4 Table. Background mortality, case fatality and mortality risk adjustment factors per event. a Lambda and Gamma are the natural logarithms of the slope of the survival hazard and age, respectively, which can be used to calculate the predicted survival at any time per age group (0-75 or >75 years) and gender. b Assumed to be equal to mortality risk adjustment factor of AF, since these events we assumed to have no additional effect on mortality risk in the period after the event. Abbreviations: AF, atrial fibrillation; CI, confidence interval; CRNMB, clinically relevant non-major bleeding; HR, hazard ratio; ICH, intracranial haemorrhage; MB, major bleeding; MI, myocardial infarction; SE, systemic embolism. (DOCX) S5 Table. Event rates per 100 patient-years for no treatment after event unrelated treatment discontinuation. a intracranial haemorrhage including haemorrhagic stroke. Abbreviations: CI, confidence interval; CRNMB, clinically relevant non-major bleeding; CV, cardiovascular; ICH, intracranial haemorrhage; MB, major bleeding; MI, myocardial infarction; PY, patient-years; SE, systemic embolism. (DOCX) S1 File. Probabilistic sensitivity analysis results. (DOCX)
Supporting information
Fig 1 .
1Representation of the Markov model. All patients entered the model in the AF state, from where they could move to another health state upon the occurrence of one of the following events: 'ischaemic stroke', 'haemorrhagic stroke', 'other ICH', 'other MB', 'CRNMB', 'SE', 'MI', 'other deaths' or 'event unrelated AC discontinuation'. The triangles show the state a patient enters after an event. These states can be transient (red squares) or absorbing (blue squares). Abbreviations: AC, anticoagulant; AF, atrial fibrillation; CRNMB, clinically relevant non-major bleeding; ICH, intracranial haemorrhage; MB, major bleeding; MI, myocardial infarction; SE, systemic embolism. https://doi.org/10.1371/journal.pone.0222658.g001 Real-world and trial based cost-effectiveness of oral anticoagulants in atrial fibrillation PLOS ONE | https://doi.org/10.1371/journal.pone.0222658 September 17, 2019
a
Based on weighted average of phenprocoumon and acenocoumarol users in the Netherlands b Assumption: weighted average of costs based on declaration codes for measurement at thrombotic service (DOT: 079995), at home (DOT: 079995 and 079986) and self-measurement/management (DOT: 190253) c Based on cost of one GP visit d Average of Dutch declaration codes for hospitalization of maximal 5 days for heart failure (DOT: 099899089), infection in the heart (DOT: 099899013) and high blood differences in total costs were observed across different types of NOACs. The total costs of AF with NOACs as well as VKA treatment are lower in real-world setting compared to the trial setting. The deterministic results of the NMA-based and RWD-based analyses are summarized in
Fig 2 .
2Probability of being the most cost-effective treatment choice per willingness-to-pay threshold for the NMAbased analysis. Abbreviations: NMA, network meta-analysis; QALY, quality adjusted life-years; VKA, vitamin K antagonist. https://doi.org/10.1371/journal.pone.0222658.g002
Fig 3 .
3Probability of being the most cost-effective treatment choice per willingness-to-pay threshold for the RWD-based analysis. Abbreviations: QALY, quality adjusted life-years; RWD, real-world data; VKA, vitamin K antagonist. https://doi.org/10.1371/journal.pone.0222658.g003
Fig 4 .
4Univariate sensitivity analysis of NMA-based analysis comparing apixaban and VKA. Figure depicts the influence of uncertainty in different input parameters on ICER. Abbreviations: AF, atrial fibrillation; HR, hazard ratio, ICER, incremental cost-effectiveness ratio; ICH, intracranial haemorrhage; INR, international normalized ratio; QALY, quality adjusted life-years; SE, systemic embolism; VKA, vitamin K antagonist.
S1
Appendix. Systematic literature search. (DOCX) S2 Appendix. Formulas. (DOCX) S1
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Table 1 .
1Utilities for different health states.Utility Value
Value, mean (standard error)
Source
Abbreviations: AF, atrial fibrillation; CI, confidence interval; CRNMB, clinically relevant non-major bleeding; CV, cardiovascular; ICH, intracranial haemorrhage; MB, major bleeding; MI, myocardial infarction; SE, systemic embolism, VKA, vitamin K antagonist.https://doi.org/10.1371/journal.pone.0222658.t001measurement (20.4%)
Table 2 .
2Drug costs, event costs and indirect costs for the different health states.Drug costs
Table 4 .
4Base-case results of the NMA-based and RWD-based analyses comparing apixaban to VKA and other NOACs.Comparator
Incremental cost
Incremental QALY
Cost per QALY gained
Incremental LY
Cost per LY gained
NMA-based analysis
VKA
€920
0.262
€3,506
0.269
€3,415
Dabigatran (110mg)
-€2,692
0.177
Dominant
0.207
Dominant
Dabigatran (150 mg)
-€819
0.131
Dominant
0.157
Dominant
Rivaroxaban
-€1,027
0.101
Dominant
0.126
Dominant
Edoxaban
-€197
0.065
Dominant
0.085
Dominant
RWD-based analysis
VKA
-€1,358
0.330
Dominant
0.352
Dominant
Dabigatran
-€3,612
0.310
Dominant
0.383
Dominant
Rivaroxaban
-€1,625
0.124
Dominant
0.154
Dominant
Abbreviations: LY, life-years; NMA, network meta-analysis; QALY, quality adjusted life-years, RWD, real-world data; VKA, vitamin K antagonist.
https://doi.org/10.1371/journal.pone.0222658.t004
PLOS ONE | https://doi.org/10.1371/journal.pone.0222658 September 17, 2019
Author ContributionsConceptualization: Jelena Stevanovic, Harrie Rila.
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Introduction
Patients with hemorrhagic stroke have high disability and mortality rates [1,2]. In addition to the direct effects of the initial bleeding event and secondary neurologic complications, patients with hemorrhagic stroke are predisposed to medical complications that can have a negative impact on patient outcomes and increase cost of care [3][4][5].
One of the most common medical complications that occur in patients with hemorrhagic stroke is infection [3,6]. The development of infections in this patient population is of significant concern as infections have been found to be one of the strongest drivers of length of stay and readmission within 30 days [7][8][9]. Vancomycin is a primarily renally eliminated antibiotic that undergoes routine drug monitoring to optimize clinical efficacy and limit toxicity in this patient population.
It has been previously reported that the vast majority of patients with hemorrhagic stroke experience a hyperdynamic state resulting in enhanced renal clearance (defined as a measured CrCl greater than that calculated based on standard equations) and/or augmented renal clearance (ARC) (defined as a measured CrCl greater than or equal to 130 mL/min/1.73 m 2 ) [10,11] However, to our knowledge, no studies exist examining the impact of these changes in renal clearance on renally eliminated medication pharmacokinetic parameters in this patient population. This is an important concept as clinicians do not routinely adjust the dosing regimen of renally eliminated medications to account for enhanced renal clearance or ARC, which may result in subtherapeutic concentrations of these medications and undertreatment of infectious complications. Therefore, the purpose of this study was to determine whether patients experiencing enhanced renal clearance or augmented renal clearance with hemorrhagic stroke had alterations in vancomycin pharmacokinetic parameters when compared with those predicted based on population-based equations.
Materials and methods
Patients
This was a prospective, observational post hoc analysis study that included adult patients with ICH or aSAH admitted to UNC Hospitals Neurosciences Intensive Care Unit [10]. Ethical approval was obtained from the institutional review board. Patients had an expected ICU length of stay (LOS) greater than 48 h, received vancomycin, and had one vancomycin peak steady-state concentration and one vancomycin trough steady-state concentration obtained. Patients were excluded if an indwelling urinary catheter was not used as part of standard management, they had pre-existing renal dysfunction (CKD stages 3-5), they had an admission serum creatinine > 1.4 mg/dL, they had a history of nephrectomy or renal transplant, they had a BMI < 18 kg/m 2 , or if they were pregnant. Management of ICH and aSAH at UNC Hospitals was consistent with published guidelines [12,13].
Vancomycin dosing regimens
Clinical pharmacists are consulted for vancomycin initiation for patients admitted to the Neurosciences Intensive Care Unit at UNC Hospitals. The consulted clinical pharmacists are responsible for determining the vancomycin dosing regimen. The goal serum vancomycin trough concentration at steady-state is 10-20 ug/mL, depending on the indication. Clinical pharmacists are also responsible for determining the optimal time to obtain steady-state vancomycin serum concentrations, which is generally considered to be at least four times the half-life of the drug after administration.
Interventions
Demographic and outcome data, including age, gender, admission diagnosis, admission height, admission weight, admission Glasgow Coma Scale (GCS) score, admission Sequential Organ Failure Assessment (SOFA) score, ICU (intensive care unit) and hospital LOS, and mortality were recorded prospectively. Specifically, for the aSAH patients, the Hunt and Hess scale grade, modified Fisher scale grade, and occurrence of symptomatic vasospasm were recorded. For the ICH patients, admission ICH score and ICH volume were collected. Serum creatinine, urine creatinine, and urine volume were recorded daily. Data collected for characterizing the vancomycin regimen include vancomycin dose, vancomycin frequency, vancomycin serum concentrations, and fluid balance.
An 8-h urine collection was the primary method of measuring renal function. CrCl was also calculated based on the Cockcroft-Gault equation. Urine was collected daily via the indwelling urinary catheter between 10:00 and 18:00, following which laboratory analysis determined the urinary volume and urinary creatinine concentration. Concurrent daily serum creatinine concentrations were obtained, following which CrCl was calculated using the following formula:
CrCl ¼ U Cr  U vol ½ = S Cr  T min ½
CrCl determined by the Cockcroft-Gault method was calculated using the following formula:
CrCl ¼ 140 À Age ð ÞÂ Weight kg ð Þ ð Þ Â0:85 if Female ð Þ ½ = 72 Â SCr mg=dL ð Þ ð Þ
CrCl and eGFR values were normalized to a body surface area of 1.73 m 2 , as per convention. Enhanced renal clearance was defined as a measured CrCl greater than the calculated CrCl via the Cockcroft-Gault equation. ARC was defined as an 8-h CrCl greater than or equal to 130 mL/min/1.73 m 2 , given the previously determined association with subtherapeutic antimicrobial concentrations when using standard doses [14].
Pharmacokinetic measures
To determine alterations in pharmacokinetic parameters of vancomycin therapy following an aSAH or ICH, predicted pharmacokinetic parameters based on population data were compared with pharmacokinetic parameters calculated based on available steady-state vancomycin serum peak and trough concentrations. Predicted pharmacokinetic parameters were calculated using the following equations [15]:
V d ¼ 0:7 L Â actual body weight kg ð Þ K e ¼ 0:00083 Â CrCl þ 0:0044
where V d is the volume of distribution, K e is the firstorder elimination rate constant, and CrCl is the estimated creatinine clearance based on the Cockroft and Gault equation [16]. Ideal body weight was used in the Cockroft and Gault equation, except if the actual body weight was less than the ideal body weight, then the actual body weight was used, or if the actual body weight was greater than 125% of the ideal body weight, then the adjusted body weight was used [17,18]. The predicted pharmacokinetic parameters calculated by the equations were then used to determine the estimated vancomycin serum trough concentration, which was compared with the measured vancomycin serum trough concentration.
The following equations were used to calculate patient pharmacokinetic parameters based on steady-state vancomycin serum peak and trough concentrations [19]:
K e ¼ ln C max=C min ð Þ ½ =Δ t V d ¼ Dose=t' ð Þ 1 À e Àket' À Á  à = ke C max− C min  e −ket 0 h i n o
where Cmax is the steady-state vancomycin serum peak concentration, Cmin is the steady-state vancomycin serum trough concentration, Δ t is difference in time between Cmax and Cmin within the same dosing interval, and t' is the infusion time.
Statistical analysis
Descriptive statistics were used to portray patient characteristics and vancomycin regimen characteristics. Continuous and ordinal variables are represented as mean ± standard deviation or median (interquartile range), and categorical variables are represented as n (%). The pharmacokinetic parameters were compared using the paired t test. Statistical significance was defined as a p value < 0.05. All analyses were performed with Stata version 14.2 (StataCorp LP, College Station, TX).
Results
The patients eligible for inclusion into the study have been previously described [10]. During the study period, 64 aSAH patients and 60 ICH patients were admitted to the Neurosciences Intensive Care Unit. Seventeen patients were included in the study and received vancomycin, and had at least one vancomycin peak steadystate concentration and one vancomycin trough steadystate concentration obtained. Table 1 displays the patient characteristics for those included within the study. The majority of patients were female (65%) with a mean age of 63.3 ± 13.3 years. The median admission GCS was 9 (6)(7)(8)(9)(10)(11)(12), and the median admission SOFA score was 4 (3)(4)(5). The majority of patients had an admission diagnosis of aSAH (71%). The mean admission serum creatinine was 0.7 ± 0.1 mg/dL.
Patient characteristics
Vancomycin characteristics
Patient vancomycin characteristics are displayed in Table 2. The mean vancomycin dosing regimen was 15.1 ± 4.2 mg/ kg every 8 (8)(9)(10)(11)(12) h. This provided a mean vancomycin serum trough concentration of 12.0 ± 3.6 ug/mL. The median daily fluid balance of 48 h prior to the vancomycin serum trough concentration was + 588.4 (− 52.2-1440.7) mL/day. The most common infection source for administration of vancomycin was pneumonia (53%). Table 3 displays the predicted pharmacokinetic parameters based on population data and the pharmacokinetic parameters calculated from steady-state vancomycin serum peak and trough concentrations. The mean creatinine clearance calculated from urine creatinine measurements on the day that the vancomycin concentrations were obtained was significantly higher than the creatinine clearance calculated from the Cockcroft-Gault equation (116.7 ± 10.3 vs. 161.6 ± 16.7 mL/min; p = 0.001). All patients experienced enhanced renal clearance on the day that the vancomycin concentrations were obtained, and 12 patients (71%) experienced ARC. The mean calculated elimination rate constant was also significantly higher than the predicted value (0.141 ± 0.02 vs. 0.087 ± 0.01 h −1 ; p = 0.004), and the mean calculated half-life was significantly lower than the predicted half-life (6.5 ± 0.9 vs. 8.7 ± 0.6 h; p = 0.03). No difference was seen between the mean calculated volume of distribution and the mean predicted volume of distribution (71.8 ± 11.3 vs. 57.6 ± 2.6 L; p = 0.23).
Vancomycin pharmacokinetic parameters
Discussion
This study is the first to our knowledge to evaluate alterations in vancomycin pharmacokinetics due to enhanced renal clearance in patents with hemorrhagic stroke. All patients with hemorrhagic stroke included in the study experienced enhanced renal clearance, and the majority experienced ARC while concomitantly receiving vancomycin. Additionally, these patients exhibited pharmacokinetic alterations favoring an increased elimination of vancomycin when compared with predicted pharmacokinetic parameters based on population data. These findings advance the theory that patients with hemorrhagic stroke may require empiric renally eliminated medication dosing regimen modifications due to experiencing enhanced renal clearance. Enhanced renal clearance and ARC are thought to be due to a hyperdynamic state experienced by some subsets of critically ill patients, resulting from systemic inflammation, administration of intravenous fluids and vasoactive medications, and increased organ blood flow [14]. There have been a few previous studies documenting enhanced and augmented renal clearance in patients with hemorrhagic stroke. In a study evaluating 20 patients with subarachnoid hemorrhage, May and colleagues found that the mean measured creatinine clearance among the patients included was 325.9 ± 135.2 mL/min/1.73 m 2 . This was significantly higher than the Cockcroft and Gault estimated creatinine clearance of 144.9 ± 42.8 mL/min/1.73 m 2 (p < 0.001) [20]. One limitation from this study was that the creatinine clearance was calculated via one 24-h urine collection. Our research team expanded on these findings by measuring creatinine clearance daily in 80 patients with hemorrhagic stroke (50 patients with aSAH and 30 patients with ICH) via an 8-h urine collection [10]. We found that patients with aSAH or ICH had mean measured creatinine clearances significantly higher over the study period than the creatinine clearance estimated via Cockcroft and Gault. Ninety-four percent of patients with aSAH and 50% of patients with ICH experienced ARC on at least one study day.
Our research team has also previously demonstrated that patients with hemorrhagic stroke exhibited pharmacokinetic alterations favoring an increased elimination of vancomycin when compared with predicted pharmacokinetic parameters based on population data [21]. In a study including 146 patients with hemorrhagic stroke (80 with aSAH and 66 with ICH), the mean calculated elimination rate constant was higher than the predicted value (0.122 ± 0.04 vs. 0.086 ± 0.03 h −1 ; p < 0.001) and the mean calculated half-life was lower than predicted (6.4 ± 2.2 vs. 8.9 ± 3.1 h; p < 0.001). These alterations in pharmacokinetic parameters resulted in vancomycin serum trough concentrations lower than predicted (10.3 ± 4.3 vs. 18.3 ± 8.6 ug/mL; p < 0.001). It has also previously been proposed that renally eliminated medications may be eliminated more quickly than expected in patients who experience augmented renal clearance, resulting in subtherapeutic medication concentrations [22][23][24][25]. However, this is the first study demonstrating a direct association between elevations in creatinine clearance in patients with hemorrhagic stroke and increased elimination of vancomycin. These data reflect an important finding, as subtherapeutic vancomycin concentrations may lead to It is reasonable to postulate that these findings also apply to other renally eliminated medications. The concern of administering inadequate renally eliminated medication regimens may be of particular concern in patients with ICH, as these patients generally empirically receive dose reductions given their older age and higher likelihood of co-morbidities associated with renal dysfunction, such as hypertension and diabetes [26]. The findings from this study suggest that it may be acceptable to empirically dose patients suspected of experiencing ARC at higher doses than calculated with traditional population-based pharmacokinetic equations. Furthermore, these findings also highlight the need for additional studies to investigate new vancomycin dosing algorithms and empiric pharmacokinetic equations for patients with aSAH or ICH. This study has several limitations worthy of discussion. There were a limited number of patients who had daily measured creatinine clearances and vancomycin serum peak and trough concentrations. Nevertheless, this is the first study to evaluate the impact of enhanced renal clearance on renally eliminated medication pharmacokinetic parameters in patients with hemorrhagic stroke. Additionally, limited information was available on follow-up vancomycin dosing regimens and subsequent serum concentrations, and the impact on infectious outcomes as vancomycin was frequently discontinued in an effort to narrow antibiotic therapy. However, this study illustrates that enhanced renal clearance in patients with hemorrhagic stroke results in vancomycin pharmacokinetic alterations, which also raises questions regarding the proper dosing of other renally eliminated medications that do not undergo routine therapeutic drug monitoring. This is an important area for future study.
Conclusions
This is the first study to evaluate the impact of enhanced renal clearance on alterations in vancomycin pharmacokinetic parameters in patients with hemorrhagic stroke. Patients with hemorrhagic stroke exhibited a mean measured creatinine clearance greater than that estimated and pharmacokinetic parameter alterations favoring a faster elimination of vancomycin, resulting in subtherapeutic concentrations. These findings indicate that it is reasonable to measure CrCl via an 8-h urine collection in this patient population in order to properly dose adjust renally eliminated medications while also highlighting the need for future studies to determine optimal renally eliminated medication dosing strategies.
Abbreviations ARC: Augmented renal clearance; aSAH: Aneurysmal subarachnoid hemorrhage; CrCl: Creatinine clearance; GCS: Glasgow coma score; ICH: Intracerebral hemorrhage; ICU: Intensive care unit; LOS: Length of stay; PK: Pharmacokinetic; SOFA: Sequential Organ Failure Assessment
Table 1
1Patient characteristics GCS, Glasgow Coma Scale; SOFA, Sequential Organ Failure Assessment; ICH, intracerebral hemorrhage; SCr, serum creatinineVariable
Hemorrhagic stroke (n = 17)
Age (years), mean ± SD
63.3 ± 13.3
Weight (kg), mean ± SD
82.3 ± 15.4
Female gender, n (%)
11 (65)
Admission GCS, median (IQR)
9 (6-12)
Admission SOFA score, median (IQR)
4 (3-5)
Subarachnoid hemorrhage, n (%)
12 (71)
Hunt and Hess scale grade, median (IQR) 3 (2-4)
Modified Fisher grade, median (IQR)
3 (3-4)
Admission ICH score, median (IQR)
3.5 (3-4)
Admission ICH volume, median (IQR)
95.9 (59.7-136.5)
Admission SCr (mg/dL), mean ± SD
0.7 ± 0.1
Selected co-morbidities, n (%)
Hypertension
11 (65)
Diabetes
1 (6)
Congestive heart failure
2 (12)
Table 2
2Vancomycin characteristicsVariable
Table 3
3Pharmacokinetic parameters treatment failure and other serious patient complications.Variable
Predicted value
(n = 17)
Calculated value
(n = 17)
p value
Creatinine clearance (mL/min), mean ± SD
116.7 ± 10.3
161.6 ± 16.7
0.001
Elimination rate constant (h −1 ), mean ± SD
0.087 ± 0.01
0.141 ± 0.02
0.004
Half-life (h), mean ± SD
8.7 ± 0.6
6.5 ± 0.9
0.03
Volume of distribution (L), mean ± SD
57.6 ± 2.6
71.8 ± 11.3
0.23
AcknowledgementsNot applicable.Authors' contributions KM and DJ participated in data collection and enrolled patients. KD participated in data collection. KM, DR, and DJ participated in the design of the study and performed the statistical analysis. All authors conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.FundingUniversity of North Carolina School of Medicine, Department of Neurology-This funding body did not participate in the design of the study or collection, analysis, and interpretation of data or in writing the manuscript should be declared.Availability of data and materialsPlease contact author for data requests Ethics approval and consent to participate The University of North Carolina at Chapel Hill Institutional Review board approved this study with reference number 16-0917.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Author details 1 Division of Practice Advancement and Clinical Education, UNC Eshelman
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| Background and Purpose ::: Ethnic disparities in stroke are well described, with a higher incidence of disability and increased mortality in Blacks versus Whites. We sought to compare the clinical outcomes between those ethnic groups after stroke endovascular therapy (ET). ::: ::: ::: Methods ::: We performed a retrospective review of the prospectively acquired Grady Endovascular Stroke Outcomes Registry between September 1, 2010 and September 30, 2015. Patients were dichotomized into two groups - Caucasians and African-Americans - and matched for age, pretreatment glucose level, and baseline National Institutes of Health Stroke Scale (NIHSS) score. Baseline characteristics as well as procedural and outcome parameters were compared. ::: ::: ::: Results ::: Out of the 830 patients treated with ET, 308 pairs of patients (n = 616) underwent primary analysis. African-Americans were younger (p < 0.01), had a higher prevalence of hypertension (p < 0.01) and diabetes (p = 0.04), and had higher Alberta Stroke Program Early CT Score values (p = 0.03) and shorter times to treatment (p = 0.01). Blacks more frequently had Medicaid coverage and less private insurance (29.6 vs. 11.4% and 41.5 vs. 60.3%, respectively, p < 0.01). The remaining baseline characteristics, including baseline NIHSS score and CT perfusion-derived ischemic core volumes, were well balanced. There were no differences in the overall distribution of 90-day modified Rankin scale scores (p = 0.28), rates of successful reperfusion (84.7 vs. 85.7%, p = 0.91), good outcomes (49.1 vs. 44%, p = 0.24), or parenchymal hematomas (6.5 vs. 6.8%, p = 1.00). Blacks had lower 90-day mortality rates (18 vs. 24.6%, p = 0.04) in univariate analysis, which persisted as a nonsignificant trend after adjustment for potential confounders (OR 0.52, 95% CI 0.26-1.03, p = 0.06). ::: ::: ::: Conclusions ::: Despite unique baseline characteristics, African-Americans treated with ET for large vessel occlusion strokes have similar outcomes as Caucasians. Greater availability of ET may diminish the ethnic/racial disparities in stroke outcomes. | NOAC monotherapy in patients with concomitant indications for oral anticoagulation undergoing transcatheter aortic valve implantation | Background: Discontinuation of oral anticoagulant (OAC) therapy in patients with non-valvular atrial fibrillation (NVAF) leaves patients unprotected from risk of stroke. We investigated OAC treatment persistence in NVAF in primary care of 3 European countries. ::: ::: Methods: Three cohorts of patients | The US Food and Drug Administration has approved only one therapy for ischemic stroke, recombinant tissue plasminogen activator (tPA), which can increase blood flow to damaged brain tissue—but it can also have severe side effects and must be administered shortly after stroke. Experiments combining the drug with activated protein C (APC) may provide a solution (pages 1379–1383). |
I schemic stroke is a complex disease with many forms and many corresponding treatments that must be tailored to the patient. The consensus guidelines from the American Heart Association and American Stroke Association regarding acute ischemic stroke treatment mentions intra-arterial therapy as one of many tools in the armamentarium of ischemic stroke therapies. 1 Intra-arterial therapy is currently applicable to a small subset of patients with ischemic stroke, but it will likely have an expanding role as new devices are introduced. These expanding applications of intra-arterial therapy for ischemic stroke lead to speculation regarding the availability of a sufficient number of operators to treat these patients in the United States. 2 To address this issue of demand and available work force for intra-arterial ischemic stroke therapy, we must analyze the number of patients with ischemic stroke who might need this technique of treatment as well as the number of physicians who might be needed to provide it. We have no direct way to measure the number of ischemic strokes that might be amenable to intra-arterial therapy in the near future. This is because there are 2 major limitations in the analysis: 1) a lack of clear definition of which patients with ischemic stroke will be candidates for intra-arterial therapy, and 2) a lack of epidemiologic data to estimate how many patients are in various subgroups that might be deemed appropriate for intra-arterial therapy. There are also challenges in estimating the number of physicians who might be needed to provide intra-arterial ischemic stroke therapy in the United States. Despite these limitations, some reasonable estimates on the basis of currently available information can be made. The following is a review of the available information that can be used to estimate both a demand for intra-arterial ischemic stroke therapy and the work force available to provide such therapy.
Who Needs Intra-Arterial Ischemic Stroke Therapy?
Intra-arterial therapy has been shown to be efficacious in opening occluded arteries in some patients with severe ischemic stroke. [3][4][5][6] However, far from all patients with ischemic stroke are candidates for intra-arterial stroke therapy. Only patients with occlusion of relatively large intracranial arteries typically undergo recanalization intra-arterially. Potential benefit of intra-arterial therapy must be balanced against potential risk. The risks are not trivial, as intra-arterial therapy has been associated with a 5% to 7% risk for clinically significant procedural complications 4,7 and a 6% to 15% risk for symptomatic intracranial hemorrhage. [4][5][6][7][8][9] For patients with less severe stroke symptoms, the risks for intra-arterial therapy almost certainly outweigh the benefits. Patients with a National Institutes of Health Stroke Score (NIHSS) of less than 10 who are treated with intravenous recombinant tissue plasminogen activator (rtPA) have an 82% chance of a good outcome (modified Rankin Scale, 0 -2), a 3% chance of symptomatic hemorrhage, and a 1% chance of death. 10 Such patients with an NIHSS of less than 10 are quite unlikely to benefit from intra-arterial therapy, as they typically have normal results on cerebral angiograms or distal or recanalizing emboli. [11][12][13] The Interventional Management of Stroke (IMS) I 8 and IMS II 5 trials treated patients with NIHSS of 10 or more, and trials of the Merci Retrieval System (Concentric Medical, Mountain View, Calif) treated patients with NIHSS of 8 or more 3,4,14 and 10 or more. 15 Major stroke centers offering ischemic stroke therapy tend to follow this practice of reserving intra-arterial therapy for major strokes. [16][17][18][19][20][21][22][23] Intra-arterial therapy has not yet been shown to be definitively better than intravenous therapy in terms of neurologic outcomes. Table 1 shows comparative data from major studies completed thus far. In IMS I and IMS II, comparisons were made between patients treated with combined intravenous and intra-arterial therapy, and patients matched for age and NIHSS from the National Institute of Neurological Disorders and Stroke trial. These outcome data for patients treated with intravenous placebo and intravenous rtPA are also included in Table 1. The data in Table 1 indicate that much improvement could be made in outcomes from ischemic stroke therapies. More data are necessary to clearly define the role of intraarterial therapy techniques in ischemic stroke therapy.
IMS I and IMS II did not include enough patients to show a statistically significant benefit to intra-arterial therapy vs intravenous therapy, nor were they designed to do so. IMS III is now underway and will randomly assign patients to receive intravenous therapy or a combination of intravenous and intra-arterial therapy, with the hope of demonstrating clear evidence of benefit of intra-arterial therapy. 24 According to data from major studies of intra-arterial therapy, only 28% to 46% of patients treated achieve a good neurologic outcome (Table 1). That means that 54% to 72% have a bad outcome despite intra-arterial therapy, including 16% to 44% who are dead at 90 days (Table 1). Some proportion of the reported low efficacy of these trials likely results from imperfect recanalization rates, ranging from 46% to 66%, but much is undoubtedly because of infarctions that were already completed before the initiation of intra-arterial therapy. Many patients presenting less than 8 hours from symptom onset clearly do not have salvageable brain because of poor collaterals. 25 If good, rapid imaging capable of identifying ischemic penumbra were available to determine salvageable brain tissue (such as diffusion-weighted or perfusion-weighted MR imaging [26][27][28][29][30][31][32][33][34][35][36] or CT perfusion 37,38 ), intra-arterial interventions for ischemic stroke could be reduced by perhaps half because it would be possible to determine that infarction is complete before these patients are moved to an interventional suite. This would be of benefit to patients because it spares them futile, aggressive interventions. This is currently a topic of intense research, which hopefully will yield clinically useful triage techniques. The Magnetic Resonance and Recanalization of Stroke Clots Using Embolectomy study is a trial underway that may give insight into the value of MR in triaging patients with ischemic stroke. 39
How Many Patients Will Need Intra-arterial Therapy?
It has been estimated that approximately 0.07% 40 to 0.17% 22 of all patients with ischemic stroke received intra-arterial therapy in the United States from 1999 and 2002, which would amount to only approximately 1100 patients. The Merci Retrieval System (Concentric Medical) 3,4 and Penumbra System (Penumbra, Alameda, Calif) 41 were recently approved by the US Food and Drug Administration for the treatment of ischemic stroke, and use of these devices may have increased the number of patients treated with intra-arterial therapy. Thus, 1100 patients per year is the absolute minimal estimate of the number of patients in the United States who would be treated with intra-arterial therapy for ischemic stroke.
To estimate the other extreme, we can start with the overall number of strokes per year in the United States, which is a reasonably well-defined number of 700,000 to 750,000 strokes per year. [42][43][44][45][46] An important point of confusion in the literature on acute ischemic stroke is the common, hyperbolic use of this number. This statistic is inappropriate in discussions specifically focused on treatment of acute ischemic stroke because it includes all types of stroke and all levels of severity and thus leads to absurd leaps in logic and assumptions that all or most of these strokes need recanalization therapy. Approximately 14% of these patients with stroke have intracranial hemorrhage, including subarachnoid hemorrhage; 4% have transient ischemic attack; and 1% have "late effects of cerebrovascular disease." 45,46 Starting with 750,000 strokes per year and subtracting 19% to correct for cases that are not due to acute ischemia yields 645,000 ischemic strokes per year. This estimate is supported by a recent study by Qureshi et al, 47 which determined that there were approximately 1,260,000 hospital admissions for ischemic stroke in the United States in 2000 and 2001 (ie, 630,000 admissions for ischemic stroke per year).
Therefore, if we settle on an estimate of 645,000 ischemic strokes per year, how many patients will need intra-arterial therapy? Approximately 17% of ischemic strokes are from lacunar infarctions, 48,49 which reduces the number of potential intra-arterial therapy cases to 535,000. However, the nonlacunar infarctions are not defined well enough to allow us to determine what fraction of these might be amenable to endovascular therapy. Another way to refine the 645,000 ischemic strokes per year is on the basis of severity. As discussed above, only patents with severe ischemic stroke (ie, NIHSS Ն 10) are likely to benefit from intra-arterial therapy. Of acute ischemic strokes evaluated by the Greater Cincinnati/Northern Kentucky Stroke Team in 2005, 20% had an NIHSS of 10 or greater (Thomas Tomsick, unpublished data, 2008). Extrapolating this percentage to the entire US population would lead to an estimate of 126,000 patients with such severe acute ischemic strokes in the United States annually. Not all of these patients would be candidates for endovascular therapy, but this would be the size of the pool of patients with severe acute ischemic stroke from which patients undergoing endovascular therapy would be drawn; therefore, it is a theoretic maximum of endovascular therapy candidates. It is not possible to narrow the number of potential interventions for intra-arterial ischemic stroke further given the limitations of available epidemiologic data. However, we can further refine our estimate by looking at available data on patients treated with intravenous rtPA for acute ischemic stroke. The treatment of stroke as an emergency in the United States has been evolving since the US Food and Drug Administration approval of intravenous rtPA for ischemic stroke in 1996. Approximately 12,000 patients received intravenous rtPA for ischemic stroke in the United States during 2004, 50 which represents 2% of all patients with ischemic stroke. Studies have shown that even with very active and organized emergency medical services and stroke teams, this number only increases to approximately 9%. 51 Many of the patients treated with intravenous lysis do fine and do not require intra-arterial intervention, but this is hard to quantify. So if 645,000 ischemic strokes per year occur in the United States, and no more than 9%, or 58,000, would qualify for intravenous rtPA in the most aggressive stroke management setting, then the number of potential candidates for intra-arterial stroke therapy is well likely to be less 58,000 per year in the United States.
Much effort has already been put into improving emergency medical services for patients with stroke. 52 Delays in presentation to a hospital that provides acute stroke therapy can certainly disqualify many patients from treatments such as intravenous rtPA and intra-arterial therapy. Approximately 25% to 59% of patients with stroke arrive at an emergency department within 3 hours of onset of symptoms, and 35% to 66% arrive within 6 hours. 53 On the basis of these numbers, programs aimed at developing the general public's awareness of stroke symptoms and at minimizing the delay in transporting the patient to an appropriate medical center might be expected to increase the number of patients who might be treatable with intra-arterial therapy. Such an aggressive educational program in Texas increased the number of patients treated with intravenous thrombolysis by a factor of 4. 51 Improving the access of patients to acute stroke centers and educating physicians and patients to respond to stroke as an emergency can increase the demand for intra-arterial thrombolysis; however, this process will be gradual and must be dealt with primarily at the local level.
Conversely, as noted above, improvements in triage with use of new imaging modalities, such as with MR imaging or CT perfusion, may indicate absence of ischemic penumbra in perhaps half of all cases for which intra-arterial therapy is contemplated. However, penumbra imaging is not yet standard in the evaluation of patients with ischemic stroke.
Who Provides Care for Patients with Acute Ischemic Stroke?
The practice of acute stroke care is dependent on patient access to skilled physicians and technology in a stroke center committed to treat acute stroke as an emergency. Intravenous thrombolysis can be administered safely and effectively in small hospitals, preferably under the guidance of stroke spe-cialists. Subsequent acute stroke care should be provided in specialized stroke centers. Stroke centers should be able to care for patients with all subtypes of stroke, including hemorrhagic strokes that require treatment by a neurosurgeon. Expert-level care has been shown to improve the care of patients with acute stroke. [54][55][56] Formal stroke center recognition can help to consolidate resources such as diagnostic capabilities and personnel trained to implement evidence-based practices, and also to bring public attention to the location of these centers of expertise. In 2000, the Brain Attack Coalition proposed 2 types of stroke centers: primary and comprehensive. 57 A primary stroke center has the staffing, infrastructure, and programs necessary to stabilize and treat most patients with acute stroke. A comprehensive stroke center is defined as a facility with the staffing, infrastructure, and programs to diagnose and treat patients with stroke who require a high intensity of medical and surgical care, specialized tests, or interventional therapies. 58 In 2003, the Joint Commission began certifying primary stroke centers. There are now 401 primary stroke centers certified by the Joint Commission. 59 This certification is based on the recommendations of the Brain Attack Coalition. 57 The requirements for primary stroke center certification do not include availability of intra-arterial ischemic stroke therapy or a neurologist. They also do not include an on-site stroke unit or on-site neurosurgery, as it is understood that some primary stroke centers will stabilize patients and then transfer them to other centers for more advanced care. This paradigm is already being developed as a "drip-and-ship" approach, 60 in which primary stroke centers initiate intravenous rtPA therapy and then transport patients to a comprehensive stroke center. With many primary stroke centers functioning with this "first stop" model, it is not reasonable to expect that all primary stroke centers will provide intra-arterial ischemic stroke therapy.
Recommendations for access to endovascular therapy were reserved for comprehensive stroke centers, which would offer a higher level of care than primary stroke centers. 58 This recommendation is also in accordance with the guidelines for the early management of adults with ischemic stroke from the American Heart Association and American Stroke Association, which states that intra-arterial therapy "will be limited to those comprehensive stroke centers that have the resources and physician expertise to perform these procedures safely." 1 The Joint Commission has not yet begun a program to certify comprehensive stroke centers. If comprehensive stroke center certification becomes a reality, it would be expected that primary stroke centers would refer more difficult patients to these centers. Many regional comprehensive stroke centers already exist in practice, but a formal certification and recognition process would be a significant advance in the development of a national system to address the needs of patients with stroke.
Epidemiologic statistics are bandied about, implying that huge numbers of patients with stroke are not getting appropriate care, 2 but such implications completely ignore the experience of leading acute stroke centers and work done at such centers to improve stroke care. Single-center reports from well-developed stroke centers doing intra-arterial ischemic stroke therapy cases demonstrate a case rate of 3 to 30 cases per year per medical center (Table 2). These centers are national and international leaders in acute stroke intervention, so it is reasonable to assume that patients are being treated with an appropriately aggressive level of care. Also, as tertiary referral centers for patients with stroke, these hospitals would be expected to receive many more patients with strokes than hospitals that do not specialize in stroke. A number of multicenter trials of intra-arterial therapies for ischemic stroke have been performed. These trials give us some insight into the quantity of patients that might be treated with intra-arterial therapy at leading stroke centers. The highest enrollment rate for any hospital in any of these trials was 27 cases per year ( Table 2). Even if rates of intra-arterial therapies were to go as high as rates of intravenous rtPA treatment of ischemic stroke, the rate would only go as high as 61 cases per year per hospital at the busiest of these tertiary centers ( Table 2).
The delivery of stroke care is analogous to the delivery of trauma care, in that a regional system is needed that provides rapid, skilled care to as many people as is practical. A regional care model has also been applied to myocardial infarctions, but this has not been implemented on a national level as in trauma. 61 Designated trauma centers have been developed throughout the United States. The number of level I trauma centers in the United States is 190. 62 Perhaps similar-level comprehensive stroke centers might be designated in the future. If each of these comprehensive stroke centers were staffed by 2 neurointerventionists, then the total number of required neurointerventionists would be approximately 400.
If each of an estimated 200 comprehensive stroke centers treated 100 patients per year with intra-arterial thrombolysis, 20,000 individuals would be treated annually in the United States. A rate of 100 patients per year per medical center is at least 3 times the rate of stroke centers that aggressively use intra-arterial therapy, and twice the rate of intravenous therapy at aggressive stroke centers ( Table 2). That translates to 3% of the 645,000 patients hospitalized for acute ischemic stroke in the United States each year who could be treated with intra-arterial methods; this rate is twice the percentage of patients with acute stroke who qualified for intra-arterial thrombolysis in the Prolyse in Acute Cerebral Thromboembolism II study. 6 Although comprehensive stroke centers have not yet been officially designated as such, many US hospitals are currently operating in this capacity unofficially. Most centers with trained, specialized neurointerventionalists would likely meet the criteria for a comprehensive stroke center. In 2002, Suzuki et al 63 Because stroke is a major health problem, it makes sense that skilled care be available from neurologic experts. Qualification standards have already been published as agreed on by multidisciplinary groups of neurologists, neurosurgeons, and neuroradiologists who perform these procedures. 64 The American College of Graduate Medical Education has also defined training standards for neurointervention. 65 It is rather naive to assume that such advanced care can be delivered by physicians without expertise in the neurosciences. Each acute stroke center that offers intra-arterial ischemic stroke therapy needs to assure that this therapy is being offered by qualified individuals.
Conclusions
The number of acute ischemic strokes in the United States that will be amenable to intra-arterial therapy can only be crudely estimated, but it is certainly less than the total number of 126,000 severe acute ischemic strokes per year and quite likely to be no more than 20,000 cases per year. The future demand for intra-arterial reperfusion techniques may change, but the number of patients who require intra-arterial thrombolysis is currently quite low, and the number of neurointerventionists Intra-arterial treatment, multicenter trials PROACT, 1998 9 1 (0-10) PROACT II, 1999 6 1 (0-17) EMS, 1999 66 5 (2-10) IMS-I, 2004 8 7 (0-27) MERCI, 2005 7 3 (0-9) IMS-II, 2007 5 2 (NA) Multi MERCI, 2008 4 5 (0-20) Intra-arterial treatment, single center Barnwell et al, 1994 19 7 Suarez et al, 1999 18 15 Jahan et al, 1999 67 5 Ernst et al, 2000 20 17 Hill et al, 2002 21 4 Ramee et al, 2004 68 3 Choi et al, 2006 22 9 Devlin et al, 2007 23 30 Kim et al, 2007 17 13 Wolfe et al, 2008 16 15 Intravenous treatment, multicenter trials NINDS, 1995 69 20 ATLANTIS, 1999 70 1 STARS, 2000 71
Table 1 :
1Outcomes of intra-arterial therapy in major trialsStudy
Mean Baseline
NIHSS
Recanalization
(TIMI 2 or 3)
Good Outcome at 90 Days
(mRS 0-2)
Mortality at
90 Days
Symptomatic
ICH
Intra-arterial therapy
PROACT 9
17
58%
NA
27%
15%
PROACT II 6
17
66%
40%
25%
10%
IMS-I 8
18
56%
43%
16%
6%
IMS-II 5
19
60%
46%
16%
10%
MERCI 7
22
46%
28%
44%
8%
Multi MERCI 4
19
68%
36%
34%
10%
Penumbra pivotal study
18
82%
25%
33%
11%
Nonarterial therapy
PROACT 9
19
14%
NA
43%
7%
PROACT II 2 control
17
18%
25%
27%
2%
NINDS placebo* 5
18
NA
28%
24%
1%
NINDS IV rtPA* 5
18
NA
39%
21%
7%
Note:-NIHSS indicates National Institutes of Health Stroke Scale; TIMI, Thrombolysis in Myocardial Infarction trial; ICH, intracranial hemorrhage; mRS, modified Rankin Scale; PROACT,
Prolyse in Acute Cerebral Thromboembolism trial; IMS, Interventional Management of Stroke trials; MERCI, Mechanical Embolus Removal in Cerebral Ischemia trials; NINDS, National
Institute of Neurological Disorders and Stroke; NA, not available.
* NINDS is subgroup of patients with an NIHSS of 10 or more.
identified 385 neurointerventionists in 238 hospitals covering 45 states. Suzuki et al 63 determined that 99% of the total US population lived within 200 miles of a neurointerventional practice, and 82% lived within a 65-mile radius. The Society of Neurointerventional Surgery (SNIS) is the largest society of practicing neurointerventionalists in the United States, with members representing neuroradiology, neurosurgery, and neurology. There were 301 senior members of the SNIS practicing in the United States in 2008. This number has increased by 50% (from 208) since 2001. Not all neurointerventionalists are members of this society, so the actual number of practicing neurointerventionalists is undoubtedly higher.The number of neurointerventionalists can be expected to increase steadily as training programs continue to provide the necessary advanced training to neuroradiologists, neurosurgeons, and neurologists.
Table 2 :
2Demand for thrombolysis in the United States based on the published literatureReport
Cases per Year
per Hospital*
Note:-PROACT indicates Prolyse in Acute Cerebral Thromboembolism trial; EMS, Emergency Management of Stroke trial; IMS, Interventional Management of Stroke trials; MERCI, Mechanical Embolus Removal in Cerebral Ischemia trials; NINDS, National Institute of Neurological Disorders and Stroke; ATLANTIS, Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke trial; STARS, Standard Treatment with Alteplase to Reverse Stroke study. * Numbers in parentheses are ranges.4
Intravenous treatment, single center
Chiu et al, 1998 72
30
Zweifler et al, 1998 73
9
Grotta et al, 2001 74
61
Kahn et al, 2005 75
26
Wolfe et al, 2008 16
45
Arenillas et al 76
46
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| 498 | Background: The first specific antidote for non-vitamin K antagonist oral anticoagulants (NOAC) has recently been approved. NOAC antidotes will allow specific tre | Pros and cons of vitamin K antagonists and non-vitamin K antagonist oral anticoagulants. | Non-vitamin K antagonist oral anticoagulants in atrial fibrillation patients undergoing elective cardioversion | The effect of vitamin K supplementation on anticoagulant treatment | Abstract 18371: Higher Treatment Persistence of Non-Vitamin K Antagonist Oral Anticoagulants than Vitamin K Antagonists at 1 Year in Non-Valvular Atrial Fibrillation in Real-World Practice | Non-vitamin K antagonist oral anticoagulants (NOACs)are a widely prescribed treatment to prevent stroke in patients with nonvalvular atrial fibrillation, and a therapy and preventative measure to prevent recurrences following venous thromboembolism. Optimal use of NOACs requires a thorough knowledge of the pharmacology of these drugs, as well as an understanding of patient factors affecting their use. The 4 NOACs-dabigatran, apixaban, edoxaban, and rivaroxaban are available in a range of doses suitable for differing indications and with a variety of dose reduction criteria. Identification of the correct dose is one of the key challenges in the individualization of treatment. Elderly patients with atrial fibrillation are at a greater risk of both ischemic and bleeding events than younger patients. Consequently, it is essential to achieve balance in anticoagulation strategies. Medication adherence to NOACs is important for safe and effective treatment, particularly in elderly populations. A growing body of evidence shows that once-daily dosing improves adherence and persistence to therapy, without having an impact on bleeding risk.6,7,10,15,16,[29][30][31][32]†This is theoretical and only true if dosing is precisely followed. Plasma concentrations have shown large variations between the 10th and 90th percentile at both peak and trough.INR, international normalized ratio. Sources:6,7,10,15,16,[20][21][22][23]Patti and Haas *In patients concomitantly taking edoxaban and the following P-gp inhibitors: ciclosporin, dronedarone, erythromycin, or ketoconazole, the recommended dose is 30 mg once daily. CrCl, creatine clearance. Sources:7,10,15,16Patti and Haas | New Oral Anticoagulants in Coronary Artery Disease. | Is There an Obesity Paradox for Outcomes in Atrial Fibrillation?: A Systematic Review and Meta-Analysis of Non–Vitamin K Antagonist Oral Anticoagulant Trials | Periprocedural Management of Direct Oral Anticoagulants Should Be Guided by Accurate Laboratory Tests |
499 | The bioreductive alkylating agent mitomycin C (mitomycin) has been shown to have greater activity under hypoxic than oxic conditions on murine cell lines such as the EMT-6 fibrosarcoma cell line. Solid tumors are known to contain hypoxic cells and are relatively resistant to ionizing radiation and some chemotherapeutic agents. We tested the cytotoxicity of mitomycin against fresh biopsies of human carcinomas under both hypoxic and oxic conditions in the human tumor clonogenic assay (HTCA). Additionally, we examined the metabolism of mitomycin by sonicates of the murine EMT-6 cells and the human WiDR colon carcinoma cells. We confirmed that under our clonogenic assay conditions the EMT-6 cell line was more sensitive to mitomycin under hypoxic than oxic conditions. Additionally, we established that EMT-6 cells also metabolize mitomycin at a more rapid rate under hypoxic than oxic conditions. However, these effects of hypoxia on mitomycin activity were not demonstrable for the human WiDR colon cancer cell line. In addition to these findings, the cytotoxicity of mitomycin was either unchanged or reduced under hypoxic conditions for ten fresh human tumors tested for mitomycin sensitivity in HTCA. Based on these observations, we conclude that the potentiating effect of hypoxia on mitomycin metabolism and biological activity may be peculiar to the murine EMT-6 and S-180 cell lines and that mitomycin C is not likely to have differential efficacy against hypoxic human carcinoma cells. | Mitomycin-C is used for the treatment of different types of tumours. In present work, a reliable method was developed for radiolabeling of Mitomycin-C with 99mTc for diagnostic purpose. 99mTc-Mitomycin-C was obtained with radiochemical yield of 100 % by adding 200 µg Mitomycin-C, 1 mL (15 mCi) of pertechnetate in 25 μg SnCl2·2H2O at pH 7. Labeling efficiency was determined by paper chromatography and ITLC. The charge on 99mTc-Mitomycin-C was determined by electrophoresis technique. HPLC analyses were performed for the determination of purity of Mitomycin-C and radiochemical purity of labeled complex. Evaluation of 99mTc-Mitomycin-C, in vitro stability, biodistribution and scintigraphic images in normal mice were performed. |
INTRODUCTION
Heterogeneous tumour oxygenation has long been recognized as a major impediment to the development of effective cancer therapies (Hockel et al., 1996;Nordsmark et al., 2005;Wilson and Hay, 2011;Marusyk et al., 2012). Given the prevalence of tumour hypoxia (Fukumura and Jain, 2007;Pries et al., 2009) and its association with treatment failure (Vaupel and Mayer, 2007), the use of hypoxia-activated prodrugs is a noteworthy approach to improve clinical outcomes. Hypoxia-activated prodrugs are designed to preferentially target regions of severe hypoxia, allowing tumour dose intensification by circumventing the normal tissue toxicities (Denny, 2005;Denny, 2010) that traditionally limit the potential for dose-escalation with conventional chemotherapeutic regimens (Rowinsky, 2000;Crawford, 2013).
Essentially, most hypoxia-activated prodrugs are comprised of a bioreductive switch or trigger unit that deactivates or masks the cytotoxic effector under aerobic conditions. Computational studies have elegantly shown that the efficacy of a hypoxiaactivated prodrug is a delicate balance between its cellular affinity (reversible binding or sequestration), metabolic stability (prodrug activation rate and O 2 -dependence), potency and diffusion capabilities of the released effector (Hicks et al., 1998;Hicks et al., 2003;Foehrenbacher et al., 2013a;Foehrenbacher et al., 2013b;Hong et al., 2018a;Hong et al., 2019). In which, the prodrug must penetrate relatively long distances through the extravascular compartment in order to reach the cells that are sufficiently hypoxic for its metabolic activation and subsequent cytotoxicity. The localization of the hypoxic target cells and the relative degree of effector redistribution (known as the bystander effect) determine the resultant cell killing. The limited success of earlier clinical candidates reflects, in part, the complexity of this design criteria (Marcu and Olver, 2006;Spiegelberg et al., 2019;Li et al., 2021).
A noteworthy example is the nitroaromatic compound PR-104A. Experimental studies showed that PR-104A is readily metabolised by endogenous one-electron reductases (e.g. cytochrome P450 oxidoreductase (POR) and other diflavin oxidoreductases) into a radical species that spontaneously converts under hypoxic conditions into DNA cross-linking metabolites (notably hydroxylamine PR-104H and amine PR-104M) (Guise et al., 2007;Patterson et al., 2007;Singleton et al., 2009;Guise et al., 2012). Computational studies indicated favourable pharmacokinetic properties for both single agent and combinatorial activity owing to its strict oxygen dependence (the half-maximal activation occurs at KO 2 0.126 µM O 2 ) and bystander efficiency in experimental models (Hicks et al., 2007;Foehrenbacher et al., 2013a;Foehrenbacher et al., 2013b). A sizeable bystander effect was shown using various multicellular layer (MCL) (Foehrenbacher et al., 2013a), spheroid co-culture (Hong et al., 2018b) and tumour xenograft models Foehrenbacher et al., 2013a). Combinatorial activity was noted with radiation (Hicks et al., 2007) as well as various systemic agents Abbattista et al., 2015).
Despite its promising preclinical activity, PR-104A, when administered to patients as its phosphate pre-prodrug form PR-104, failed to demonstrate significant clinical efficacy due to its myelotoxicity profile. The principal toxicities of neutropenia and thrombocytopenia aligned with those of conventional therapies resulting in additive toxicity rather than efficacy upon co-administration (Jameson et al., 2010;Abou-Alfa et al., 2011;Mckeage et al., 2011;Mckeage et al., 2012). A retrospective study identified an alternative route of PR-104A activation involving two-electron metabolism by aldo-keto reductase 1C3 (AKR1C3) that bypasses the oxygen-dependent step in the activation schematic (Guise et al., 2010). The off-target activation of PR-104A was specific to the human AKR1C3 orthologue explaining the clear oversight during its preclinical development (Guise et al., 2010;Van Der Wiel et al., 2021).
CP-506 is a second-generation PR-104A analogue that is rationally designed to be resistant to AKR1C3 activation in human tissues (Van Der Wiel et al., 2021). The mechanism of activation involves an initial one-electron reduction by endogenous oxidoreductases to a nitro radical anion intermediate that acts as a direct oxygen sensor, as previously described for PR-104A . Experimental studies have shown that this reaction is catalysed efficiently by cytochrome P450 oxidoreductase (POR), although a number of other flavoenzymes can also contribute (Van Der Wiel et al., 2021). Further reduction leads to the formation of various DNA cross-linking metabolites selectively under hypoxic conditions. While CP-506 shows similar potency to PR-104A in antiproliferative assays, the oxygen-dependence of prodrug activation and cytotoxicity is more favourable with complete inhibition above 1 µM O 2 and through the physiological O 2 concentration range (Van Der Wiel et al., 2021).
In the present study, we investigate the extravascular transport properties and bystander efficiency of CP-506 in tumour tissues by coupling various experimental and computational modelling approaches. The experimental framework involves a series of novel in vitro systems to measure reaction-diffusion in cells and 3D tumour models. Then, a well-validated in silico spatiallyresolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling approach (Hicks et al., 1997;Hicks et al., 2006;Hicks et al., 2010;Foehrenbacher et al., 2013b) was applied to estimate parameters for the cellular uptake, metabolism and diffusion of CP-506 and its active metabolites in the tumour tissue (Foehrenbacher et al., 2013a;Hong et al., 2018b). A cellular PK model was first developed by quantifying the extracellular (C e ) to intracellular (C i ) partitioning ratio for CP-506 and its formed metabolites in monolayer cultures treated under normoxic (21% O 2 ) and anoxic conditions (<1 ppm O 2 ). The derived reaction and diffusion terms were then scaled to tissuelike densities based on the transport of the prodrug and its effectors across multicellular layer (MCL) cultures maintained under supraoxic (95% O 2 ) and hypoxic conditions (<1 ppm O 2 ). Next, the parameter estimates were validated by comparing theoretical predictions to experimental determinations of clonogenic cell killing in spheroid co-cultures by agent-based modelling (ABM) (Hong et al., 2018b). Differences in fluorescent protein expression (mRuby vs. EGFP), antibiotic resistance genes (puromycin vs. geneticin) and POR expression (high vs. null) were used to delineate between the subpopulations of metabolically competent "activator" and metabolically defective "target" cells to interpret experimental findings. We conclude by simulating the spatial gradients of CP-506 and its active metabolites in a transverse section of a representative spheroid. Our findings illustrate the superior tissue pharmacokinetic properties of CP-506 relative to PR-104A and support its future clinical evaluation in patients with advanced solid malignancies.
MATERIALS AND METHODS
Compounds carbonyl]-2-(methylsulfonyl)-4-nitro anilino]ethyl methanesulfonate), CP-506H (2-((2-bromoethyl)(5-(4-ethylpiperazine-1-carbonyl)-4-(hydroxyamino)-2-(methylsulfonyl)phenyl)amino)ethyl methanesulfonate) and CP-506M (2-((4-amino-5-(4ethylpiperazine-1-carbonyl)-2-(methylsulfonyl)phenyl)(2bromoethyl)amino)ethyl methanesulfonate) and their respective deuterated (D8) standards (prepared from D8-1ethylpiperazine) were manufactured by Mercachem (Nijmegen, the Netherlands) employing a synthetic route developed at the University of Auckland as previously described (Van Der Wiel et al., 2021). The downstream metabolites CP-506H-(OH) 2 (5-(bis(2-hydroxyethyl)amino)-2-(hydroxyamino)-4-(methylsulfonyl)phenyl)(4-ethylpiperazin -1-yl)methanone), CP-506M-(OH) 2 (2-amino-5-(bis(2hydroxyethyl)amino)-4-(methylsulfonyl)phenyl)(4-ethylpiperazin-1-yl)methanone), CP-506H-Cl 2 (5-(bis(2-chloroethyl)amino)-2 -(hydroxyamino)-4-(methylsulfonyl)phenyl)(4-ethylpiperazin-1-yl)methanone), CP-506M-Cl 2 (2-amino-5-(bis(2-chloroethyl) amino)-4-(methylsulfonyl)phenyl)(4-ethylpiperazin-1-yl) methanone) and their respective deuterated (D8) standards (prepared from D8-diethanolamine) were synthesized at the University of Auckland as described in the supplementary methods. Stock solutions were prepared in DMSO (CP-506, CP-506H and CP-506M) or acetonitrile (CP-506H-Cl 2 , CP-506H-(OH) 2 , CP-506M-Cl 2 , CP-506M-(OH) 2 ) to concentrations of up to 100 mM and stored at −80°C until use. Working solutions were made by dilution into culture medium immediately before drug addition on the day of the experiment.
Cell Culture
The HCT-116 cell line was purchased from the American Type Culture Collection (Manassas, VA). A POR-overexpressing clone (POR-R) was generated by transfecting the parental cell line with a pBRP expression vector encoding an N-terminal truncated soluble version of the human POR gene (Foehrenbacher et al., 2013a;Guise et al., 2020). Ectopic expression is driven by the human cytomegalovirus (CMV) major immediate-early promoter. Downstream of POR is a human elongation factor-1 alpha (EF-1 alpha) promoter, which drives expression of mRuby and the puromycin resistance gene pac (puromycin N-acetyltransferase). A POR-null clone (PORG-ko) was generated as previously described (Su et al., 2013). POR-null clones were then transfected with a pEGFP-N1 plasmid encoding enhanced green fluorescent protein (EGFP) and geneticin resistance to permit identification upon co-culture (Hong et al., 2018b). Cell lines were maintained by weekly subculture in T-flasks containing alpha minimal essential medium (αMEM) with 5% foetal bovine serum (FBS) for a maximum of 24 passages or 90 days (whichever came first). The culture medium was further supplemented with 1 mg/ml geneticin or 2 µM puromycin for the routine maintenance of the POR-null and POR-overexpressing clones respectively. All cultures were reestablished from STR-authenticated, mycoplasma negative frozen stocks.
Anti-Proliferative IC 50 Assays
Cells (700-1,000 per well) were seeded with 100 µl culture medium into 96-well plates and left to attach for 2 h in a humidified 37°C incubator. Compounds were prepared in culture medium and serially diluted (1:3) in the well volume. Plates were returned to a humidified 37°C incubator for the duration of the drug exposure (4 h). Aerobic experiments were performed under standard tissue culture conditions (21% O 2 ), and the anoxic experiments were performed in parallel in a 5% H 2 /Pd catalyst anaerobic chamber (Shellab Bactron, Sheldon manufacturing Inc., Cornelius, OR) using pre-equilibrated media and plasticware. All anoxia experiments were conducted using a catalyst-scrubbed anaerobic chamber, unless otherwise specified, with gas oxygen measurements of <1 ppm O 2 . Cells were then washed three times and left to grow for 96 h before cell density determination using a sulforhodamine B (SRB) assay (Vichai and Kirtikara, 2006). To this end, the cells were fixed by layering 50 µl of trichloroacetic acid (40% w/v in MilliQ water) on top of the culture media (150 µl) in each well for 1 h at 4°C. Plates were rinsed under running tap water and stained with 50 µl of SRB (0.4% w/v in MilliQ water containing 1% v/v acetic acid) for 30 min at room temperature. Plates were rapidly destained by rinsing three times in tap water containing 1% (v/v) acetic acid. The stain was solubilized by adding 100 µl of 10 mM unbuffered Tris base to each well with gentle rocking for 1 h. Plates were read on a Biotek ELx808 Absorbance Microplate Reader at 490 nm using KC4 software. The IC 50 value (concentration required to reduce staining to 50% of the untreated control on the same plate) for each compound was determined by interpolation using a 4parameter logistic regression model.
Monolayer Clonogenic Assays
Cells (4.8 x 10 5 cells) were seeded with 2 ml culture medium into 6-well plates and left to attach for 2 h in a humidified 37°C incubator. Compounds were then prepared in culture medium and added in a 500 µl volume to each well. Plates were returned to a humidified 37°C incubator for the duration of the drug exposure (4 h). Aerobic and anoxic experiments were performed as described above. Plates were then removed from the anaerobic chamber and processed alongside the aerobic cultures. Cells were detached from monolayer by trypsinisation and plated at serial dilutions in 6-well plates. Cells were left to grow in a standard 37°C incubator for 10 days until the controls formed discrete Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602 colonies comprised of at least 50 cells. Colonies were visualized by staining with methylene blue (2 g/L in 50% aqueous ethanol) for 30 min. The number of colonies with at least 50 cells was manually counted to determine the plating efficiency. The surviving fraction was determined as the ratio of plating efficiency of treated cells to that of the controls.
Cellular Metabolism Assay
Aerobic and anoxic conditions were achieved as described above. Cells (5 x 10 5 per well) were seeded with 350 µl medium in duplicate into 24-well plates and left to attach for 2 h in a humidified 37°C incubator. A working solution of CP-506 was prepared in culture medium to achieve a 100 µM concentration in each well after the addition of a 500 µl volume. Plates were exposed to drug for 1 h in a humidified 37°C incubator. Metabolism was then halted by the addition of twovolumes of ice-cold acetonitrile containing 2 µM of internal standard for each analyte. Samples were stored at −80°C until LC-MS/MS analysis.
Media Stability Studies
The stability of each compound was determined in stirred medium (without serum) maintained under aerobic conditions by the constant supply of a humidified gas mixture comprised of 21% O 2 , 5% CO 2 with balance N 2 . Vials were spiked with 100 µM of the relevant compound, and samples were collected using a positive displacement pipettor for up to 3 h thereafter. The collected samples were then treated with two-volumes of icecold acetonitrile containing 2 µM of the relevant deuterated (D8) internal standard and stored at −80°C until LC-MS/MS analysis.
Cellular Uptake Studies
The intracellular/extracellular partitioning ratio for CP-506 and its major metabolites was determined in monolayer cultures of POR-overexpressing HCT-116 cells, as described previously (Hong et al., 2018b). Briefly, cells (5 x 10 5 per well) were seeded with 350 µl medium into 24-well plates and left to attach for 2 h in a humidified 37°C incubator. Stock solutions of CP-506 were diluted and added with 50 µl medium to the well volume to achieve a final concentration of 100 µM. Samples were harvested at various time points for up to 3 h thereafter. Aerobic experiments were performed under standard tissue culture conditions (21% O 2 ), and the anoxic experiments in parallel in a H 2 /Pd-catalyst-scrubbed anaerobic chamber as described. The well volume was spiked with [ 3 H]-mannitol immediately before sample harvest and treated with two-volumes of ice-cold acetonitrile containing 2 µM of the deuterated (D8) internal standards for each CP-506, CP-506H and CP-506M. The cell monolayer was then treated with 100 µl of this crashing solution.
Samples were stored at −80°C until LC-MS/MS analysis. The cellular exclusion of [ 3 H]-mannitol was used to account for the contribution of the extracellular medium toward determinations of the intracellular concentration, as described previously (Foehrenbacher et al., 2013a).
Multicellular Layer Flux Studies
The extravascular transport properties of CP-506 and its formed metabolites were investigated using a custom-designed diffusion apparatus, as described previously (Hicks et al., 2006). Briefly, MCL cultures were established by seeding 1 x 10 6 POR-R cells onto collagen-coated Millicell-CM inserts for 3 days. Established MCLs were placed at the interface of the two media-filled compartments of diffusion chambers. Diffusion chambers were placed in a 37°C water bath and gassed with a supraoxic (95% O 2 , 5% CO 2 ) or anoxic (5% CO 2 , bal N 2 ) mixture for at least 1 h prior to drug addition. Flux was initiated by the addition of internal standard ([ 14 C]-urea) and 20 µM CP-506 to the donor compartment. Samples (100 µl) were collected from both the donor and receiver compartments for up to 5 h thereafter. At each time point, a 25 µl aliquot was mixed with emulsifier-safe wateraccepting scintillant and assayed for radioactivity using a Perkin Elmer Tricard 2910 TR liquid scintillation analyser. The remaining sample was treated with two volumes of ice-cold acetonitrile (containing 2 µM of the relevant deuterated internal standards) and stored at −80°C until LC-MS/MS analysis. Similar experiments were performed using collagencoated inserts without MCLs to deduce the chemical stability and diffusion coefficient of CP-506 across the bare support membrane.
Spheroid Growth and Survival Assays
Spheroid cultures were established by seeding 3 x 10 3 cells with 100 µl medium into ultra-low attachment, round bottom 96-well plates (Corning Inc, Corning, NY). Plates were briefly centrifuged (200 g, 5 min) to facilitate cell aggregation and grown in a standard incubator (37°C, 21% O 2 , 5% CO 2 ) for 4 days before experiments. The culture medium was then aspirated, and the established spheroids taken into a H 2 /Pd-catalyst anaerobic chamber. Spheroids were washed three times with anoxia preequilibrated medium and left to settle for 1 h in a humidified 37°C incubator. Spheroids were removed from the anaerobic chamber after a 4 h drug exposure and washed three times with fresh culture medium. The growth of representative spheroids was monitored for 10 days using a published method of image analysis for volume determination (Mao et al., 2018). The remainder were enzymatically dissociated and plated at serial dilutions in 6-well plates containing 2 µM puromycin or 1 mg/ml geneticin for positive selection of POR-R and PORko-G cells, respectively. Colonies were visualized after 10 days of drug-free proliferation by staining with methylene blue (2 g/L in 50% aqueous ethanol) for 30 min. The number of colonies with at least 50 cells was manually counted to determine the plating efficiency. The surviving fraction was determined as the ratio of the plating efficiency of treated cells to that of the controls.
Flow Cytometric Detection of EF5 Binding in Multicellular Spheroids
Spheroid cultures were established by seeding 3 x 10 3 cells into ultra-low attachment, round bottom 96-well plates, as described above. The culture medium was then aspirated, and the established spheroids taken into the H 2 /Pd-catalyst anaerobic chamber. Spheroids were washed three times with anoxia preequilibrated medium and left to settle for 1 h in a humidified 37°C incubator. Spheroids were then treated with 100 µM EF5 for 4 h at 37°C. Plates were removed from the anaerobic chamber and washed three times with fresh culture medium. Spheroids were then dissociated by trypsinisation, washed three times with 0.1 M PBS and fixed in 4% formaldehyde. Samples were blocked with PBS containing 0.3% Tween-20 (PBS-T) and 10% FBS for 1 h at 4°C. EF5 adducts were then stained with 10 μg/ml Alexa Fluor 488-conjugated Elk3-51 antibody (supplied by Prof. Cameron Koch, University of Pennsylvania) diluted in incubation buffer (5% FBS, PBS-T v/v) overnight at 4°C. After a further three washes in PBS-T, cells were stored in 500 μl of PBS at 4°C until analysis on a BD Accuri flow cytometer (BD Biosciences, Franklin Lakes, NJ). A primary gate was set based on forward and side scatter to exclude cellular debris, dead cells and doublets. A secondary gate was set based on the selective metabolism and binding of EF5 in monolayer cultures treated under anaerobic conditions (<1 ppm O 2 ) compared to standard tissue culture conditions (37°C, 21% O 2 , 5% CO 2 ) to define EF5-positive events.
Flow Cytometric Analysis of Fluorescent Protein Expression
The relative proportion of POR-R ("activator") and PORko-G ("target") cells in spheroid co-cultures was quantified by the flow cytometric detection of EGFP expression in paired samples. Established spheroids were enzymatically dissociated and resuspended in 500 µl of 0.1 M PBS for analysis on a BD Accuri flow cytometer (BD Biosciences, Franklin Lakes, NJ). A primary gate was set based on forward and side scatter to exclude cellular debris, dead cells and doublets. A secondary gate was set based on the basal fluorescence of the parental HCT-116 cell line to define GFP-positive events.
Western Immunoblotting
Cellular lysates were prepared from log-phase cultures using modified radioimmuno-precipitation assay (RIPA) buffer (50 mM Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1mM EDTA, 1 mM Na 3 VO 4 , 1 mM NaF, 1:100 protease inhibitor cocktail (Roche, Basel, Switzerland)). Cellular lysates were then diluted in sample buffer (4 x LDS sample buffer containing 5% β-mercaptoethanol) and loaded with equal amounts of protein (30 µg) into BOLT precast gels (Thermofisher Scientific, Carlsbad, CA). Proteins were separated at 100 V for 1 h using BOLT MES SDS running buffer (Thermofisher Scientific, Carlsbad, CA) and transferred at 100 V for 1 h onto polyvinylidene difluoride (PVDF) membranes (pre-soaked in 100% methanol for 5 min) using ice-cold transfer buffer (14.4 g glycine, 3 g Trisma base, 200 ml methanol, 800 ml MilliQ water). Membranes were incubated with blocking solution for 1 h followed by the relevant primary antibody overnight at 4 o C. Membranes were then washed three times with 0.1 M TBS-T (48 g Tris-HCl, 11.2 g Trisma base, 176 g NaCl, 20 ml Tween-20, 1800 ml MilliQ water (w/ v)) and incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. After a further three washes with 0.1 M TBS-T, bands were visualized with Supersignal West Pico Chemiluminescent substrate (Thermofisher Scientific, Carlsbad, CA) using a ChemiDoc MP Imaging System (Biorad Laboratories, Hercules, CA). Band densitometry was performed using Image J software (Schneider et al., 2012). Additional details regarding the antibodies and blocking solutions used in this study can be found in Supplementary Table S1.
LC-MS/MS Analysis of CP-506 and Metabolites
The LC system for tandem MS consisted of an Agilent 1200 autosampler (4°C) and binary pump, a 3.0 × 50 mm, 1.8-micron Zorbax SB-C18 column (Agilent; PN: 827975-302) and 2.1 × 7 mm guard column (Alltech, IL) maintained at 35°C, at a pressure of <400 bar and flow rate of 0.3 ml/min. The mobile phase comprised a gradient of 0.01% formic acid in 80% acetonitrile -20% water, v/v (solvent A), and 0.01% formic acid in water (solvent B). Initial mobile phase composition was 20% solvent A and 80% solvent B, increasing to 60% A (0-3 min) then 80% A (3-5.45 min), returning to initial conditions (5.5-6 min). Post-run time was 1 min, and the total run time was 9 min. Mass spectrometric detection was carried out using an Agilent 6410 triple quadrupole mass spectrometer equipped with a multimode ionization (MMI) source. The mass spectrometer was operated in electrospray positive ionization mode using multiple reaction monitoring (MRM), with Q1 & Q3 set to unit resolution (0.7 μm). The electrospray ionization parameters, optimized for the parent molecular ion ([M+H]+) abundance were: drying gas temperature 325°C, vaporiser temperature 150°C, drying gas flow 5 L/min, nebuliser pressure 50 psi, capillary voltage 1.8 kV, Delta EMV 300 V. Ultra-pure nitrogen was used as collision gas. The divert valve feature of the Agilent 6410 triple quadrupole mass spectrometer was utilised to prevent the eluate from entering the ionisation source chamber during the first 0.5 min of each run. For CP-506 samples, positively charged ions representing the [M+H]+ for CP-506 and CP-506-D8 were collisionally dissociated to form specific product ions which were monitored in MS2 (unit resolution). The MRM transition, ionization mode, collision energy, dwell time, fragmentor voltage and retention time for all compounds is provided in Supplementary Table S2. Agilent MassHunter software (v.4.04.00) was used for data acquisition and chromatographic peak integration.
Cellular Pharmacokinetic Model
An agent-based model (ABM) employing a one-dimensional approximation for monolayers was used to develop a cellular PK model, as described in further detail elsewhere (Hong et al., 2018b;Mao et al., 2018). The mathematical framework assumes that solute concentrations in the medium are dependent only on the depth (on the z coordinate) and not the lateral position (x,y) so that all cells are exposed to the same concentration of nutrients and drugs within the monolayer culture. Moreover, all cells have the same rate of metabolism and volume growth at any instance with variability allowed in cell volume, rate of cell division and cell death. Monolayer ABM simulations were performed on a desktop Windows PC (Intel core I7 processor) from a Qt (Qt Company, https://www.qt.io/) graphical user interface passing parameters to a DLL built with Fortran95. The monolayer ABM uses a finite difference method to solve reaction-diffusion equations for each compound (N) in the medium and cells. Parameters for the cellular uptake and metabolism were fitted to the concentration-time profiles of CP-506 and its metabolites in monolayer cultures of POR-R cells using a linear (first order) cell transport and metabolism model. Terms for the extracellular instability of CP-506 and its metabolites were set at their respective values in media stability studies, and media diffusion coefficients were estimated from the support membrane diffusion coefficients described below. Experimental data describing the kinetics of CP-506 uptake under aerobic conditions was fitted by adjusting the permeability coefficients k in and k out for each compound. These values were fixed for anoxic experiments to allow for parameter estimation of the rate of intracellular CP-506 metabolism (k met0 ) as well as the intracellular/extracellular partitioning ratio (k in and k out ) and metabolic instability (k met0 ) for its cytotoxic metabolites.
Pro(drug) Transport Model
Drug transport in MCLs was modelled as a one-dimensional diffusion with reaction across four consecutive compartments (stirred donor, POR-R MCL, collagen-coated teflon support membrane and stirred receiver) using a custom designed MatLab routine (Foehrenbacher et al., 2013a). Model parameters were derived by fitting Eq. 1 and Eq. 2 to the measured concentration-time profile of internal standard [ 14 C]-urea, CP-506, CP-506H and CP-506M in MCL flux studies. The flux of CP-506 and its metabolites across POR-R MCLs was described using Fick's second law with a reaction term:
zC eN zt D N z 2 C eN zx 2 − k lossN C eN − φ( k inN C eN − k outN C iN ) (1) zC iN zt k met0(N−1) C i(N−1) + φ(k inN C eN − k outN C iN ) − k met0N C iN (2)
where C is the concentration of the diffusing substance at position x and time t in the extracellular (e) or intracellular (i) compartment, D is the diffusion coefficient through either a bare support membrane (without cells) (D suo ) or extracellular space of a POR-R MCL (D MCL ), k loss is the rate constant for first-order loss (instability) in culture medium, k in and k out are the rate constants for transmembrane transport and k met0 is the rate constant for intracellular metabolism and instability for each compound (N). For metabolites, N-1 represents the previous compound from which the current compound is produced by metabolism. Experimental data was fitted iteratively to derive a tissue diffusion coefficient (D MCL ) and metabolic scaling factor (φ i ) to restrain parameter estimates from the cellular PK model (K in , K out , K met0 ). The diffusion coefficient for CP-506 across the bare support membrane (D sup ) was determined by solving Eq. 1 with a reaction term only for chemical instability. MCL thicknesses were estimated by fitting the measured [ 14 C]-urea concentrations to those predicted by Eq. 1 and Eq. 2 without instability or metabolism by using the known diffusion coefficient (D MCL ) for [ 14 C]-urea in HCT-116 MCLs (Wilson et al., 2004). The diffusion coefficient (D MCL ) for CP-506 in POR-R MCLs was then fitted from the measured concentrations in the donor and receiver compartments of supraoxic experiments by assuming onedirectional diffusion in the extracellular space. The metabolic scaling factor (φ i ) was then fitted from the measured concentrations in the donor and receiver compartment of anoxic experiments by fixing parameter estimates to the diffusion coefficient (D MCL ) from supraoxic experiments.
Simulations of CP-506 Diffusion and Efficacy in Spheroid Cultures
The spheroid ABM is described in further detail elsewhere (Hong et al., 2018b;Mao et al., 2018). The model employs two 3D grids and two solvers, a coarse grid represents the culture medium and is embedded with a much finer grid that represents the spheroid in a small cubic region at the bottom of the well. The spheroid grows in a specified volume and depth of unstirred medium containing dissolved oxygen. The geometry of the model is lattice-based, in which cells occupy cubic sites on a regular 3D lattice and grow in volume at a rate dependent on the local oxygen concentration. Autonomous cell mobility and cell-cell forces are not simulated. Cell motion occurs only as a result of cell division. Cells divide when the volume reaches a pre-set value (V div ) and the resultant daughter cell occupies an adjacent empty lattice site. If a vacancy does not exist, cells are moved radially outward to create one. The oxygen concentration at the air-medium boundary is defined by the gas phase of the culture environment. As the spheroid grows and total oxygen consumption increases, concentration gradients within the culture medium and spheroid interior steepen with significantly lower concentrations within the spheroid core. When oxygen concentrations fall below a critical level (<0.15 µM), cells are tagged to die and cytolysis occurs after a further 24 h leading to the central necrosis observed in in vitro spheroids. Spheroid ABM simulations were performed on a desktop Windows PC (Intel core I7 processor) from a Qt (Qt Company, https://www.qt.io/) graphical user interface passing parameters to a DLL built with Fortran95. The solution of the coarser grid provides the boundary conditions for the solution of the intracellular and extracellular concentrations within the spheroid lattice. Oxygen and drug are transported by diffusion from the boundary into the spheroid interior through the intercellular space in parallel with their uptake into cells. The model allows specification of two mass transfer constants to characterise the uptake (K in ) and efflux (K out ) rates for each compound. Prodrug metabolism is restricted to the intracellular compartment and dependent on the local oxygen concentration. Parameter estimates for the cellular uptake and metabolism of CP-506 in monolayer cultures were used to fit clonogenic survival data to estimate the kill probability rate constant (k d ), as described previously for PR-104A (Hong et al., 2018b). The kill probability (model 4 in the ABM program (Mao et al., 2018)) was assumed proportional to the intracellular concentration of the bioreductive metabolites (k d C N ) and was set to zero for the prodrug. The same k d was assumed for each of the major metabolites (CP-506H, CP-506M and CP-506M-Cl 2 ), and that the intrinsic sensitivity of the cell lines was identical with differences only in the oxygen-and POR-dependent first order rate constant for the metabolic consumption (k met0 ) of CP-506. Parameter estimates were used alongside the in vitro determined drug pharmacokinetic terms to predict the killing of "activator" (POR-R) and "target" (PORko-G) cells in spheroid co-cultures.
Statistical Analysis
All statistical tests were performed using GraphPad Prism 8. Individual tests used are specified in the relevant figures.
RESULTS
The Hypoxia-Dependent Metabolism and Cellular Cytotoxicity of CP-506 is Influenced by Cytochrome P450 Oxidoreductase Expression
Given the primary role of POR in CP-506 metabolism (Van Der Wiel et al., 2021), a panel of isogenic cell lines with variable levels of POR expression was used to investigate the extravascular transport properties of CP-506 and its metabolites in an in vitro setting. Consistent with the genetic modifications, protein expression was elevated by 11-fold in the PORoverexpressing cell line (POR-R) relative to wild-type cells and was undetectable in the POR-knockout line (PORko-G) ( Figure 1A). The impact of POR expression on the sensitivity of HCT-116 cells to CP-506 was examined using both an anti-proliferative ( Figure 1B) and clonogenic endpoint ( Figure 1C). Both assays demonstrate hypoxia-selective cellular cytotoxicity, with a marked increase in the sensitivity of HCT-116 cells to CP-506 under anoxic conditions. A 13-fold differential in anoxic sensitivity was noted between the PORko-G and POR-R cell lines by anti-proliferative assay. Aerobic IC 50 values were 240 ± 26, 257 ± 37 and 120 ± 13 µM for the PORko-G, HCT-116 WT and POR-R cell lines, respectively (p-value ≤ 0.01). Values under anoxic conditions were 7.3-fold, 20.1-fold and 48.6-fold lower at 33.1 ± 5.6, 12.8 ± 3.7 and 2.5 ± 0.3 µM, respectively (p-value ≤ 0.01). Clonogenic assays showed a similar trend with a 22-fold differential in anoxic sensitivity between the PORko-G and POR-R cell lines. Clonogenic IC 50 values under anoxia were 17.1 ± 3.4, 7.4 ± 0.5 and 0.8 ± 0.3 µM for the PORko-G, HCT-116 WT and POR-R cell lines respectively. The mass spectrometric detection of reduced metabolites accompanied the selective cytotoxicity of CP-506 in hypoxic cultures. Although a number of species were detected (Figure 2), the hydroxylamine CP-506H, amine CP-506M and dichloro-amine CP-506M-Cl 2 were the major metabolites present at concentrations sufficient for comparison between cell lines. Rates of metabolite formation reflected differences in POR expression, with metabolite concentrations 5.5-(CP-506H) to 8.6-fold (CP-506M-Cl 2 ) higher in POR-R cells and 4.1-(CP-506M) to 17.7-fold (CP-506M-Cl 2 ) lower in PORko-G cells than HCT-116 WT cells after a 1 h treatment period ( Figure 3A). Authentic standards of the six identified metabolites (Figure 2) were synthesized (see Supplementary Material) and shown to have marked differences in intrinsic stability in stirred medium at 37°C ( Figure 3B). While the prodrug CP-506 showed no loss of mass balance for over 360 min in stirred medium, half-lives (T 1/2 ) for the reduced metabolites were significantly shorter at 2.5, 10.5, 7.6, 141.3, 271.1 and 393.9 min for the diol-hydroxylamine CP-506H-(OH) 2 , hydroxylamine CP-506H, amine CP-506M, dichlorohydroxylamine CP-506H-Cl 2 , dichloro-amine CP-506M-Cl 2 and diol-amine CP-506M-(OH) 2 respectively. Anti-proliferative IC 50 assays were performed to estimate the relative contribution of each metabolite toward the cellular cytotoxicity of CP-506 under hypoxic conditions ( Figure 3C). Both diol-containing metabolites were essentially inactive (IC 50 > 30 μM), consistent with the absence of appropriate mustard leaving groups required for formation of the cytotoxic intermediates. The dichloro-hydroxylamine CP-506H-Cl 2 metabolite also exhibited limited potency. The major cytotoxic metabolites (IC 50 < 10 μM) were the hydroxylamine CP-506H, amine CP-506M, and dichloro-amine CP-506M-Cl 2 . The former two metabolites were short-lived (T 1/2 of 7-11 min) while the latter had relatively high stability (T 1/2 of 271 min).
Cellular Pharmacokinetic Model for CP-506 in Tumour Tissues
A cellular PK model was developed based on the extracellular and intracellular concentration-time profile for CP-506 and its reduced metabolites in monolayer cultures. Experiments were performed using POR-R cells to allow for greater sensitivity in metabolite detection. Under aerobic conditions, the cellular uptake of CP-506 was described by a simple two compartment model with the loss of mass balance in the extracellular medium (C e ) coupled with a respective increase in the intracellular concentration (C i ) ( Figure 4A). The rate of cellular uptake was fastest during the first 30 min after drug addition and plateaued thereafter. The cellular uptake of CP-506 was not concentration-dependent, with steady-state C i /C e ratios of 50-fold across the C o range of 0.3-30 µM initial concentrations. No net metabolism nor intrinsic instability were observed under aerobic conditions. In anoxic experiments, the cellular uptake of CP-506 was accompanied by the appearance of metabolites CP-506H and CP-506M in the intracellular compartment ( Figure 4B). The loss of mass balance was reiterated in the lower steadystate C i /C e ratio of 40-fold. The formed metabolites were released from the cell of origin into the extracellular medium indicating bystander potential. However, the mass balance was dominated by the intracellular fraction indicating that the cellular efflux of these metabolites is a passive process.
A first order kinetic model describing the cellular uptake and metabolism of CP-506 ( Figure 4C) was fitted to the in vitro data with excellent correlation between the experimental data and model-predicted fits (R 2 0.9596) (lines in Figure 4B). The model assumed that the high intracellular concentrations of CP-506 are driven by its large cell uptake factor, and that metabolic loss of CP-506 is O 2 -dependent and restricted to the intracellular compartment. Once formed, the downstream metabolites can diffuse freely between the two compartments. The stability varies between compounds with the half-life (T 1/2 ) of CP-506 significantly longer than that of its metabolites. The rate of CP-506 metabolism and metabolite uptake is higher than the rate of cellular efflux resulting in the high intracellular concentrations observed under anoxic conditions. Model estimates of the uptake and metabolism parameters for CP-506 and its metabolites are provided in FIGURE 2 | Schematic representation of the activation sequence for CP-506. CP-506 is metabolised by endogenous one-electron reductases into a nitro radical anion that is readily back-oxidised in the presence of molecular oxygen. Under hypoxia, the nitro radical anion is further metabolised to its corresponding hydroxylamine (CP-506H) and amine (CP-506M) metabolites. The major metabolites are highly reactive. Each arm of the bromomesylate mustard can react with chloride ions and/or undergo aqueous hydrolysis providing the major observed downstream metabolites, CP-506H-Cl 2 , CP-506H-(OH) 2 , CP-506M-Cl 2 , and CP-506M-(OH) 2 .
Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602 Supplementary Table S3. Cell uptake studies considered only the major metabolites (CP-506H and CP-506M) due to the retrospective identification and synthesis of the downstream metabolites and deuterated internal standards thereof. For inclusion in the cellular PK model, the rate constants for the cellular uptake, cellular efflux and extracellular instability of CP-506M-Cl 2 were fixed to the fitted values for the stable downstream metabolites of PR-104A (termed "metabolite 2") (Foehrenbacher et al., 2013a;Hong et al., 2018b). Parameter estimates were later refined based on the accrued stability and MCL transport data for spheroid simulations as described below.
Extravascular Transport Model for CP-506 in Tumour Tissues
The cellular uptake and metabolism parameters from monolayer assays were scaled to tissue-like densities based on the concentration-time profile of CP-506 and its formed metabolites in the donor and receiver compartment of diffusion chambers containing multicellular layers maintained under supraoxic (95% O 2 , 5% CO 2 ) and anoxic conditions (5% CO 2 , bal N 2 ). Initial experiments were performed using collagencoated inserts without cells to determine the chemical stability and diffusion coefficient of CP-506 across the bare support membrane (D sup ) ( Figure 5A). Irrespective of oxygen tension, the extravascular transport of CP-506 across the bare support membrane was described using a simple Fickian diffusion model without loss of mass balance between the media compartments of the diffusion chambers (donor, receiver). Differences in stirring rate resulted in a lower D sup for anoxic experiments ((0.717 ± 0.01) x 10 −6 cm 2 s −1 ) compared to supraoxic experiments ((1.32 ± 0.19) x 10 −6 cm 2 s −1 ), however this discrepancy was accounted for in D MCL calculations. MCL cultures were established from POR-R cells to allow for greater sensitivity in metabolite detection. The predicted thickness of the MCL cultures (L MCL ) was 101.7 ± 6.9 µm, as estimated from the acquired flux data and established D MCL (3.67 x 10 −7 cm 2 s −1 , (Wilson et al., 2004)) for internal standard [ 14 C]-urea. Drug transport in the MCL was modelled as a one-dimensional diffusion with reaction in the extracellular and intracellular compartments, with the integrated reaction term used to describe the loss of mass balance due to cellular uptake and metabolism. The cellular uptake of CP-506 slowed extravascular transport across the MCL, as indicated by the estimated D MCL of (1.93 ± 0.011) x 10 −7 cm 2 s −1 for supraoxic experiments ( Figure 5B). The rate of diffusion was further reduced by reductive metabolism with its major metabolites detected in both the donor and receiver compartments of anoxic experiments ( Figure 5C). A metabolic scaling factor (φ i ) of 0.3 was derived to scale the metabolic parameters from the previous monolayer-based assays. A summary of the determined extravascular transport parameters for CP-506 across POR-R MCLs is provided in Supplementary Table S4.
Bystander Potential of CP-506 in Spheroid Co-cultures
Predictions based on the tissue pharmacokinetics of CP-506 in experimental model systems were compared to determinations of clonogenic cell survival and growth delays in spheroid co-cultures. Experiments were performed under strict anoxia to eliminate oxygen gradients to ensure that the established concentration gradients of CP-506 and its reduced metabolites reflect their respective pharmacokinetic properties rather than metabolic potential due to oxygen availability. This notion was confirmed by the flow cytometric detection of EF5 adducts (<1 µM O 2 ) upon the enzymatic dissociation of anoxic spheroids ( Figure 6A). Preliminary dose ranging experiments identified a 22-fold differential in anoxic sensitivity between PORko-G and POR-R spheroids by clonogenic endpoint reflecting the intrinsic differences in metabolic potential between the cell lines ( Figure 6B). Spheroid co-cultures were established by seeding "activator" POR-R cells and "target" PORko-G cells at differing proportions growing for 4 days in ultra-low attachment, round bottom 96-well plates. At this point, fluorescent imaging showed intimate mixtures of red and green fluorescent cells in proportions broadly consistent with the respective seeding densities of activator and target cells ( Figure 6C). A flow cytometric analysis of representative spheroids showed that activator cells were overrepresented by 5-10% due to . Spheroids were then enzymatically dissociated and plated in parallel in media supplemented with 2 µM puromycin or 1 mg/ml geneticin to select for activator and target cells respectively. Values are the mean ± SEM for three independent experiments. (F) Growth delay after exposure of spheroid co-cultures to CP-506 for 4 h under anoxic conditions (<1 ppm O 2 ). Spheroid volumes were quantified at endpoint (day 14 post-treatment) using a previously described method of image analysis (Mao et al., 2018). Values are the mean ± SEM for three independent experiments.
Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602 the longer doubling time of the target cells (21.7 ± 0.04 vs. 22.95 ± 0.38 h, respectively) ( Figure 6D). Established spheroids were exposed to CP-506 for 4 h under anoxic conditions. Treated spheroids were then enzymatically dissociated for a clonogenic endpoint or tracked using an image analysis method of volume determination. Clonogenic survival assays showed increased killing of both target and activator cells with the proportion of activator cells in the co-culture ( Figure 6E). Growth delay assays illustrated a similar trend with an increase in the proportion of activator cells leading to greater spheroid growth inhibition at each concentration relative to the DMSO-only control ( Figure 6F). IC 50 values for inhibition of spheroid growth were >40 µM, 13.6 µM, 5.6 µM and 2.4 µM for spheroid co-cultures comprised of 0%, 10%, 50% and 100% activator cells respectively.
A summary of the parameters used for ABM simulations is provided in Table 1. Here, the in vitro determined terms for the cellular uptake, metabolism and transport of CP-506 were used to simulate the killing of activator and target cells in anoxic spheroids. The clonogenic cell survival data from monolayer-based assays was used to estimate the kill probability parameter (k d ) and was well-fitted with the monolayer ABM (R 2 0.9685; Figure 7A). This assumed uniform solute concentrations and equipotent metabolites (same k d ) with a lower anoxic first-rate constant for metabolic consumption (k met0 ) fitted for target compared to activator cells (0.0019 min −1 vs. 0.12 min −1 respectively) ( Figure 7B). However, a higher k d was required to model the experimental data from spheroid co-cultures (0.01 vs. 0.0256 respectively). The spheroid ABM assumed that diffusion occurs through the unstirred medium and extracellular spaces with the metabolic activation of CP-506 restricted to the intracellular space ( Figure 7C). CP-506 and its metabolites can exchange across the plasma membrane with the reduced metabolites sufficiently stable to diffuse from the cell of origin. Simulated spheroids were exposed to CP-506 for 4 h under anoxic conditions with oxygen gradients restored upon treatment withdrawal. Best efforts recapitulated trends in target cell killing (R 2 0.9733) but slightly underestimated activator cell killing (R 2 0.7703; Figure 7D).
To understand how its pharmacological properties may impact tissue distribution in in vivo tumours, spatial gradients of CP-506 and its reduced metabolites were modelled in a transverse section of a representative spheroid comprised of 50% activators (Figure 8). Simulations show intracellular concentrations as a function of penetration depth after a 4 h exposure under anoxic conditions. The model predicts a rapid decline in the intracellular concentration of CP-506 from the spheroid periphery toward the spheroid core (613-fold range). Concentrations are negligible beyond a penetration depth of 190 µm. Concentrations of the formed metabolites CP-506H and CP-506M largely reflect the spatial gradient of CP-506 within the spheroid, with concentrations naturally higher in the peripheral cells. The decline in the intracellular concentration is however less pronounced compared Parameter estimates were assumed equivalent for the active metabolites due to their similar physiochemical properties -Refer to Figure 5. b D sup fitted to the concentration-time profile of CP-506 in the donor and receiver compartment of diffusion chambers bearing bare support membranes maintained under supraoxic conditions. Parameter estimates were assumed equivalent for the active metabolites due to their similar physiochemical properties -Refer to Figure 5. c Parameters estimated from the extracellular/intracellular partitioning of CP-506 and its active metabolites in monolayer cultures maintained under supraoxic or anoxic conditions -Refer to Figure 4. d Extracellular/intracellular partitioning parameters of CP-506M-Cl 2 were assumed equivalent to the measured metabolites or derived from published estimates for PR-104A metabolites (Foehrenbacher et al., 2013a;Hong et al., 2018b). e Stability of CP-506 and its active metabolites in stirred culture medium at 37°C -Refer to Figure 3. to CP-506, with concentrations in the innermost cells only 3-(CP-506M) to 10-fold (CP-506H) lower than that of the peripheral cells. Intracellular concentrations of the downstream metabolite CP-506M-Cl 2 mirror the spatial gradients of CP-506H and CP-506M, with concentrations markedly higher in the spheroid core. Collectively, these findings indicate that the reduced metabolites of CP-506 are sufficiently stable to diffuse from their hypoxic activating cells to neighbouring populations to potentiate cell killing by means of a bystander effect.
DISCUSSION
This study demonstrates that CP-506 exhibits favourable tissue pharmacokinetic properties, with a high cell uptake factor, maximal activation under extreme hypoxia, long half-life and good aqueous stability; all properties consistent with the observed anti-tumour efficacy of CP-506 (Van Der Wiel et al., 2021). These optimal properties are comparable or superior to the first generation analogue PR-104A Patel et al., 2011) but Exchange of the extracellular prodrug (P e ) and intracellular prodrug (P i ) between cell membranes is defined by permeability rate constants k in and k out with metabolism of P i to intracellular metabolites (M i ) defined by the first order rate constant k met0 and is strictly oxygendependent. (B) Model predictions (lines) compared to experimental determinations (symbols) of clonogenic survival in monolayer cultures of target (PORko-G) and activator (POR-R) cells exposed to CP-506 for 4 h under anoxic conditions (<1 ppm O 2 ) determined from the monolayer ABM by fitting k d for POR-R cells and the k met0 parameter for PORko-G cells (data redrawn from Figure 2C). (C) Schematic representation of the cellular uptake, metabolism and diffusion of CP-506 in spheroid cocultures. Diffusion of extracellular prodrug (P e ) and intracellular prodrug (P i ) across cell membranes is defined by permeability rate constants k in and k out . P i is metabolized to intracellular metabolites (M i ) in activator cells (red) defined by a first order rate constant k met0 which is oxygen-dependent. M i can diffuse to nearby target cells (green) by the extracellular route to elicit bystander killing and can diffuse out into the extracellular compartment, M e . In the extracellular compartment P e and M e can diffuse as defined by their diffusion coefficients D P and D M respectively. (D) Model predictions (lines) compared to experimental determinations (symbols) of clonogenic survival in spheroid co-cultures comprised of differing proportions of activator (act) and target cells (data re-drawn from Figure 6E). without the potential for off-target aerobic activation by AKR1C3 in normal tissues. The clinical development of PR-104 was halted due to dose-limiting myelotoxicity at suboptimal plasma exposures (Jameson et al., 2010;Abou-Alfa et al., 2011;Mckeage et al., 2012) with AKR1C3 expression in human bone marrow progenitor cells implicated as the probable cause (Birtwistle et al., 2009;Guise et al., 2010;Van Der Wiel et al., 2021). CP-506 thus represents an optimised PR-104 analogue designed to undergo metabolic activation exclusively under hypoxic conditions (Van Der Wiel et al., 2021).
Here, we employ a series of novel experimental systems to solve reaction-diffusion equations to define parameters for the cellular uptake, metabolism and diffusion of CP-506 and its active metabolites ( Figure 2) in tumour tissue. Model predictions were validated by comparing theoretical predictions to experimental determinations of clonogenic cell survival in spheroid co-cultures comprised of varying proportions of metabolically competent "activator" and metabolically defective "target" cells.
The cellular uptake of CP-506 in monolayer cultures was described by a first order kinetic model with a constant cell uptake (C i /C e ) ratio of 50-fold ( Figure 4A). The physiochemical properties of CP-506 imply that the high affinity cellular uptake is due to lysosomal sequestration reflecting ionization of the piperazine nitrogen (pKb 7.79). The developed prodrug transport model suggests that the high cell uptake factor is offset by the intrinsic stability of CP-506 in the absence of reductive metabolism ( Figure 5B (Guise et al., 2007;Guise et al., 2012). Here, we reaffirm the role of POR in CP-506 metabolism using paired cell lines with variable levels of protein expression. Forced overexpression of soluble POR heightened protein expression levels by 11-fold with corresponding increase in the rate of CP-506 metabolite formation ( Figure 3A) and cellular cytotoxicity ( Figure 1B) under hypoxic conditions. POR gene knockdown had the converse effect, with metabolite production suppressed up to 151-fold (CP-506M-Cl 2 , Figure 3A) relative to the PORoverexpressing cell line resulting in a 20-fold differential in anoxic sensitivity by clonogenic endpoint in both monolayer ( Figure 1C) and spheroid-based assays ( Figure 6B).
The hypoxia-selective metabolism of CP-506 leads to the formation of a series of reactive cytotoxic species (Figure 2). By direct exogenous exposure of cell monolayers to each authentic metabolite, we identify the amine CP-506M, hydroxylamine CP-506H and dichloro-amine CP-506M-Cl 2 as the major cytotoxic metabolites (Figure 3). Our spheroid-based assays provide the first direct evidence of a bystander effect, with reduced growth kinetics ( Figure 6F) and increased target and activator cell killing ( Figure 6E) correlating with the proportion of activator cells in the co-culture. These findings are consistent with metabolite redistribution via efflux of the formed metabolites from metabolically enhanced "activator" cells followed by rapid uptake into neighbouring POR-null "target" cell populations leading to widespread cell killing. Moreover, heightened "activator" cell killing with escalating proportions of activators indicates that metabolic consumption of CP-506 does not impede its extravascular transport to the most distal regions of the tumour tissue resulting in minimal spatial heterogeneity in metabolite distribution. This interpretation is supported by the appearance of the active metabolites in the media compartments of our experimental model systems (Figure 4, Figure 5), although the attained parameters slightly underestimate activator cell killing ( Figure 7D). This implies that the extravascular transport properties of CP-506 are likely superior to that anticipated by our experimental model systems. It may also indicate missing variables in our bystander model, analogous to the implied requirement for metabolites with low potency but high stability for PR-104A (Foehrenbacher et al., 2013a). Despite these caveats, our simulations predict a striking bystander efficiency at tissue-like densities ( Figure 8). Although reaction (cellular uptake and metabolism) within the tumour tissue completely depletes intracellular concentrations of CP-506 beyond a penetration depth of 190 µm, concentrations of the formed metabolites were fairly homogenous throughout the simulated spheroid. In fact, intracellular concentrations of the major metabolites were only 3-(CP-506M) to 10-fold (CP-506H) lower at a penetration depth of 300 µm, essentially compensating for the CP-506 concentration gradient.
CP-506 is metabolically converted under hypoxic conditions into a series of short-lived reduced metabolites (Figure 2), but the relative contribution of each toward the bystander efficiency of CP-506 is not yet known. In principle, the bystander potential of each metabolite is dependent on multiple factors including its rate of intracellular production, stability, potency and membrane permeability. Of the metabolites examined, CP-506H-Cl 2 and CP-506M-Cl 2 demonstrated at least a 10-fold longer half-life than CP-506H or CP-506M ( Figure 3B), reflecting the slower leaving group efficiency of the bis-chloro-mustard metabolites. Analogous to the dichloro metabolites of PR-104A (Hong et al., 2018b), the dichlorohydroxylamine CP-506H-Cl 2 and dichloro-amine CP-506M-Cl 2 are predicted to be approximately 10-fold more lipophilic than their corresponding Br/OMs mustard metabolites CP-506H and CP-506M, indicating that they will more readily permeate membranes and exit the cell of origin. Notably, the stability of CP-506H-Cl 2 was relatively poor in comparison to CP-506M-Cl 2 , reflecting the tendency of the hydroxylamine moiety to undergo further reduction to its amine derivative, CP-506M-Cl 2 . In an analogous manner, the diol-hydroxylamine CP-506H-(OH) 2 was undetectable in anoxic cultures indicating rapid conversion to its sixelectron reduction product CP-506M-(OH) 2 ( Figure 3B). Despite the long half-life of CP-506M-(OH) 2 , the cell line panel was not sensitive to concentrations up to 100 µM ( Figure 3C), consistent with the hydroxyl moieties failing to act as leaving groups (and the hydrophilic nature of the metabolite). This identifies CP-506M-(OH) 2 as an inert terminal metabolite. Collectively these data also indicate the reduction from CP-506H-Cl 2 to CP-506M-Cl 2 proceeds faster than its hydrolysis to CP-506H-(OH) 2 . Taken together, the superior physicochemical properties of CP-506M-Cl 2 are consistent with the simulated accumulation of CP-506M-Cl 2 in the spheroid Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602 core and identify it as the major bystander mediator of CP-506, perhaps with CP-506H and CP-506M playing minor secondary roles. While the concentration-dependence of "activator" and "target" cell killing with CP-506 is comparable to previous determinations for PR-104A (Hong et al., 2018b), it is important to note any difference in plasma pharmacokinetics are not accounted for in the current modelling paradigm. Although PR-104A showed favourable activity in murine xenograft models , safe human exposure levels were only 10-29% of those observed in mice (Guise et al., 2010), most likely due to off-target aerobic activation of PR-104A by human AKR1C3 in myeloid progenitor cells. The resistance of CP-506 to AKR1C3 bioactivation predicts for significantly higher plasma exposures in human trial without dose-limiting myelotoxicity. When coupled with the favourable extravascular transport properties of CP-506 and its bystander metabolite(s), as described in this study, marked single agent activity would be anticipated. Substantial anti-tumour activity of CP-506 has indeed been reported using clinically-relevant doses and schedules in murine xenograft models of diverse cancer types with variable hypoxic fractions reflecting the superior physicochemical properties and murine pharmacokinetics of CP-506 (Van Der Wiel et al., 2021). Current research efforts are exploring this further using a well-validated in silico spatially-resolved pharmacokinetic/ pharmacodynamic modelling approach (Foehrenbacher et al., 2013a), where experimental inputs are used to model the spatial gradients of a prodrug and its cytotoxic effectors with respect to molecular oxygen in a representative mapped tumour microvascular network.
Overall, this study uses a series of novel experimental systems along with computational modelling to investigate the extravascular transport properties and bystander efficiency of nitrogen mustard prodrug CP-506 and its active metabolites in the tumour tissues. We show that CP-506 possesses favourable tissue pharmacokinetic properties for anti-tumour activity including a long half-life, high cell uptake factor, tissue diffusion, selective activation under severe hypoxia and a substantial bystander potential. Our findings endorse the ongoing clinical development of CP-506 (NCT04954599) for use in the treatment of patients with advanced solid malignancies.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.
FIGURE 1 |
1Hypoxia-dependent cellular cytotoxicity of CP-506 is influenced by POR expression. (A) Western blot analysis of POR expression in HCT-116 cells upon gene knockout (PORko-G) and ectopic overexpression (POR-R) as compared to the parental cell line (HCT-116 WT). (B) Anti-proliferative potency after exposure of monolayer cultures (700 -1000 cells per well) to CP-506 for 4 h under normoxic (21% O 2 ) and anoxic conditions (<1 ppm O 2 ). IC 50 values were determined after 96 h of drug-free proliferation as the drug concentration required to inhibit cellular proliferation by 50% of the untreated controls. Values are the mean ± SEM of triplicate wells for three independent experiments. ANOVA tests were performed using GraphPad Prism 8 (p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001). (C) Clonogenic survival after exposure of monolayer cultures (4.8 x 10 5 cells per well) to CP-506 for 4 h under normoxic (21% O 2 ) and anoxic conditions (<1 ppm O 2 ). IC 50 values were interpolated from the fitted dose response curve as the concentration required to reduce the clonogenic surviving fraction to 50% of the untreated controls (shown by the reference line). Values are the mean ± SEM of duplicate wells for three independent experiments. Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602
FIGURE 3 |FIGURE 4 |
34Hypoxia-dependent metabolism of CP-506 leads to the formation of a multitude of short-lived species with variable cytotoxicity. (A) Bioreductive metabolism of CP-506 under normoxic (21% O 2 ) and anoxic conditions (<1 ppm O 2 ). Concentrations of CP-506 and its metabolites (CP-506H, CP-506M and CP-506M-Cl 2 ) were quantified by mass spectrometry with reference to the appropriate deuterated (D8) internal standard. Values are the mean ± SEM for three independent experiments. (B) Stability of CP-506 metabolites in culture medium (without cells) at 37°C. Concentrations were determined by mass spectrometry using standard curves generated from authenticated stocks. (C) Anti-proliferative potency after exposure of monolayer cultures to CP-506 metabolites for 5 days under normoxic conditions (21% O 2 ). IC 50 values are the mean ± SEM of duplicate wells for three independent experiments. T-tests were performed using GraphPad Prism 8 (ns, p-value > 0.05; *, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001).Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602 Cellular PK model of CP-506 in the tumor tissues. (A) Cellular uptake of CP-506 in monolayer cultures of POR-R cells after exposure to a range of initial concentrations (C o ) under normoxic conditions (21% O 2 ). Concentrations were determined in the extracellular medium and cellular extracts by mass spectrometry. Graphs show the concentration-time profile of CP-506 in the extracellular volume (C e ), intracellular volume (C i ) and resultant C i /C e ratio over the experiment duration (3 h). Values represent the mean ± SEM of three independent experiments. Lines show the corresponding estimates from the cellular PK model as fitted to the experimental data. (B) Hypoxia-selective metabolism of CP-506 and release of CP-506 metabolites into the extracellular medium of POR-R cells under anoxic conditions (<1 ppm O 2 ). Graphs show the concentration-time profile of CP-506 and its metabolites CP-506H and CP-506M in the extracellular volume (C e ), intracellular volume (C i ) and resultant C i /C e ratio over the experiment duration (3 h). Data points have been censored if below the lower limits of quantification (LLOQ, 0.01 µM). Lines show the corresponding estimates from the cellular PK model. (C) Cellular PK model for CP-506. The concentration of CP-506 and its metabolites in the extracellular and intracellular compartments is defined by the permeability rate constants (k in and k out ). The bioreductive metabolism of CP-506 (k met0 ) is O 2 -dependent and restricted to the intracellular compartment. The stability of each compound is defined by their respective half-life (T 1/2 ). Model estimates for the given parameters are provided in Supplementary
FIGURE 5 |
5Extravascular transport of CP-506 and its formed metabolites (CP-506H, CP-506M and CP-506M-Cl 2 ) across support membranes with or without a MCL culture. Flux was initiated by the addition of CP-506 (C o 17.4 ± 1.3 µM) and internal standard [ 14 C]-urea into the donor compartment of diffusion chambers maintained under supraoxic (95% O 2 , 5% CO 2 ) and anoxic (5% CO 2 , bal N 2 ) conditions. The concentration-time profile of CP-506 in the donor and receiver compartments of diffusion chambers was determined by mass spectrometry. Mass balance was calculated as the sum of the donor and receiver compartments. Lines are the concentrations predicted from the developed (pro)drug transport model. Values are mean ± SEM of three independent diffusion chambers.(A) Concentration-time profile of CP-506 across bare support membranes (without cells) maintained under supraoxic and anoxic conditions. (B) Concentration-time profile of CP-506 across POR-R MCLs maintained under supraoxic and anoxic conditions. (C) Concentration-time profile for the formed metabolites of CP-506 (CP-506H, CP-506M and CP-506M-Cl 2 ) in POR-R MCLs under anoxic conditions. Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602
FIGURE 6 |
6Bystander efficiency of CP-506 in spheroid co-cultures. Multicellular spheroids were established by seeding differing proportions of target (PORko-G) and activator cells (POR-R) and growing for 4 days in ultra-low attachment, round bottom 96-well plates. (A) Flow cytometric analysis of EF5 binding upon the enzymatic dissociation of anoxia pre-equilibrated POR-R spheroids. The hypoxic population was defined by the hypoxia-selective metabolism and binding of EF5 in monolayer controls. (B) Clonogenic survival after exposure of spheroid cultures to CP-506 for 4 h under anoxic conditions (<1 ppm O 2 ). IC 50 values were interpolated from the fitted dose response curve as the concentration required to reduce the plating efficiency to 50% of the untreated controls (shown by the reference line). Values are the mean ± SEM for three independent experiments. (C) Fluorescent microscope images of representative spheroids showing target (GFP-positive) and activator cells (mRuby-positive) at the anticipated proportions based on their initial seeding densities. Images were acquired at 4 X objective using a JuliStage Real-Time Cell History Recorder (NanoEnTek, Seoul, Korea). Scale bar indicates 1 mm. (D) Flow cytometric detection of target and activator cells in spheroid co-cultures based on GFP expression. The parental cell line (HCT-116 WT) was used as a negative control. Values are the mean ± SEM for three independent experiments. (E) Clonogenic survival of target and activator cells (Act) in spheroid co-cultures treated with CP-506 for 4 h under anoxic conditions (<1 ppm O 2 )
the clonogenic survival of monolayer or spheroid cultures following exposure to CP-506 under anoxic conditions -Refer to Figure 7. Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602
FIGURE 7 |
7ABM simulations of clonogenic cell survival in monolayer and spheroid cultures following CP-506 treatment. (A) Schematic representation of the cellular uptake, metabolism and diffusion of CP-506 in monolayer cultures.
FIGURE 8 |
8ABM simulations of the intracellular concentration gradients of CP-506 and its reduced metabolites in a transverse section of a multicellular spheroid. Model predictions of the intracellular concentration gradients of CP-506 and its reduced metabolites CP-506H, CP-506M and CP-506M-Cl 2 as a function of penetration depth in a spheroid co-culture comprised of 50% activators after exposure to 20 µM CP-506 for 4 h under anoxic conditions (<1 ppm O 2 ).
), where the extended half-life affords the long equilibrium time required for delivery of effective concentrations to distal, metabolically active hypoxic cells (<1 µM O 2 ) (Van Der Wiel et al., 2021). The reliance on severe hypoxia (<1 µM O 2 ) for CP-506 metabolism and cytotoxicity (Van Der Wiel et al., 2021) ensures selective activation in tumour tissues. A number of flavoenzymes have been implicated in the nitroreduction of CP-506 in an in vitro setting (Van Der Wiel et al., 2021), including POR which is a major enzyme responsible for the hypoxia-selective metabolism of PR-104A in cancer cell lines
Frontiers in Pharmacology | www.frontiersin.org February 2022 | Volume 13 | Article 803602
Table S3 .
S3Frontiers in Pharmacology | www.frontiersin.org
February 2022 | Volume 13 | Article 803602
TABLE 1 |
1CP-506 PK/PD parameters used for ABM simulations.Parameter
(unit)
Description
Estimate
CP-506
CP-506H
CP-506M
CP-506M-Cl 2
D s (cm 2 s −1 ) a
Diffusion coefficient in spheroid
1.93 × 10 −7 ± 1.8 × 10 −8
1.93 × 10 −7 ± 1.8 × 10 −8
1.93 × 10 −7 ± 1.8 × 10 −8
1.93 × 10 −7 ± 1.8 × 10 −8
D M (cm 2 s −1 ) b
Diffusion coefficient in medium
1.32 × 10 −6 ± 0.19 × 10 −6 1.32 × 10 −6 ± 0.19 × 10 −6 1.32 × 10 −6 ± 0.19 × 10 −6 1.32 × 10 −6 ± 0.19 × 10 −6
k in (min −1 ) c
Rate constant for transfer from the
extracellular to intracellular volume
3.7
0.9
0.9
0.9 d
k out (min −1 ) c
Rate constant for transfer from the
intracellular to extracellular volume
0.06
0.25
0.4
0.3 d
Half-life (h) e
Time required to reduce the
concentration of drug to half of its
initial value
30
0.18
0.13
4.52
Activator k met0
(min −1 ) c
Rate constant for the maximum rate
of metabolism in POR-R cells under
anoxia
0.12
0.09
0.1
0.1 d
Target k met0
(min −1 ) f
Rate constant for the maximum rate
of metabolism in PORko-G cells
under anoxia
0.0019
0.09
0.1
0.1 d
k d M
f
Kill probability rate constant in
monolayer cultures
0
0.01
0.01
0.01
k d S
f
Kill probability rate constant in
spheroid cultures
0
0.0256
0.0256
0.0256
a D MCL fitted to the concentration-time profile of CP-506 in the donor and receiver compartment of diffusion chambers bearing POR-R MCLs maintained under supraoxic conditions.
AUTHOR CONTRIBUTIONSSUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2022.803602/ full#supplementary-material
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Conflict of Interest: JS and AP have previously served as scientific consultants to Convert Pharmaceuticals. JS, AP, and AA are co-inventors on patents assigned to Health Innovation Ventures (PCT: WO2014031012A1. Granted patents: EP2888227B1, US10202408B2, CA2886574C, US9873710B2, AU 2013/306514B2, US9505791B2)Conflict of Interest: JS and AP have previously served as scientific consultants to Convert Pharmaceuticals. JS, AP, and AA are co-inventors on patents assigned to Health Innovation Ventures (PCT: WO2014031012A1; Granted patents: EP2888227B1, US10202408B2, CA2886574C, US9873710B2, AU 2013/306514B2, US9505791B2).
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Introduction
Vancomycin is a glycopeptide antibiotic with activity against gram-positive bacteria, such as Staphylococcus aureus [1]. Since the emergence of methicillin-resistant Staphylococcus aureus, vancomycin is increasingly used in clinics with prolonged duration and elevated dosages. However, vancomycin harbors the risk of several adverse effects, which include ototoxicity [2], thrombocytopenia [3], and neutropenia [4]. Additionally, higher dosages of vancomycin are associated with an increased risk of acute kidney injury [5]. Hence, Du et al. used physiologically-based pharmacokinetic modeling and simulation and verified their results with a human specimen [6]. This study found vancomycin levels in the kidney which were up to 50-fold higher than plasma vancomycin levels. Therefore, vancomycin associated acute kidney injury is highly clinically relevant and the mechanisms behind it have only partially been unraveled. Wang and colleagues found that vancomycin activates microRNA-301a-5p in a methyl-CpG-binding domain protein 2 dependent manner, ultimately driving proximal tubule cells into apoptosis [7]. Similarly, Chen et al. uncovered stimulation of p53-dependent apoptosis by microRNA-192-5p in a human renal epithelial cell line upon vancomycin treatment [8]. Another mechanism to activate apoptosis in kidney cells used by vancomycin is the suppression of complex I, which leads to elevated levels of mitochondrial superoxide [9]. The production of reactive oxygen species (ROS) enhances the permeabilization of the mitochondrial membrane, resulting in apoptosome activation [10,11]. ROS also contribute to apoptosis by the formation of cardiolipin peroxides, a mitochondria specific phospholipid [12]. Hence, oxidative species also mediate expression of pro-inflammatory cytokines, as treatment with antioxidant species ameliorate vancomycin-induced nephrotoxicity [13].
A common consequence of mitochondrial redox imbalance is the activation of other redox dependent pathways. Specifically, the pharmacological inhibition of complex I results in an increase of glucose metabolization to lactic acid [14]. Complex I oxidizes reduced nicotinamide adenosine dinucleotide (NADH) and thus the inhibition of it causes increased levels of NADH [15]. Accumulated NADH is then oxidized by lactate dehydrogenase and replenishes the redox pool necessary to drive glycolysis [16]. Another redox dependent glucose metabolizing pathway is the polyol pathway, in which glucose is reduced to sorbitol, which is then oxidized to fructose [17]. The latter reaction also leads to the production of NADH [18]. Therefore, when the redox pool is shifted to elevated NADH, sorbitol cannot be oxidized to fructose anymore, resulting in an accumulation of sorbitol.
Despite the apparent role of mitochondria in vancomycin-induced acute kidney injury, no metabolomics studies of vancomycin treated kidney cells have been conducted so far. Du et al. used kidney cells' supernatant after a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay (XTT-assay) for an exometabolic screen based on reversed-phase chromatography mass spectrometry. They aimed at discovering potential bio-markers for vancomycin-induced nephrotoxicity and found several lyso-phospholipids in the supernatant of cells with low viability [6].
In regard with other nephrotoxins, metabolomics is widely used. The well-known chemotherapeutic drug cis-diamminedichloroplatinum II (cisplatin) is nephrotoxic [19] and has a high impact on renal metabolism. Among others, we showed accumulation of glucose and down-regulation of glycolysis intermediates together with a loss of amino acids [20]. Cyclosporine A is an immunosuppressive drug with adverse activity against the kidney [21]. Metabolic profiling of cultured kidney cells treated with different doses of cyclosporine A revealed a stark induction of glutathione metabolism, as well as alterations in amino acids and tricarboxylic acid cycle (TCA-cycle) intermediates [22]. Hence, the radiocontrast agent diatrizoic acid was also shown to induce an oxidative stress response [23]. Administration of the antiviral drug acyclovir, commonly causing acute kidney injury to rats, revealed excessive excretion of nitrogen containing metabolites to the urine [24]. Metabolomics is also capable of detecting alterations induced by non-toxic pathological stress conditions, such as high glucose and protein levels present in diabetic kidney disease [25].
In the presented study, we analyzed for the first time cellular changes in the metabolome and lipidome of kidney cell lines exposed to clinically relevant doses of vancomycin. We uncovered increased glycolysis and sorbitol levels, which might stem from the known mitochondrial disturbances caused by vancomycin.
Results and Discussion
Vancomycin Is Taken Up in a Dose-Dependent Manner
To assess metabolic alterations induced by highly concentrated vancomycin, we treated mouse inner medullary collecting duct cells (mIMCD-3 cells) and Madin-Darby canine kidney cells (MDCK cells) with control media and media containing 0.25 mg/mL, 1 mg/mL or 4 mg/mL vancomycin. During these experiments, no major changes regarding morphology or cell number were observed (data not shown). As displayed in Figure 1, both cell lines have taken up vancomycin in a dose-dependent manner.
To assess metabolic alterations induced by highly concentrated vancomycin, we treated mouse inner medullary collecting duct cells (mIMCD-3 cells) and Madin-Darby canine kidney cells (MDCK cells) with control media and media containing 0.25 mg/mL, 1 mg/mL or 4 mg/mL vancomycin. During these experiments, no major changes regarding morphology or cell number were observed (data not shown). As displayed in Figure 1, both cell lines have taken up vancomycin in a dose-dependent manner.
Metabolic Profiling of Vancomycin Treated Kidney Cell Lines
After proving the uptake of vancomycin by the used kidney cell lines, we subjected metabolite extracts to untargeted metabolic profiling by gas chromatography-mass spectrometry (GC/MS). Additionally, glutathione levels and small chain acyl-carnitines were acquired by targeted LC/MS analysis. In total, 88 metabolites were thoroughly identified using both mass spectra and retention time/index information. The data set was first analyzed by principal component analysis (PCA) as shown in Figure 2. Treatment with 0.25 mg/mL vancomycin did not result in global alterations, neither in mIMCD-3 cells nor in MDCK cells. Such alterations were visible with 1 mg/mL only in mIMCD-3 cells, and in both cell lines, when a concentration of 4 mg/mL was applied.
Metabolic Profiling of Vancomycin Treated Kidney Cell Lines
After proving the uptake of vancomycin by the used kidney cell lines, we subjected metabolite extracts to untargeted metabolic profiling by gas chromatography-mass spectrometry (GC/MS). Additionally, glutathione levels and small chain acyl-carnitines were acquired by targeted LC/MS analysis. In total, 88 metabolites were thoroughly identified using both mass spectra and retention time/index information. The data set was first analyzed by principal component analysis (PCA) as shown in Figure 2. Treatment with 0.25 mg/mL vancomycin did not result in global alterations, neither in mIMCD-3 cells nor in MDCK cells. Such alterations were visible with 1 mg/mL only in mIMCD-3 cells, and in both cell lines, when a concentration of 4 mg/mL was applied. Statistical analysis revealed 35 significantly altered metabolites (one-way analysis of variance (ANOVA), corrected for multiple testing by false discovery rate (FDR), q-value < 0.05) in mIMCD-3 cells and 11 significantly altered metabolites in MDCK cells. Detailed results of statistical analyses can be found in supplementary Table S1. These numbers also reflect the more profound impact of vancomycin on mIMCD-3 cells in comparison to MDCK cells as seen in the PCA ( Figure 2). In Figure 3, these significant alterations are displayed in heat maps, in which range-scaled z-scores are shown. In both cell lines, distinct clustering is visible between control and 4 mg/mL vancomycin treatment.
In mIMCD-3 cells, small-chain acyl carnitines were down-regulated upon high-dose vancomycin treatment together with sugars and some sugar alcohols. Upregulated metabolites included key metabolites of glycolysis and the tricarboxylic acid cycle. In contrast to myo-inositol and meso-erythritol, the sugar alcohol sorbitol was up-regulated upon vancomycin exposure. A cluster of amino acids, which were mainly essential amino acids, trended to increase in 4 mg/mL vancomycin compared to the control, but they peaked in the 1 mg/mL vancomycin condition. An up-regulation of glycolysis and lactic acid in particular can point to a decreased activity of oxidative phosphorylation [26]. A disturbed activity in oxidative phosphorylation was already described and substantiates our finding [9]. A decreased TCA-cycle flux might also explain accumulating citrate levels. The decrease in acyl-carnitines further supports this hypothesis, since fatty acid oxidation is a predominant energy source in tubule cells [27]. An elevated usage of glucose also fits with the accumulation of sorbitol in tubule cells [25]. In line with that, we detected higher levels of sorbitol along with increased intermediates of glycolysis in 4 mg/mL vancomycin treated mIMCD-3 cells. In addition, sorbitol acts as an osmolyte in the kidney [28]. Thus, higher doses of vancomycin in the cell culture medium could also contribute to an increase of sorbitol. However, this is probably a minor contribution given the high osmolar pressure normally applied to renal epithelium [29]. Activation of sorbitol accumulation is corroborated by a dose-dependent decline in myo-inositol levels, a known effect of high sorbitol pathway activity [30]. The fact that fructose was decreased might suggest sorbitol accumulation was caused by redox imbalance [31]. Indeed, glutathione and glutathione disulfide increased in parallel, which points to an increased synthesis in response to redox imbalance (Figure 3 left). Elevated glutathione synthesis was already associated with another nephrotoxin, namely cyclosporine A [22]. Statistical analysis revealed 35 significantly altered metabolites (one-way analysis of variance (ANOVA), corrected for multiple testing by false discovery rate (FDR), q-value < 0.05) in mIMCD-3 cells and 11 significantly altered metabolites in MDCK cells. Detailed results of statistical analyses can be found in supplementary Table S1. These numbers also reflect the more profound impact of vancomycin on mIMCD-3 cells in comparison to MDCK cells as seen in the PCA (Figure 2). In Figure 3, these significant alterations are displayed in heat maps, in which range-scaled z-scores are shown. In both cell lines, distinct clustering is visible between control and 4 mg/mL vancomycin treatment.
In mIMCD-3 cells, small-chain acyl carnitines were down-regulated upon high-dose vancomycin treatment together with sugars and some sugar alcohols. Upregulated metabolites included key metabolites of glycolysis and the tricarboxylic acid cycle. In contrast to myo-inositol and meso-erythritol, the sugar alcohol sorbitol was up-regulated upon vancomycin exposure. A cluster of amino acids, which were mainly essential amino acids, trended to increase in 4 mg/mL vancomycin compared to the control, but they peaked in the 1 mg/mL vancomycin condition. An up-regulation of glycolysis and lactic acid in particular can point to a decreased activity of oxidative phosphorylation [26]. A disturbed activity in oxidative phosphorylation was already described and substantiates our finding [9]. A decreased TCA-cycle flux might also explain accumulating citrate levels. The decrease in acyl-carnitines further supports this hypothesis, since fatty acid oxidation is a predominant energy source in tubule cells [27]. An elevated usage of glucose also fits with the accumulation of sorbitol in tubule cells [25]. In line with that, we detected higher levels of sorbitol along with increased intermediates of glycolysis in 4 mg/mL vancomycin treated mIMCD-3 cells. In addition, sorbitol acts as an osmolyte in the kidney [28]. Thus, higher doses of vancomycin in the cell culture medium could also contribute to an increase of sorbitol. However, this is probably a minor contribution given the high osmolar pressure normally applied to renal epithelium [29]. Activation of sorbitol accumulation is corroborated by a dose-dependent decline in myo-inositol levels, a known effect of high sorbitol pathway activity [30]. The fact that fructose was decreased might suggest sorbitol accumulation was caused by redox imbalance [31]. Indeed, glutathione and glutathione disulfide increased in parallel, which points to an increased synthesis in response to redox imbalance (Figure 3 left). Elevated glutathione synthesis was already associated with another nephrotoxin, namely cyclosporine A [22]. In MDCK cells, some essential amino acids, tended to decrease with higher vancomycin doses. Four metabolites were significantly elevated in the 4 mg/mL vancomycin condition in comparison to the control cells. Three of them were catabolites of glucose, and lactic acid and sorbitol were likewise regulated as in the mIMCD-3 cell line. Together with the low levels of one small-chain acyl carnitine, these results confirm the finding from the mIMCD-3 cells: an impaired mitochondrial oxidative phosphorylation might have increased glycolysis, with parallel sorbitol accumulation.
Lipidomics of Vancomycin Treated Kidney Cell Lines
Next, the lipophilic extracts were analyzed by targeted lipidomic analysis. Marked differences were unveiled in the baseline lipidomic profile of the two cell lines (Supplementary Figure S1). Additionally, the two cell lines behaved differently towards vancomycin exposure: only slight differences were observed in the PCA of MDCK cells between the control and 4 mg/mL vancomycin, whereas in mIMCD-3 cells, these conditions were clearly separated from each other (Figure 4). In MDCK cells, some essential amino acids, tended to decrease with higher vancomycin doses. Four metabolites were significantly elevated in the 4 mg/mL vancomycin condition in comparison to the control cells. Three of them were catabolites of glucose, and lactic acid and sorbitol were likewise regulated as in the mIMCD-3 cell line. Together with the low levels of one small-chain acyl carnitine, these results confirm the finding from the mIMCD-3 cells: an impaired mitochondrial oxidative phosphorylation might have increased glycolysis, with parallel sorbitol accumulation.
Lipidomics of Vancomycin Treated Kidney Cell Lines
Next, the lipophilic extracts were analyzed by targeted lipidomic analysis. Marked differences were unveiled in the baseline lipidomic profile of the two cell lines (Supplementary Figure S1). Additionally, the two cell lines behaved differently towards vancomycin exposure: only slight differences were observed in the PCA of MDCK cells between the control and 4 mg/mL vancomycin, whereas in mIMCD-3 cells, these conditions were clearly separated from each other (Figure 4).
In line with that, no significant alterations in lipid species upon vancomycin treatment were detected in MDCK cells, while 22 lipids were significantly altered in mIMCD-3 cells: in accordance with the metabolite analysis, two long chain acyl-carnitines were decreased in the 4 mg/mL condition ( Figure 5). This confirms a reduced fatty acid oxidation in this cell line. Several lipids were up-regulated in mIMCD-3 cells, mainly glycosphingolipids and phosphatidylethanolamines ( Figure 5). Activation of glycosphingolipid synthesis was already found in cisplatin induced acute kidney injury [32]. Four of the up-regulated glycosphingolipids are hexosyl-ceramides. Glucosyl-ceramides are known to be altered in several kidney diseases [33] and are involved together with other glycosphingolipids in apoptosis signaling [34,35]. Although we did not observe huge differences of cell mass, this might already prime the cells to enter into apoptosis. In line with that, no significant alterations in lipid species upon vancomycin treatment were detected in MDCK cells, while 22 lipids were significantly altered in mIMCD-3 cells: in accordance with the metabolite analysis, two long chain acyl-carnitines were decreased in the 4 mg/mL condition ( Figure 5). This confirms a reduced fatty acid oxidation in this cell line. Several lipids were up-regulated in mIMCD-3 cells, mainly glycosphingolipids and phosphatidylethanolamines ( Figure 5). Activation of glycosphingolipid synthesis was already found in cisplatin induced acute kidney injury [32]. Four of the up-regulated glycosphingolipids are hexosyl-ceramides. Glucosyl-ceramides are known to be altered in several kidney diseases [33] and are involved together with other glycosphingolipids in apoptosis signaling [34,35]. Although we did not observe huge differences of cell mass, this might already prime the cells to enter into apoptosis.
Phosphatidylethanolamines were increased upon high-dose vancomycin ( Figure 5). Previous studies showed a decrease in phosphatidylethanolamines using aristolochic acid I, celiptium, and cisplatin [36][37][38], but in the latter case, cisplatin led to an increase of certain phosphatidylethanolamines in the renal medulla. This indicates that the regulation of phosphatidylethanolamines might be more complex in acute kidney injury and more toxins have to be tested to evaluate whether or not this lipid species is commonly regulated. Most of the lipids altered in mIMCD-3 cells upon vancomycin treatment were glycosphingolipids, which were increased. These lipids already had a higher basal level in the MDCK cell line, which might explain why these lipids did not further increase when vancomycin was applied. However, the lipidomic results from the mIMCD-3 cells should not be overinterpreted since they were not observed in MDCK cells.
Conclusions
To the best of our knowledge, this is the first metabolomics study of kidney cells treated with clinically relevant concentrations of the nephrotoxin vancomycin. These concentrations were only recently modelled by Du et al. and were found to exceed typical plasma concentrations up to 50-fold. We showed that these high concentrations were still Phosphatidylethanolamines were increased upon high-dose vancomycin ( Figure 5). Previous studies showed a decrease in phosphatidylethanolamines using aristolochic acid I, celiptium, and cisplatin [36][37][38], but in the latter case, cisplatin led to an increase of certain phosphatidylethanolamines in the renal medulla. This indicates that the regulation of phosphatidylethanolamines might be more complex in acute kidney injury and more toxins have to be tested to evaluate whether or not this lipid species is commonly regulated. Most of the lipids altered in mIMCD-3 cells upon vancomycin treatment were glycosphingolipids, which were increased. These lipids already had a higher basal level in the MDCK cell line, which might explain why these lipids did not further increase when vancomycin was applied. However, the lipidomic results from the mIMCD-3 cells should not be overinterpreted since they were not observed in MDCK cells.
Conclusions
To the best of our knowledge, this is the first metabolomics study of kidney cells treated with clinically relevant concentrations of the nephrotoxin vancomycin. These concentrations were only recently modelled by Du et al. and were found to exceed typical plasma concentrations up to 50-fold. We showed that these high concentrations were still dose-dependently taken up by the two kidney cell lines used. Further, although MDCK cells responded less than mIMCD-3 cells, both seemed to upregulate anaerobic glycolysis. This might be due to disturbances of the redox pool, which were already shown by Arimura et al. [9]. In line with an altered redox state and a consequent malfunctioning of mitochondria, is an increase in anaerobic glycolysis [26]. In addition, glucose metabolism towards fructose is disturbed in parallel to redox imbalance, which might explain increased sorbitol levels in both cell lines, and resulting from that, a decrease of myo-inositol [25]. The latter metabolite was however only altered in mIMCD-3 cells. A reduced functionality of mitochondria can also be assumed by the decrease in acyl-carnitines in high-dose vancomycin cells. Acyl-carnitines serve as shuttles for fatty acids, making them available for oxidation in mitochondria [39].
Lipid profiling revealed marked differences between the two cell lines and might have contributed to the decreased vulnerability of MDCK cells towards vancomycin on the lipidomic level. Therefore, the detected differences of glycosphingolipids should be critically evaluated, as they were only observed in one cell line. Future studies should investigate the metabolic effects of vancomycin in animal models to further understand the pathological mechanisms underlying vancomycin-induced acute kidney injury.
In conclusion, this study provides a new data set of several altered and unaltered metabolites and lipids in two established kidney cell lines treated with high concentrations of vancomycin. This might aid in developing new hypotheses of mechanisms of action in vancomycin induced nephrotoxicity.
Materials and Methods
Cell Culture
Mouse inner medullary collecting duct cells (mIMCD-3, ATCC ® CRL-2123™, ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco™) containing penicillin/streptomycin, and 10% fetal bovine serum (FBS). Madin-Darby canine kidney cells (MDCK, ATCC ® CCL-34™, ATCC, Manassas, VA, USA) were grown in DMEM (Gibco™) containing penicillin/streptomycin, and 10% FBS. Both cell lines were seeded in 6-well plates and grown until confluency. Cells were washed once with phosphate buffer saline before treatment with vancomycin.
For treatment, 48 mg vancomycin hydrochloride (Hikma Pharmaceuticals, London, UK) were dissolved in 12 mL of the respective cell culture medium to yield a 4 mg/mL solution. This solution was sterile filtered and diluted with a corresponding cell culture medium to 1 mg/mL and 0.25 mg/mL. The cells were incubated with 2 mL of vancomycin containing (4, 1 and 0.25 mg/mL) or the control cell culture medium for 24 h.
Cell Harvest
The cells were harvested as previously described [40]. In brief, cell culture medium was centrifuged to remove cell debris, transferred to a new vial and snap frozen in liquid nitrogen. Cells were washed twice with 0.9% NaCl and quenched with 1 mL ice-cold methanol:water (1:1, v:v) containing internal standards. Cells were scraped off, transferred to a new vial, and snap frozen. Cell culture medium and cells were stored at −80 • C until analysis.
Analysis of Vancomycin in Cell Culture Media
100 µL of cell culture medium were mixed with 900 µL ice-cold acetonitrile:methanol (3:1, v:v), vortexed and centrifuged (45 min, 20,000× g, 4 • C). 100 µL of the supernatant were evaporated in a speedvac and the pellets reconstituted in 100 µL ddH 2 O. 70 µL were transferred into an LC-Vial and 20 µL was used to prepare a mixed quality control sample. Vancomycin was analyzed by reversed-phase chromatography coupled to mass spectrometry using a gradient of water/0.1% formic acid with acetonitrile/0.1% formic acid (Waters: Acquity Hsst3 2.1 × 100 mm, 1.8 µm. Agilent Technologies, Waldbronn, Germany: G4220A, G4226A, G1316A, G6460A triple-quadrupole mass spectrometer). Gas temperature was set to 350 • C, gas flow was 8 L/min, and sheath gas temperature was 250 • C with 5 L/min flow. The nebulizer pressure was maintained at 30 psi. Capillary voltage was 3000 V in positive ionization mode with 500 V nozzle voltage. Samples were injected in a randomized order with regular injections of quality control samples to monitor possible analytical drifts.
Cell Lysis for Metabolomics and Lipidomics
500 µL chloroform (containing heptadecanoic acid as internal standard) was added to the methanol:water cell suspension and lysed by rigorous vortexing. Afterward, phases were separated by centrifugation. 300 µL of the upper phase were evaporated for GC/MS analysis, 300 µL of the upper phase were evaporated for LC/MS analysis and 200 µL of the lower phase were evaporated for lipidomics analysis.
Analysis of Glutathione and Small Chain Acyl-Carnitines
Glutathione and glutathione disulfide were analyzed as described by Schlimpert et al. [41] Authentic standards of small chain acyl-carnitines were used to determine retention times and multiple reaction monitoring (MRM) transitions and added to the existing method. MRM-transitions were determined with the MRM-optimizer software by Agilent Technologies.
Untargeted GC/MS Profiling
Untargeted metabolic profiling was conducted as previously described [20]. In brief, pellets were derivatized by methoxyamination and silylation and injected on an HP5-MS column. Gas chromatography was coupled with an electron ionization mass spectrometer. Data files were deconvoluted and peak picking performed by AMDIS [42]. Features were aligned with the online tool SpectConnect [43]. Metabolites were identified by mass spectra and retention indices from three different libraries [44][45][46] and an in-house data base.
Targeted Lipid Profiling by LC/QqQ-MS
Lipids were analyzed by targeted LC/MS MRM-analysis (Waters: BEH C18 2.1 × 100 mm, 1.8 µm. Agilent Technologies, Waldbronn, Germany: G4220A, G4226A, G1316A, G6460A triple-quadrupole mass spectrometer). Chromatographic separation was as previously described [40]. The gas temperature was set to 290 • C with a flow rate of 10 L/min. The sheath gas flow rate was 11 L/min at 370 • C. The nebulizer pressure was 25 psi. The mass spectrometer was operated with +5 kV/−4 kV and 500 V nozzle voltage. Details about transitions and collision energies are shown in Supplementary Table S2. The samples were kept at 15 • C and 5 µL were injected in randomized order with regular quality control samples in between.
Data Processing and Statistical Analysis
Intensities of metabolites or lipids were normalized to an internal standard and by the sum of all peaks [47]. MetaboAnalyst 5.0 were used for statistical analyses [48]. Missing values were replaced by one fifth of the minimal values of each feature. Samples with >25% relative standard deviation in the quality control samples were excluded from analysis. For principal component analysis and heat map generation, values were rangescaled. One-way ANOVA was used to determine significance, followed by false discovery rate-based multiple testing correction. A q-value cut-off of 0.05 was used. ANOVA was followed by Tukey's post-hoc test. Results of statistical analyses are show in Supplementary Table S1.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms221810111/s1, Figure S1: heat map of all detected lipids, Table S1: Results of statistical analyses, Table S2: MRM transitions of lipids, Table S3: Results of metabolic analysis, Table S4: Results of lipidomic analysis.
Figure 1 .
1Vancomycin in cells and cell culture medium. Extracellular (top) and intracellular (bottom) vancomycin levels in mIMCD-3 cells (left) and MDCK cells (right) are shown. Y-axes show intensities which were acquired by liquid chromatography-mass spectrometry (LC/MS) and normalized to phenol red or O-methyl-L-tyrosine in cell culture media or within the cells, respectively. Error bars indicate standard deviation. N = 3.
Figure 1 .
1Vancomycin in cells and cell culture medium. Extracellular (top) and intracellular (bottom) vancomycin levels in mIMCD-3 cells (left) and MDCK cells (right) are shown. Y-axes show intensities which were acquired by liquid chromatography-mass spectrometry (LC/MS) and normalized to phenol red or O-methyl-L-tyrosine in cell culture media or within the cells, respectively. Error bars indicate standard deviation. N = 3.
Figure 2 .
2Principal component analysis of metabolites in vancomycin treated kidney cells. (Left): mIMCD-3 cells showed a dose-dependent impact on the metabolome by vancomycin. (Right): in MDCK cells, high-dose vancomycin was necessary to induce global alterations. PC: principal component. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Dots represent samples, shaded area: the confidence interval. N = 3.
Figure 2 .
2Principal component analysis of metabolites in vancomycin treated kidney cells. (Left): mIMCD-3 cells showed a dose-dependent impact on the metabolome by vancomycin. (Right): in MDCK cells, high-dose vancomycin was necessary to induce global alterations. PC: principal component. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Dots represent samples, shaded area: the confidence interval. N = 3.
Figure 3 .
3Heat map analysis of endometabolites. Only significantly altered metabolites (one-way ANOVA, corrected for multiple testing by FDR, q-value < 0.05) are displayed in mIMCD-3 cells (left) and MDCK cells (right). Cluster analysis after Euclidian and Ward are displayed on top for samples and on the left side for metabolites. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Range scaled z-scores are displayed. N = 3.
Figure 3 .
3Heat map analysis of endometabolites. Only significantly altered metabolites (one-way ANOVA, corrected for multiple testing by FDR, q-value < 0.05) are displayed in mIMCD-3 cells (left) and MDCK cells (right). Cluster analysis after Euclidian and Ward are displayed on top for samples and on the left side for metabolites. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Range scaled z-scores are displayed. N = 3.
Figure 4 .
4Principal component analysis of lipids in vancomycin treated kidney cells. (Left), mIMCD-3 showed dose-dependent alterations of lipids after vancomycin application. (Right): Lipids in MDCK cells were only impacted to a minor extent by vancomycin treatment. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Dots represent samples, shaded area the confidence interval. N = 3.
Figure 4 .of 12 Figure 5 .
4125Principal component analysis of lipids in vancomycin treated kidney cells. (Left), mIMCD-3 showed dosedependent alterations of lipids after vancomycin application. (Right): Lipids in MDCK cells were only impacted to a minor extent by vancomycin treatment. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Dots represent samples, shaded area the confidence interval. N = 3. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7Heat map analysis of significantly altered lipids in mIMCD-3 cells. Significance was determined by ANOVA (one-way ANOVA, corrected for multiple testing by FDR, q-value < 0.05). Cluster analysis after Euclidian and Ward revealed marked differences in the high-dose vancomycin condition. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Range scaled z-scores are displayed. N = 3.
Figure 5 .
5Heat map analysis of significantly altered lipids in mIMCD-3 cells. Significance was determined by ANOVA (one-way ANOVA, corrected for multiple testing by FDR, q-value < 0.05). Cluster analysis after Euclidian and Ward revealed marked differences in the high-dose vancomycin condition. Turquois: control, red: 0.25 mg/mL vancomycin, yellow: 1 mg/mL vancomycin, blue: 4 mg/mL vancomycin. Range scaled z-scores are displayed. N = 3.
Author
Contributions: Conceptualization, S.L., R.P. and B.K.; methodology, S.L.; formal analysis, S.L., G.V., J.G., F.B., A.S., D.K. and D.A.P.; investigation, S.L., G.V., J.G., F.B., A.S. and D.K.; resources, B.K., R.P. and S.S.L.; writing-original draft preparation, S.L.; writing-review and editing, R.P., B.K., S.S.L. and D.A.P.; visualization, S.L., D.A.P.; supervision, B.K., S.S.L. All authors have read and agreed to the published version of the manuscript. Funding: R.P. is supported by the Berta-Ottenstein-Programme for Clinician Scientists, Faculty of Medicine, University of Freiburg. S.S.L. is supported by European Research Council (grant agreement No 804474, DiRECT), and the Swiss National Science Foundation (NCCR Kidney.CH, and project 310030_189102). The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding program Open Access Publishing. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Metabolomics and Lipidomics results are provided in supplementary Tables S3 and S4, respectively.
Acknowledgments:We thank Angelina Bogus for outstanding technical support.Conflicts of Interest:The authors declare no conflict of interest.
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| Objective To compare the inhibitory effects of inulicin on the cell growth of mouse hepatoma cell line(H22),mouse sarcoma cell line(S180),human lung adenocarcinoma cell line(A549),human ovarian cancer cell line(SK-OV3) and human cervical cancer cell line(Hela).Methods MTT method was used to detect the inhibitory effects of inulicin on the cell growth of the 5 cell lines mentioned above,and the IC50 was calculated.The effects of inulicin on morphology of the 5 cell lines were observed by using laser scanning confocal microscope and inverted microscope.Results Inulicin inhibited the cell growth of the 5 cell lines in vitro,but the inhibitory effects of inulicin on S180,SK-OV3,H22 and A549 were more obvious than those on Hela cell,with obvious morphological changes being showed by laser scanning confocal microscope and inverted microscope.Conclusion The inulicin has anti-tumor effect and the effect is selective for different cell lines. | Comparison of the cytotoxic activities of chemotherapeutic drugs using a human bladder cancer cell line | In this study, the commercially available secnidazole was successfully converted to secnidazole xanthate (SNXT), in which the xanthate group can act as a bifunctional chelator to coordinate with 99mTc. 99mTc-nitrido complex of SNXT(99mTcN-SNXT) and 99mTc-oxo complex of SNXT(99mTcO-SNXT) were prepared with high radiochemical purity. Both of the complexes were found to be stable in vitro and to exhibit similar hydrophilicity. In addition, comparative in vitro cell uptake studies under anoxic and normoxic conditions demonstrated that both agents were preferentially taken up by hypoxic cells. Biodistribution studies in mice bearing S180 tumor showed 99mTcO-SNXT exhibited a higher tumor uptake and tumor-to-muscle ratio than 99mTcN-SNXT. Furthermore, in SPECT imaging study, 99mTcO-SNXT exhibited a clear accumulation in tumor at 2 h post-injection, suggesting its potential to be a novel hypoxia imaging agent. |
INTRODUCTION
Cancer is one of the greatest serious diseases everywhere throughout the world. Cancer therapy and its managing depend fundamentally on the utilization of chemotherapeutic agents. These agents mainly target the rapidly proliferating neoplastic cells in cancer patients. But chemotherapy is always associated with a severe form of toxic manifestations [1]. Solid and the ascetic types of Ehrlich ascites carcinoma (EAC) are undifferentiated tumors usually used to assess different lines of cancer therapeutics [2].
Cyclophosphamide (CPA) is used as a chemotherapeutic drug [3]. Numerous antineoplastic conventions require high doses of CPA that are correlated with acute side effects, such as hepatotoxicity, cardiotoxicity, nephrotoxicity, and immunotoxicity [4]. Preceding studies have exhibited that CPA could produce substantial reactive oxygen species (ROS) and add to succeeding raised concentrations of highly cytotoxic oxidative stress-resulting lipid peroxidation aldehydes, such as acrolein and malondialdehyde (MDA) [5]. CPA was demonstrated that the imponderables among oxidant and antioxidant condition lead to the creation of pro-inflammatory mediators which participate in liver damage after CPA injection [6]. Hepatotoxicity has been recorded in patients injected intravenously with a low dose of CPA [7]. Preceding studies have indicated that liver toxicity is a significant issue related to cyclophosphamide application [8].
Antioxidant agents are believed useful to alleviate oxidative stress. In like manner, a blend of a treatment program with potent and safe antioxidants could be an advantageous approach to lessen CPA-induced toxicity [9]. Mesenchymal stem cells (MSCs) are multipotent stem cells that were initially isolated from the bone marrow (BM-MSCs). BM-MSCs can differentiate into cells of the 3 germ layers, including endothelial cells of blood vessels, smooth muscle cells, adipocytes, nerve cells, chondrocytes, cardiomyocytes, and hepatocytes. Furthermore, they can reduce the immune response brought about by T lymphocytes in allogeneic transplantation [10]. The therapeutic effects of transplanted MSCs have been associated with the following mechanisms: (1) differentiation to parenchymal cell; (2) production of trophic features that encourage proliferation and differentiation of local progenitor cells; (3) neovascularization; and (4) immunomodulation [11]. Therefore, the present work was designed to investigate the protective role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on CPA induced hepatotoxicity in EAC mice and to test whether BM-MSCs influences the antitumor properties of the CPA.
MATERIALS AND METHODES
Chemicals
CPA was supplied from Sigma-Aldrich Chemicals Co., (USA) and was prepared in sterile physiological saline (SPS). All the other chemicals were of high purity grade in agreement with international standards.
The mice were left for one week before to the experiments initiated to adapt to the animal house environments under normal dark/light cycle and were provided diets and water ad libitum. The experimental protocols were carried out corresponding to the guidelines for the animal experiment, which was approved by the Ethical Committee of Medical Research of National Research Centre, Giza, Egypt (registration number: 13/165).
Tumor cell line
The Ehrlich Ascites Carcinoma (EAC) cells were obtained from the National Cancer Institute (NCI) (Cairo, Egypt). The initial inoculation of EAC cells was kindly supplied by the National Cancer Institute (Cairo University, Egypt). EAC cells were thereafter propagating in our laboratory by weekly intraperitoneal (IP) injection of 2.5 x 10 6 cells per mouse, which was carried out according to the method recommended by the Egyptian National Cancer Institute, Cairo University. Cells were counted before injection using the bright-line hemocytometer and dilutions were made by physiological saline, and the desired number of cells was injected in a volume of 0.5 ml per mouse.
Cell viability
On the 7th day of the initial inoculation of EAC, ascitic fluid was collected and subjected to a trypan blue dye exclusion method for estimation of cell viability [13]. The number of viable cells (unstained) was counted under a microscope and average from 5 squares was taken. The cells which took trypan blue were deemed non-viable ( fig. 1). The concentration of viable cells (expressed as follows: C= T x D x 10 4 ), where C= concentration of viable cells/ml, and T= Average no. of viable cells per square, and D= dilution factor.
Fig. 1: (a) Showing the culture of EAC in vivo (in abdominal cavity of female mouse). (b) Showing the normal EAC cells X200 Preparation and isolation of BM-derived MSCs
Bone marrow was collected by flushing the tibiae and femurs of 6 w old male Wistar rats with phosphate-buffered saline (PBS). Bone marrow cells were plated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Nucleated cells were isolated with a density gradient and resuspended in complete culture medium supplemented with 1% penicillin-streptomycin. Cells were incubated at 37 °C in 5% humidified CO2 for 12e14 d as primary culture or upon the formation of large colonies. When large colonies developed (80e90% confluence), cultures were washed twice with phosphate buffer saline (PBS) (pH 7) and the cells were trypsinized with 0.25% trypsin in 1 mmol EDTA for 5 min at 37 °C. After centrifugation at 3000 rpm for 10 min, cells were re-suspended in serumsupplemented medium and incubated in 50 cm2 culture flasks. The resulting cultures were referred to as first-passage cultures [14]. Cells were identified as being MSCs by their morphology or fusiform shape, adherence [15] and Florescent Analysis Cell Sorting (FACS) by detection of CD29 + , CD90 + , and CD34 -specific to MSCs.
FACS analysis
Following short centrifugation, cells were re-suspended in wash buffer (BD Biosciences, Germany). 300 ml of cell suspension was incubated with antibodies compared to CD29, CD34 and CD90 conjugated with Allophycocyanin (APC), Cyanine 5 (CY5), Phycoerythrin (PE) and Fluorescein isothiocyanate (FITC) dyes respectively for 45 min at room temperature. Flow cytometry was executed on a FACS Caliber (BD Biosciences, Germany) and Cell Quest software was sourced for analysis.
Tracking of stem cells
MSCs cells were gathered through the 4th passage. Then, cells were trypsinized and were placed into a single cell suspension. 2 x 107 single cells were placed in a falcon tube, washed once applying culture medium free of serum then cells were centrifuged (400xg) for 5 min. Finally, cells were labeled with PKH26 fluorescent linker dye (according to the manufacturer's protocol) and examined using fluorescence microscopy (Sigma-Aldrich, Saint Louis, USA).
Ehrlich solid carcinoma induction
Ehrlich Solid Carcinoma (ESC) was induced into forty female mice using Ehrlich Ascites Carcinoma (EAC) cells obtained from the ascitic fluid of female mice bearing EAC. On day zero about 2.5 × 10 6 viable EAC cells present in 0.2 ml of diluted ascitic fluid (1:10) with saline was injected intramuscularly in the left thigh of each mouse to induce solid tumors [16].
Experimental protocol
Anti-tumor studies
The anti-tumor activities of CPA or/and BM-MSCs were assessed on 192 mice bearing solid Ehrlich Carcinoma that were classified into two main groups, the first group 32 mice which were used to determine the mortality rate and subdivided into four groups (n=8) as flow:
Gr.1: female mice inoculated with EAC cells (2.5 × 10 6 viable EAC) intramuscularly in the left thigh of each mouse to induce solid tumors and served as (EAC group).
Gr.2: female mice inoculated with EAC cells (2.5 × 10 6 viable EAC), Ten days after inoculating the EAC cells (when the tumor becomes papale), the animals treated with CPA (10 mg/kg/d i. p.) for one month [17] and served as (EAC+CPA) group.
Gr.3: female mice inoculated with EAC cells (2.5 × 106viable EAC), Ten days after inoculating the EAC cells (when the tumor becomes papale), the animals injected with BM-MSCs i. v. by a single dose of 100 µl of a cell suspension containing allogenic BM-MSCs from rats at the moment of the boost (when BM-MSCs were collected) [18] then lifted untreated for one month and served as (EAC+BM-MSCs) group.
Gr4: female mice inoculated with EAC cells (2.5 × 10 6 viable EAC), Ten days after inoculating the EAC cells (when the tumor becomes papale), the animals treated with both CPA and BM-MSCs at the same previous doses and served as (EAC+CPA+BM-MSCs) group.
The second group 160 mice were used to calculate the mean tumor weight (MTW) until the end of the study and were classified into four equal-sized groups (n=40) as flow: b Gr1: EAC group, Gr2: EAC+CPA group, Gr3: EAC+BM-MSCs and Gr4: EAC+CPA+BM-MSCs (the animals in these groups were injected as mentioned before). Then, these groups were left untreated until the end of the experiment. The weight of the solid tumor was determined by killing three animals every week and by taking the mean tumor weight of control (MTWc) and mean tumor weight of test (MTWt). The cumulative mean survival time of control (MSTc), the cumulative mean survival time of test (MSTt) the increase in life span (ILS, %) and the tumor growth inhibition ratio (T/C %) were recorded as given by Fahim et al. [19], where: MST is the days in which animals were lived only.
Biochemical and histopathological studies
40 animals were assigned into five groups, each containing 8 mice as follow: Gr1: mice received i. p. injection of physiological saline and served as a normal control group (Con), Gr2: EAC group, Gr3: EAC+CPA, Gr4: EAC+BM-MSCs and Gr5: EAC+CPA+BM-MSCs (the animals in these groups from Gr2-Gr5 were injected as mentioned before).
Samples collection
At the end of the experiment, the mice were fasted overnight, subjected to anesthesia by diethyl ether and sacrificed by cervical dislocation, and a midline abdominal incision was performed, and whole liver of each animal was rapidly dissected out, thoroughly washed with ice-cold isotonic saline, blotted dry and then weighed. Each liver was divided into two portions. One portion was immediately homogenized to give 10% (w/v) homogenate in icecold medium containing phosphate buffer (pH: 7.4). The homogenate was centrifuged at 1800 ×g for 10 min at 4 °C. The supernatant (10%) was separated and stored at −20 °C for the biochemical determinations. The second portion of the liver was fixed in 10% formalin saline for histopathological investigation.
Biochemical determinations
Hepatic total protein level was determined by the colorimetric method of Lowry et al. [20]. Hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity were determined by the colorimetric method using the Salucea kit (The Netherlands) according to the method described by Young [21]. Hepatic gamma-glutamyl transferase (GGT) was measured by standard methodology using a commercially available kit provided by the Biodiagnostic Company (Giza, Egypt).
Assessment of oxidative stress
Oxidative stress was estimated using the following parameters: Hepatic MDA content was determined by a colorimetric method using the Biodiagnostic kit (Egypt) following the method of Satoh [22]. The assay of reduced glutathione (GSH) level in liver tissue was performed using the Biodiagnostic kit purchased from the Biodiagnostic Company (Giza, Egypt). Which is based on the spectrophotometric method of Beutler et al. [23]. Hepatic superoxide dismutase activity (SOD) was determined by a colorimetric method as described by Nishikimi et al. [24] using a diagnostic kit supplied by Biodiagnostic Company (Giza, Egypt).
Determination of inflammatory and anti-inflammatory markers
The levels of biochemical parameters in liver homogenate were measured by ELISA assay using a commercially available IL-6 and IL-10 ELISA kits provided from (eBioscience Inc., San Diego, CA, USA), following the manufacturer's instructions.
Estimation of apoptotic and anti-apoptotic markers
The activity of Caspase-3 was measured in hepatic tissue by assay (ELIZA) technique using Gscience kits purchased from Glory Science Co., Ltd, USA, according to the manufacturer's instruction. Hepatic Bcl2 content was estimated by using ELISA kit for quantitative detection of human Bcl-2 purchased from eBioscience Co., Vienna, Austria, Europe according to the method of Barbareschi et al. [25].
Histopathological investigation
After the fixation of liver specimens in formalin saline (10%) for 24 h., washing in tap water was done, and then the liver samples were subjected to serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) for dehydration. Afterward, liver specimens were cleared in xylene and embedded in paraffin wax at 56 ° in a hot air oven for 24 h. Paraffin wax tissue blocks were submitted for sectioning at 4 μm by Sledge microtome. The obtained tissue sections were collected on glass slides, deparaffinized, and stained with hematoxylin and eosin [26]. After staining, the slides were viewed with an Olympus CH (Japan) light microscope [27].
Statistical analysis
In the present study, all results were expressed as mean±standard error of the mean. Statistical Package for the Social Sciences program, version 14.0 was used to compare significance between every two groups. The difference was considered as statistically significant when p˂0.05. Percentage difference representing the percent of variation for the corresponding control group was calculated according to the following formula: . 3). Moreover, the most elevation of the tumor growth inhibition ratio (T/C %) was noted in the CPA+BM-MSCs group as compared to the EAC group ( fig. 4, 5).
RESULTS
Effects of treatment with CPA or/and BM-MSCs on Liver functions in EAC mice treated with CP
The EAC mice (table 2) showed a generalized disturbance in all measured hepato-specific markers comparable to control (P<0.05). Treatment of EAC mice with CPA resulted in an increment in hepatic levels of AST, ALT, and GGT compared to tumor group. These levels of liver enzymes were declined in EAC mice treated with BM-MSCs. Otherwise, treatment of EAC mice with both CPA and BM-MSCs resulted in significant depletion (P<0.05) in the hepatic levels of AST, ALT, and GGT verses to both of EAC and EAC+CPA groups.
Effects of treatment with CPA or/and BM-MSCs on oxidative stress markers in EAC mice
Data in
Effects of treatment with CPA or/and BM-MSCs on Inflammatory and Anti-inflammatory mediators in EAC mice
The result of this study showed that hepatic IL-6 level displayed significant elevation (P<0.05) in the EAC treated group correlated with Maximum amelioration in the level of IL-10 as compared with the control groups proving increased inflammatory response (table 4). However, CPA treatment induced upregulation in the level of IL-6 and downregulation in the level of IL-10 as compared with the EAC group.
In contrast, treatment of EAC group with BM-MSCs or CPA+BM-MSCs significantly restored the levels of IL-6 and IL-10 near to normal levels compared to both EAC and EAC+CPA groups (table 4).
Effects of treatment with CPA or/and BM-MSCs on apoptotic and antiapoptotic markers in EAC mice
It is clear from
Histopathological investigation
Histopathological studies showed that inoculation of EAC into mice induced massive liver damage including severe congestion of erythrocytes and leucocytes, leucocytic infiltration, and vacuolar degeneration when compared to the control rat liver ( fig. 6B). Also, dilatation in the portal vein and Thickening in the bile ductule wall was observed due to the accumulation of fibers around the wall lining ( fig. 6C). On the other side, treatment with CPA revealed some changes in the liver tissue demonstrated by dilatation in the portal vein and destruction in its wall beside leucocytic infiltration surrounded by necrotic lesions was noted. Moreover, different types of leucocytes appeared scattered in the tissue besides giant phagocytic Kupffer cells in addition to large foci of necrosis ( fig. 6D and E). The pathological changes induced by EAC were improved greatly in the liver of rats treated with BM-MSCs ( fig. 6F). However, after the treatment with CPA+BM-MSCs showed the hepatic lobules appeared more like normal and hepatic cells are arranged in a series of branching plates ( fig. 6G).
Fig. 6: (A) photomicrographs of H and E stained liver sections of control mice demonstrated the normal hepatic architecture. (X200). (B) hepatic Section of EAC mice displayed distortion in hepatic architecture manifested by leucocytic infiltration area (LIA), necrotic areas (NA) and degeneration of the liver cells that looked empty of nucleus (X400). (C)Liver Section of EAC mice showing swollen in lining epithelia of the bile ductule (arrows), congestion of erythrocytes and leucocytes in portal vein (PV) and leucocytic infiltration (X200). (D) Liver Section of EAC mice treated with CP showing dilatation in portal vein (PV), Leucocytic infiltration (In) and necrotic foci (Nf) (X100). (E) Liver Section of EAC mice treated with CP displayed large necrotic foci (Nf) with scattered inflammatory cells and giant Kupffer cells (KC) (X400). (F) Hepatic section of EAC mice treated with BM-MSCs showed almost normal hepato-cellular architecture (X100). (G) Liver
Section of EAC mice treated with CP+BM-MSCs displayed normal healthy hepatocytes around normal central vein (CV) (X200).
DISCUSSION
The current study showed that CPA, BM-MSCs or CPA and BM-MSCs administration significantly reduced the solid tumor burden in mice and enhanced the survival time of EAC bearing mice, especially in the co-administration of CPA and BM-MSCs which caused an increase in the life span of animals and the tumor inhibition ratio [28]. The present findings revealed that both CPA and BM-MSCs exerted antitumor activity through interference with cell metabolism and inhibiting tumor proliferation. CPA undergoes metabolic activation by hepatic enzymes and forms 4-hydroxy cyclophosphamide, which converts into two cytotoxic metabolites acrolein and phosphoramide mustard. These cytotoxic metabolites on enzymatic activation form covalent bonds with DNA and proteins, causing DNA damage, This DNA damage could render tumor cells more apt to undergo apoptosis causing cell death, hence reducing the proliferation, migration and invasive properties of the cell line [29]. BM-MSCs repressed tumor proliferation and metastasis via altitude T-cell responses likely through MSC-secreted T-cell chemokines. All through increasing T-cell responses, the excessive levels of T-cell cytokines subsequently turn on the negative feedback mechanisms in MSCs to suppress excessive T-cell reactivity.
A key Tcell cytokine in this regulation is IFNγ, which acts independently or with inflammatory mediators including tumor necrosis factor-α (TNF-α), IL-1 and interleukin 17 to functionally transform MSCs from a resting state to a highly immunosuppressive stage [30]. Such immunosuppression is attained via cytokine-induced manifestation of the immunosuppressive molecules inducible nitric oxide synthase (iNOS) in human and mouse MSCs [31].
The results of the present study also demonstrated that the coadministration of CPA and BM-MSCs resulted in a significant decrease in MTW and increases the mean survival time of tumorbearing mice. This decrease in MTW may attribute to the action of CPA in the development of free radicals-mediated oxidative stress in tumor cells. This ROS production is believed to be the main effect in sensitizing tumor cells towards apoptosis [32]. Compatible with our results, a preceding study found that exosomes derived from microRNA-143-overexpressing mesenchymal stem cells inhibiting proliferation, invasion, and migration while enhancing apoptosis of hepatoma cells via regulating the NF-κB pathway [33].
In the present study, the elevations recorded in serum ALT, AST and GGT activities in EAC and CPA-treated groups may be due to drastic conditions induced by the toxic activity of tumor cells and CPA accumulations in the liver and in turn this might cause cellular destruction or increase hepatic cells permeability [34]. In contrast, the EAC group treated with BM-MSCs recorded a significant decline in serum ALT, AST and GGT activities as versus to the EAC group. Moreover, the double treatment of EAC with both of CPA and BM-MSCs resulted in a significant improvement in serum ALT, AST and GGT as compared to both EAC group and EAC+CPA group, showing the protective effects of BM-MSCs against liver injury and hepatic fibrosis. Several studies demonstrated that transplantation of stem cells has been demonstrated to exert therapeutic effects in cases of liver fibrosis, as well as after repeated partial hepatectomy, and significantly lowering levels of AST, ALT, and GGT [35]. The acquired results exhibited a significant increase of hepatic MDA level in tumor-bearing mice associated with a significant decline in GSH and SOD contents as versus to that of the normal control group. Such finding is in agreement with Fahim et al. [19] who stated that the increase in MDA and decrease of GSH and SOD due to the oxidative stress on the host in response to the continual production of free radicals by the expanding tumor load. The present work demonstrated a significant increase in hepatic MDA level correlated with a significant decrease in SOD activity as well as GSH level in the EAC+CPA group as compared to EAC group. Decreasing of the antioxidant levels may be due to malfunction of the antioxidant defense mechanisms that probably result from the consumption of antioxidants by chemotherapy stimulated-oxidative stress and/or renal loss of low molecular weight, water soluble antioxidants. Maruthanila et al. [36] stated that the prooxidant properties of chemotherapy may be implicated in its capability to induce tumor cell apoptosis, which is achieved partly through direct oxidative damage of DNA, RNA, and/or protein in the cells. Current study revealed an improvement by BM-MSCs treatment in the hepatic MDA, GSH and SOD levels in the both of EAC group and EAC+CPA+BM-MSCs versus to EAC group and EAC+CPA group. BM-MSCs rise GSH via increased efflux of cysteine with enhanced GSH synthesis which facilitated by secretion of soluble growth factors by BM-MSCs or by their interaction with host cells or both [37]. Likewise, our results show that BM-MSCs infusion is associated with expansion of SOD activity which reflects the efficiency of BM-MSCs in combating the induced oxidative stress [38]. This study is built on the fact that stem cells can be chemotactically attracted to the location of injury [39].
In the contemporary study, implantation of EAC resulted in a significant increment in IL-6 associated with a significant reductions in IL-10 versus to the control group which was in a similar line with other studies that stated that proinflammatory cytokines such as IL-6 have an important role in cancer development [40]. IL-6 is possibly the best characterized protumorigenic cytokine that was initially supposed to be complicated in cancer due to their capability to activate the oncogenic transcription factors such as activator protein 1 (AP-1), signal transducers and nuclear factor kappa B (NF-kB) and activators of transcription 3 (STAT3) in epithelial cells [41].
The kindness impacts of IL-10 in cancer include inhibition of angiogenesis either directly on tumor cells or indirectly boosts antitumor immunity by influencing infiltrating immune cells [42] and modulation of apoptosis [43]. IL-10 may wield pro-tumorigenic effect via repression of adaptive immune replies that caused tumor escape from immune surveillance [44]. These opposite effects of IL-10 might depend on relations with either cytokines or factors found in the tumor microenvironment. In this perspective, IL-10 has been stated to repress NO production and control the inducible NO synthase (iNOS) activation [45]. The iNOS-derived NO has been identified as one of the extremely versatile players in the immune system and pathogenesis of numerous diseases as well as cancer [46].
Administration of CPA resulted in hepatotoxicity manifested by a significant increase in IL-6 associated with a significant decrease in the IL-10 as compared to the EAC group. These results reveal the existence of liver inflammation in mice treated with CPA. Inflammation is the most important cause of liver injury, as various pieces of substantiation indicate that high levels of inflammatory cytokines can damage the liver [47]. Pro-inflammatory cytokines, for example, interleukin (IL)-1b and IL-6 can injure the hepatocytes via the NF-kB pathway. Simultaneously, the NF-kB pathway controls the synthesis and expression of numerous inflammatory factors and then produces apoptosis of the hepatocytes. Studies have demonstrated that CPA can promote the IL-1b, IL-6 and TNF-α levels [48]. Furthermore, apart from the influence of CPA and its metabolites on the IL-6 production the diminishing of IL-10 production by macrophages [49].
On the other hand, the treatment of the EAC group and EAC+CPA+BM-MSCs with BM-MSCs recorded a significant reduction in the IL-6 level associated with significant increment in IL-10 level as compared to the EAC group and EAC+CPA group. MSCs are recognized for their powerful anti-inflammatory functions, as they habitually transplanted into inflammatory environments where they capable to survive and regulate host immune responses [50]. The gained findings of the existing study declare that the protective influence of BM-MSCs is multifactorial as well as oxidative stress, inflammatory response, and tissue repair. It has been stated that MSCs are qualified for delivering an assortment of cytokines and hematopoietic soluble growth factors [51].
In this study, EAC inoculation produced a significant increment in hepatic caspase 3 and significant reduction in the hepatic Bcl2 level versus to normal control group. It is known that damaged cells could tend to turn into cancer if they did not choose apoptosis [52]. Caspase 3 activity likewise raised in human hepatocarcinoma in vitro and in vivo studies by S. marianum treatment [53]. EAC apoptotic cellular death stimulated by activating caspase 3 utilizing Synadenium umbellatum Pax [54]. In view of our findings, Badr El-Din et al. [55] documented that significant variations in the expression of apoptotic regulators (activation of caspase-3, upregulation in the expression of proapoptotic proteins Bax and p53, downregulation in the expression of the antiapoptotic protein Bcl-2, and decreased Bcl2: Bax ratio) in mice that were inoculated with Ehrlich ascites carcinoma. The treatment of EAC group with CPA was also up-regulating the hepatic level of caspase-3 and down-regulating the hepatic level of Bcl-2 as compared to the EAC group. Related studies Shokrzadeh et al. [8] reported that the CPA-induced liver damage was associated with the ability to regulate hepatocyte apoptosis, including DNA insertion, p53 protein activation, and ROS production. Initiation of p53 further begins a cascade of enzymatic reactions that push along to stimulate downstream members of the family (such as caspase-3 and caspase-9) even though a positive feedback process. Stimulated caspase-3 specifically cleaves its substrate and fragments DNA to produce apoptosis [56]. Caspase-3 and caspase-9 can be initiated, and apoptosis can be caused by regulating the expression of mitochondrial apoptosis-related proteins (eg. Bcl-2 or Bax) or causing apoptosis-related complexes [57].
In contrast, the treatment of the EAC group and EAC+CPA+BM-MSCs with BM-MSCs recorded a significant reduction in the caspase-3 level associated with significant increment in Bcl-2 level as compared to the EAC group and EAC+CPA group. BM-MSCs considerably diminished caspase-3 activity, this finding verified their role in mitigating inflammatory reply and apoptosis to healing damaged cells and BM-MSCs are of a certain therapeutic effect for hepatic dysfunction [38]. It has been described that apart from cytokine/chemokine secretion, MSCs also exhibit a powerful capacity for mitochondrial transfer and microvesicle (exosomes) emission in response to injury with consequent advancement of tissue regeneration [58]. Exosomes have a most important role in the paracrine impact of transplanted cells where they be able to carry a complex content that involves nucleic acids proteins long noncoding RNAs and microRNA [59]. Feng et al. [60] displayed that MSCs-derived exosomes include miR-22, which silence methyl CpG binding protein 2 and lessened apoptosis in cardiac tissue. Additionally, numerous studies should be performed to establish the vital role of cytokines, growth factor and MSCs released vesicles (exosomes) in purpose repair of MSCs in addition to establishing their role in hepatic microenvironment.
The present study showed that Ehrlich solid tumor-bearing mice showed harmful effects on liver tissue displayed by degeneration of the liver cells and dilatation in the portal vein beside marked congestion of erythrocytes and leucocytes surrounded by Aggregations of inflammatory cells. The infiltrations of Ehrlich tumor cells are thought to be the proliferation and migration of tumor cells into the internal organs and the presence of Aggregations of inflammatory cells might occur to degeneration of the mitochondria or disorganization of the cytoplasm [61]. Meanwhile, CPA showed major impairments dilatation in the portal vein and marked necrotic area surrounded by inflammatory cells and giant Kupffer cells in the tissue. This observation coincided with the results of Rahmouni et al. [62] who found that hepatic centrilobular necrosis, degeneration, and infiltration in the liver of CPA-intoxicated animals is associated with A higher level of liver marker enzymes in the bloodstream. On the other side, co-treatment of BM-MSCs with CPA compensated the conspicuous hepatic alterations induced by EAC and CPA and restored the liver tissue to normal hepato-cellular architecture indicating to anti-inflammatory, antiproliferative and anti-fibrotic effect of BM-MSCs [63].
CONCLUSION
In conclusion, these findings demonstrated that BM-MSCs might be significantly reducing CPA-induced hepatotoxicity in EAC mice. This protection of BM-MSCs was demonstrated by the induction of the antioxidant enzyme systems to increase the disposal of overproduction reactive free oxygen radicals by blocking the lipid peroxidation in liver tissue after CPA treatment. Moreover, this study revealed that BM-MSCs significantly ameliorate the inflammation and apoptosis in liver tissue characterizing hepatotoxicity in EAC mice treated with CPA due to its antiinflammatory effect as well as its antiapoptotic effect. In addition, BM-MSCs enhance the antitumor properties of CPA by increase the inhibitory effect of CPA on tumor growth.
FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
AUTHORS CONTRIBUTIONS
Asmaa M Zaazaa and Bosy A Abd El-Motelp created, designed the study, analyzed the data, wrote and revised the manuscript.
Cell surface marker expression analysis, characterization using flow cytometry and tracking of stem cellsThe immunophenotype of BM-MSCs cells was analyzed by flow cytometry. BM-MSCs cells were negative for the hematopoietic marker CD34 (fig. 2A), while powerfully positive for mesenchymal stem cell markers including CD29 (fig. 2B) and CD90 (fig. 2C). The blue histograms represent antibody labeled cells while the grey histogram shows the profile of the isotype control. BM-MSCs were labeled by PKH26 to track its engraftment in the liver tissue (fig. 2D and 2E).
Fig. 2 :
2Immunophenotype of BM-MSCs was examined by flow cytometry. BM-MSCs cells were negative for the hematopoietic marker CD34 (A), while strongly positive for mesenchymal stem cell-specific markers including CD29 (B) and CD90 (C). The blue histograms represent antibody labeled cells while the grey histogram shows the profile of the isotype control. EAC+BM-MSCs group showing PKH26-labelled injected stem cells engrafted in liver tissue (D) and EAC+CPA+BM-MSCs group showing PKH26-labelled injected stem cells engrafted in liver tissue (E)The anti-tumor studies
Fig. 3 :Fig. 4 :Fig. 5 :
345Showing the anti-tumor activities of CPA or/and BM-MSCs on solid EAC compared to the control group (after forty days from inoculation of EAC). (a) normal control mouse, (b) mouse bearing tumor, (c) mouse bearing tumor and treated with CPA, (d) mouse bearing tumor and treated with BM-MSCs and (e) mouse bearing tumor and treated with CPA and BM-MSCs Showing the morphological changes in the legs bearing tumor after treatment with CPA or/and BM-MSCs compared to control leg (after forty days from inoculation of EAC). (a) normal leg of control mouse, (b) leg of a mouse bearing tumor, (c) leg of a mouse bearing tumor and treated with CPA, (d) leg of a mouse bearing tumor and treated with BM-MSCs and (e) leg of a mouse bearing tumor and treated with CPA and BM-MSCs Showing the reduction in the tumor size after treatment with CPA or/and BM-MSCs compared to the EAC without any treatment (after tumor isolation from the leg) after forty days from inoculation of EAC. (a) tumor of a mouse bearing EAC without treatment, (b) tumor of a mouse bearing EAC and treated with CPA, (c) tumor of a mouse bearing tumor and treated with BM-MSCs and (d) tumor of mouse bearing EAC and treated with CPA and BM-MSCs
The treatment of EAC mice with the CPA, BM-MSCs or CPA and BM-MSCs together resulted in an increase in the life span (ILS %), which reached to 153.8 %, 123%, and 169.2 %, respectively, compared to the EAC mice(table 1). No marked difference in the mean tumor weight was found in the EAC group treated with BM-MSCs (5.25g) compared to the EAC group (5.48 g). Meanwhile, Treatment involving CPA and CPA+BM-MSCs revealed highly growth retardation in the mean tumor weight (3.41g) and (2.98 g), respectively(table 1and fig
Table 1 :
1The anti-tumor activities of CPA or/and BM-MSCs solid Ehrlich carcinoma mice PTI= Post tumor inoculation. M= Mortality. MTW= Mean tumor weight. MST (days)= Mean survival time. ILS= Increase of life span. T/C= Tumor inhibition ratio.PTI
(days)
EAC
EAC+CPA
EAC+BM-MSCS
EAC+CPA+BM-MSCS
M
MTW±SE
M
MTW±SE
M
MTW±SE
M
MTW±SE
10
0/8
1.19±0.02
0/8
1.13±0.08
0/8
1.01±0.07
0/8
1.21±0.03
17
0/8
2.11±0.03
0/8
1.62±0.21
0/8
1.93±0.16
0/8
1.31±0.14
24
0/8
3.23±0.21
0/8
2.33±0.24
0/8
2.84±0.16
0/8
1.47±0.06
30
2/8
4.55±0.33
0/8
2.58±0.21
0/8
3.71±0.03
0/8
1.72±0.19
36
3/8
6.54±0.41
0/8
2.94±0.08
1/8
4.22±0.21
0/8
2.23±0.13
42
5/8
7.13±0.36
0/8
3.25±0.42
2/8
6.57±0.26
0/8
2.82±0.19
48
6/8
8.52±0.25
0/8
3.82±0.29
3/8
7.25±0.18
0/8
3.22±0.18
54
8/8
9.82±0.45
2/8
4.23±0.32
5/8
7.91±0.20
0/8
3.68±0.28
60
3/8
4.60±0.08
6/8
8.22±0.26
1/8
3.91±0.12
66
5/8
5.29±0.20
8/8
8.92±0.31
3/8
4.32±0.12
72
8/8
5.81±0.36
6/8
4.71±0.32
78
8/8
5.23±0.32
MTW
5.48
3.41
5.25
2.98
MST (days)
39
60
48
66
ILS%
-
153.8
123
169.2
T/C%
-
37.77
4.05
45.62
Table 2 :
2Effects of treatment with CPA or/and BM-MSCs on liver enzymes in liver tissue of EAC mice Effects of BM-MSCs on EAC mice treated with cyclophosphamide (CPA)Parameters
Groups
ALT
(U/l)
AST
(U/l)
GGT
(U/g)
Con
66.17±7.28
235.62±12.35
25.83±4.05
EAC
97.73±8.35 a
a (47.69%)
283.25±11.82 a
a (20.21%)
132.62±12.82 a
a (413.43%)
EAC+CPA
120.62±9.13 b
b (23.42%)
348.25±15.31 b
b (22.94%)
118.23±4.21 b
b (-10.85%)
EAC+BM-MSCs
82.08±6.71 b
b (-16.01%)
268.30±12.62 b
b (-5.27%)
62.82±6.23 b
b (-52.63%)
EAC+CPA+BM-MSCs
72.56±8.23 bc
b (-25.75%)
c (-39.84%)
252.82±13.12 bc
b (-10.74%)
c (-27.40%)
38.71±4.32 bc
b (-70.81%)
c (-67.25%)
a: Significant change at P<0.05 when it compared to the normal control group (Con); b: Significant change at P<0.05 contra EAC group. c: Significant
change at P<0.05 contra EAC+CPA group.
Table 3 :
3Effects of treatment with CPA or/and BM-MSCs on oxidative stress markers in liver tissue of EAC miceParameters
Groups
MDA
(nmol/gm)
GSH
(mg/g tissue)
SOD
(µg/gm)
Con
86.28±9.12
56.43±6.12
338.81±10.12
EAC
165.14±10.52 a
a (91.40%)
26.32±4.41 a
a (-53.35%)
226.82±11.21 a
a (-33.05%)
EAC+CPA
282.76±17.32 a
b (71.22%)
16.11±2.12 b
b (-38.79%)
118.32±9.24 b
b (-47.85%)
EAC+BM-MSCs
132.27±11.35 b
b (-19.90%)
33.02±5.94 b
b (25.45%)
272.11±12.61 b
b (19.96%)
EAC+CPA+BM-MSCs
99.82±10.81 bc
b (-39.55%)
c (-64.69%)
45.51±5.11 bc
b (72.91%)
c (182.49%)
315.25±13.03 bc
b (38.98%)
c (166.44%)
a: Significant change at P<0.05 when it compared to the normal control group (Con); b: Significant change at P<0.05 contra EAC group. c: Significant
change at P<0.05 contra EAC+CPA group.
Table 4 :
4Effects of treatment with CPA or/and BM-MSCs on inflammatory and anti-inflammatory markers in liver tissue of EAC miceParameters
Groups
IL-6
Pg/mg protein
IL-10
Pg/mg protein
Con
36.52±5.21
146.82±10.31
EAC
111.24±10.32 a
a (204.60%)
96.92±9.34 a
a (-33.92%)
EAC+CPA
203.62±11.32 b
b (83.04%)
42.82±5.12 b
b (-55.81%)
EAC+BM-MSCs
72.53±9.62 b
b (-34.79%)
116.87±11.52 b
b (20.58%)
EAC+CPA+BM-MSCs
56.32±8.63 bc
b (-49.37%)
c (-72.34%)
132.04±12.53 bc
b (36.23%)
c (208.36%)
a: Significant change at P<0.05 when it compared to the normal control group (Con); b: Significant change at P<0.05 contra EAC group. c: Significant
change at P<0.05 contra EAC+CPA group.
Table 5 :
5Effects of treatment with CPA or/and BM-MSCs on apoptotic and anti-apoptotic markers in liver tissue of EAC miceParameters
Groups
Caspase-3
ng/mg protein
Bcl2
ng/mg protein
Con
3.08±0.40
8.52±1.08
EAC
8.71±0.53 a
a (182.79%)
4.32±0.53 a
a (-49.29%)
EAC+CPA
11.56±2.22 b
b (32.72%)
2.61±0.18 b
b (-39.58%)
EAC+BM-MSCs
6.11±0.32 b
b (-29.85%)
5.68±0.81 b
b (31.48%)
EAC+CPA+BM-MSCs
4.50±0.62 bc
b (-48.33%)
c (-61.07%)
7.12±1.12 bc
b (64.81%)
c (172.79%)
a: Significant change at P<0.05 when it compared to the normal control group (Con); b: Significant change at P<0.05 contra EAC group. c: Significant
change at P<0.05 contra EAC+CPA group.
Int J Pharm Pharm Sci, Vol 12, Issue 4, 53-62
CONFLICT OF INTERESTSThe authors declare that they have no conflicts of interest that influenced this work.
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Background
Hypoxia is a ubiquitous phenomenon in most malignant tumors, which is the main reason for the tumors refractory and resistance to chemoradiotherapy (1,2). Noninvasive detection of the extent of hypoxic areas of tumors is essential for delineation of radiotherapy target areas and dose. In the oxygen-enriched area, enough radiation doses could be given to kill malignant tumor cells. Then, oxygen-enriched cells experience an apoptotic die and hypoxic cells get enough oxygen and transform into oxygenenriched cells, in which, enough doses could be given too. Therefore, monitoring the range and extent of hypoxic area can guide the radiation dose segmentation and the timing of radiotherapy. At present, there are many methods for the detection of tumor hypoxia, but most of them are invasive. Thus, their applications are restricted. Non-invasive detection of hypoxic areas has been improved continuously, and radionuclide hypoxic imaging is currently one of the most concerned detection methods of tumor hypoxia (3), which is beneficial to the diagnosis and treatment of tumor (4).
In 2009, Zha et al. (5) reported a new synthetic hypoxic imaging agent 99 Tc m -metronidazole phosphate ester [2-(2-methyl-5-nitro-1H-imidazol-1-yl) phosphate ethyl ester], also namely 99 Tc m -MNLS. A preliminary animal study has shown 99 Tcm-MNLS has rapid clearance in blood and early tumor imaging. It is mainly excreted through the kidneys.
Objectives
The aim of this study was to investigate the feasibility of 99 Tc m -MNLS in monitoring the hypoxic status of tumor after radiotherapy and to confirm whether it could accurately reflect the tumor hypoxic status by immunohistochemical staining of hypoxia-inducible factor 1α (HIF-1α) in tumor cells.
Materials and Methods
Quality Control 99 Tc m -MNLS
A freshly rinsed 99 Tc m liquid adjusted to 7.4 MBq/0.1mL with saline was added into a MNLS lyophilized product (Beijing Xinkesida Pharmaceutical Technology Co., Ltd., China), and then shaken manually and stood at room temperature for 10 minutes. Polyamide as the stationary phase and 10% hydrochloric acid solution as a developing solvent, the radiochemical purity of 99 Tc m -MNLS kept at room temperature and 37°C for 30 minutes, 1, 2, 3, 4, 6 and 8 hours was determined.
Establishment of Tumor-Bearing Mouse Model
Ninety Kunming mice (clean grade, half male and half female), aged 6 -8 weeks old and weighing 20 -25 g, were provided by the Experimental Center. 0.1 mL H22 hepatoma cells (provided by the Experiment Center) were subcutaneously inoculated in the right forelimb armpit of each mouse at a concentration of 2 × 10 8 /mL. Then, the mice were fed in a clean environment for eight days. The size of the tumor was measured each day. The largest diameter of the tumor was about 1 cm at the last observing day. The tumor-bearing mice with well-growing, regular shape and almost similar size of tumors were selected for further experiments.
99 Tc m -MNLS Imaging and Biological Distribution in Tumor-Bearing Mice
Forty-two tumor-bearing mice were equally divided into seven groups according to the imaging time after the injection by random number table: 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours and 8 hours groups. Mice were treated with tail vein injection of 7.4 MBq/0.1 mL 99 Tc m -MNLS and received single photon emission computed tomography (SPECT) (Millennium VG Hawkeye, GE Company, the United States) imaging at the corresponding time point of each group. The image acquisition parameters were set as: SPECT equipped with high resolution collimator with low energy, each frame image acquisition was 200 K and the matrix was 256 × 256 with 4 times magnification. The region of interest (ROI), such as the tumor site and the contralateral site, was sketched out on the images obtained from each group to get their respective radiocounting. The ratio of the radiocounting in tumor and non-tumor (T/NT) was calculated. Immediately after the imaging, the eyeballs of each tumor-bearing mouse were taken out for collecting blood and then the mouse was killed by dislocating the neck. Tissues such as tumor, heart, lung, liver, stomach and hind limb muscles were taken out for measuring the radiocounting per minute of each tissue using γ-counting instrument (ZD-6000 well type counting technetium analyzer, Xi'an Zhida Company, China) and the percent of injected dose per gram (% ID/g) of each tissue at each time point was calculated. The standard source of 99 Tc m -MNLS was reserved.
99 Tc m -MNLS Imaging on Tumor-Bearing Mice
Forty-eight successfully established tumor-bearing mice were randomly divided into eight groups according to random number table: imaged at 24 hours before radiotherapy group (A 24 h before ) and its control (C 24 h before ), imaged immediately after radiotherapy (A immediately ) and its control (C immediately ), imaged at 24 hours after radiotherapy (A 24 h ) and its control (C 24 h ) and imaged at 48 h after radiotherapy (A 48 h ) and its control (C 48 h ). Radiotherapy was given to the mice in A immediately , A 24 h and A 48 h groups, but not to A 24 h before and all the control groups. After radiotherapy, mice in each group were injected with 99 Tc m -MNLS at the corresponding time points and imaged at 2 hours after the injection. The ROIs of the tumor region and its counter-lateral region were sketched out on the images obtained from each group to obtain their own radiocounting. Then the radiocounting ratio between tumor and non-tumor (T/NT) was calculated. Two doctors with over 10-year experience delineated the coverage of ROI independently and compared. If the ROIs were mismatched between the two doctors, they would discuss and reach a consensus about the ROI; then one of the doctors re-delineated the ROI according to the consensus.
Immunohistochemistry (IHC)
Immediately after the imaging, mice were sacrificed by dislocating the neck. The tumor tissues were isolated and made into paraffin sections. The expression of HIF-1α was determined by IHC using HIF-1α primary antibody and streptavidin-peroxidase ligation kit (BIOWORLD Co., USA). The cytoplasm of tumor cells stained brown-yellow was positive for HIF-1α. The percentage of positive cells in each field of vision was regarded as the HIF-1α expression in this field of vision. Five non-overlapping fields of vision at 400 × magnification were selected on each slice from top to bottom and from left to right to calculate the average percentage of HIF-1α positive cells, which was taken as the expression of HIF-1α in this slice. Every field was counted twice, and the average percentage of HIF-1α positive cells was taken as the expression of HIF-1α in this field. 2 Iran J Radiol. 2019; 16(4):e86127.
Statistical Analysis
Data were expressed as mean ± standard deviation (SD) and analyzed using statistical software SPSS version 17.0. The normality of data were assessed before analysis.
If the data were normality, comparisons of measurement data were carried out using one-way ANOVA, pairwise comparison least significant difference (LSD) -t test, two independent sample t-test and non-parametric Spearman analysis. If not, the Rank sum test was used. The difference was statistically significant if P < 0.05 was considered.
Results
Quality Control of 99 Tc m -MNLS and Tumor-Bearing Mice
The labeled 99 Tc m -MNLS was a colorless and transparent solution. Its radiochemical purity at room temperature for 10 minutes was 97%. Moreover, it could be stable for at least 8 hours after it was labeled. Its radiochemical purities were all greater than 90% at room temperature and 37°C for 30 minutes, 1, 2, 3, 4, 6 and 8 hours (5). There was no significant difference in the weight and diameter of tumors among the groups (F = 4.539, t = 1.980, P > 0.05).
99 Tc m -MNLS Imaging and Its Biological Distribution in Tumor-Bearing Mice
All tumor-bearing mice developed clear tumor images at the right forelimb armpit. The retention of imaging agents in tumors (T) and non-tumorous tissues (NT) was highest at 1 hour, and excreted with time, which was more significant in non-tumorous tissues, T/NT ratio was the highest and the imaging was the best at 2 hours after the injection of 99 Tc m -MNLS. 99 Tc m -MNLS could be absorbed quickly by tumor (0.243 ± 0.026 % ID/g at 30 minutes). The percentages of injected dose per gram tumor (% ID/g) at different time points were compared with that at 2 hours (Table 1). 99 Tc m -MNLS was mainly excreted via the kidneys. The ratio of % ID/g in tumor and muscle (T/M) was increased significantly over time within 2 hours, and reached 5.368 ± 2.624 %ID/g at 2 hours, then it began to decrease gradually over time from 3 hours.
99 Tc m -MNLS Imaging
The 99 Tc m -MNLS imaging of tumor-bearing mice after radiotherapy showed that the T/NT ratio changed with a trend of A immediately > A 24 h > A 48 h (F = 16.031, t = 5.640 and 7.674, P < 0.05). No statistical significance was found in the T/NT ratio between A 24 h before and A 24 h or its control group (t = 1.967 or t = 0.635, P > 0.05), but it changed following the trend of A immediately > A 24 h before > A 48 h (F = 15.481, t = 6.732 and 7.589, P < 0.05) (Figure 1). The T/NT ratio in A immediately group was significantly higher than that in its control group C immediately (
Expression of HIF-1α
The expression of HIF-1α in A 24 h before , A immediately , A 24 h and A 48 h groups was 45.43 ± 2.35%, 87.52 ± 0.84%, 49.12 ± 3.78%, and 21.58 ± 0.89%, respectively; while in the C 24 h before , C immediately , C 24 h and C 48 h groups, it was 40.52 ± 3.24%, 48.26 ± 1.70%, 62.38 ± 0.89%, and 74.57 ± 2.98%, respectively ( Table 2 and Figure 2). The comparisons of the expression of HIF-1α among the groups after radiotherapy displayed that A immediately > A 24 h > A 48 h (F = 28.056, t = 6.701 and 4.911, P < 0.05). No statistical significance in the expression of HIF-1α was found between A 24 h before and A 24 h or its control group (t = 0.935 or t = 2.513, P > 0.05), but its changes showed a trend of A immediately > A 24 h before > A 48 h (F = 29.189, t =6.693, 4.877, P < 0.05). The expression of HIF-1α in A immediately was remarkably higher than that of C immediately (t = 3.563, P < 0.05), but there was no significant difference in the expression of HIF-1α between A 24 h and C 24 h (t = 2.125, P > 0.05), while that in A 48 h group was markedly lower than that in C 48 h group (t = 3.279, P < 0.05) ( Table 2).
Correlation Between 99 Tc m -MNLS Hypoxia Imaging and the Expression of HIF-1α
There was a positive correlation between T/NT ratio and the expression of HIF-1α in the radiotherapy groups (R s = 0.793, P < 0.05). The correlation was also found in the control groups (R s = 0.756, P < 0.05).
Discussion
99 Tc m -MNLS is a positive hypoxic imaging agent. Its imaging mechanism, similar to other nitroimidazole hypoxic imaging agents (6, 7), is associated with its high permeability and high redox potential. High permeability is conducive to its rapid access to mitochondria within the cell, while high redox potential is to promote its redox reaction. Thus, it is an ideal positive hypoxic imaging agent.
In this study, the distribution of 99 Tc m -MNLS in tumorbearing mice showed that the uptake of 99 Tc m -MNLS by the tumor was higher at early stage. Its distribution was mainly in the blood, liver and kidney but less in the brain, spleen, stomach and intestine. With extension of time, radioactivity was gradually increased in the tumor, while that in other organs (except kidneys and bladder) and tissues was gradually decreased. The T/NT ratio was increased gradually with time and reached the highest level at 2 hours. The reason may be that 99 Tc m -MNLS is continuously diffused from the blood to the hypoxic area and absorbed by the hypoxic cells, and the background in blood is gradually cleared with time. In contrast, the low T/NT ratio in the tumor, kidney and bladder may result from the excretion 99 Tc m -MNLS mainly by the kidneys and bladder. 99 Tc m -MNLS hypoxia imaging showed that the tumor site could be visualized as early as within 30 minutes, and clearly at 2 hours with low background and high T/NT ratio. Then, the imaging of the tumor site and background were both reduced with time, so was the T/NT ratio. No obvious imaging was found in the abdomen of tumor-bearing mice except the kidneys, suggesting that 99 Tc m -MNLS can display tumor lesions well and may have greater advantages on displaying the abdominal neoplasms than other hypoxia imaging agents (such as 99 Tc m -HL91, which dis-plays enriched radioactivity in all the abdomen). However, it needs more studies to confirm. After single high-dose radiotherapy, apoptosis occurs in most of the oxygen-rich tumor cells, but the functional blood vessels of the tumor were less affected. Cells in part of the hypoxic tissue were relieved from the hypoxic state and transferred into oxygen-rich cells. Such process is called re-oxygenation of the hypoxic tumor cells. Researches (8) have reported hypoxic fraction of radiationsensitive solid tumors is close to 100% immediately after radiotherapy due to the rapid reduction of oxygen-rich cells, and then it is gradually decreased to the initial value after a period of time. In this study, the T/NT ratio at 24 hours before radiotherapy was significantly lower than that immediately after radiotherapy because the killing of oxygen-rich cells is an oxygen consumption process, which increases the degree of tumor hypoxia, and the death of oxygen-rich cells leads to the relative increase of the proportion of hypoxic cells. The changes of T/NT ratio in the ra-Iran J Radiol. 2019; 16(4):e86127. 5 diotherapy groups showing a trend of increasing first and then decreasing gradually were the re-oxygenation process of the hypoxic cells in the tumor. Different types of tumors have different sensitivity to radiotherapy. For instance, lymphomas and seminoma are sensitive to radiotherapy with significant effect, while melanoma and glioma are not sensitive to radiotherapy with poor curative effect. Nevertheless, most of the remaining malignant solid tumors have moderate sensitivity to radiotherapy and easily relapse and metastasize (9). Hypoxic cells in the tumors mainly contribute to this phenomenon because the percentage of hypoxic cells determines the sensitivity of the tumor to radiotherapy. The ionizing radiation of radiotherapy induces DNA to generate free radicals, combining with oxygen to form peroxides, which cause strand break of DNA and lipid peroxidation of the cell membrane, thereby leading to tumor cell death (10). Low oxygen content in hypoxic cells leads to insufficient combination with free radicals generated after radiotherapy to cause tumor cells death, thus, the dose of radiotherapy should be improved. Studies have shown that if the tumors are all hypoxic cells, the radiation dose should be three times of aerobic cells to kill most of the cells (> 90%), but a simple increase of the radiation dose will inevitably increase the chance of damaging the surrounding normal tissues (11). Therefore, assessing the proportion of hypoxic to oxygen-rich cells in the tumor is of great significance for determining the appropriate timing of radiotherapy. Adjusted fractionated irradiation should be considered basing on the extent of tumor re-oxygenation. The re-oxygenation time of hypoxic tumor cells provides a basis for clinicians to make a decision of re-radiation (12). In this experiment, the degree of hypoxia in the tumor site of 48 hours group was lower than that of A immediately and A 24 h groups, indicating that re-oxygenation occurred in the hypoxic cells of the tumor. Regarding this, one more radiotherapy for the tumor site at this time can achieve better results than at 24 hours after the first radiotherapy.
HIF-1α is an important transcription factor in tissues that responds to the hypoxia microenvironment. HIF-1α can let tumor cells adapt to hypoxia microenvironment formed due to the rapid proliferation by regulating various biological processes including glycolysis, angiogenesis factor, vascular endothelial growth factor (VEGF) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) (13)(14)(15)(16)(17). Its content can also predict the efficacy of radiotherapy on tumor (18,19). HIF-1α has an oxygen-dependent degradation region in the polypeptide region and is strongly dependent on oxygen. HIF-1α is easily degraded in oxygenrich condition, so it is almost undetectable in normal cells. But the hypoxia condition increases the protein stability of HIF-1α, resulting in the block of HIF-1α degradation and its continuous accumulation in tumor hypoxic cells. Reoxygenation of hypoxic cells can effectively inhibit HIF-1α production, thus the amount of HIF-1α can also reflect the re-oxygenation state of hypoxia within tumor (20,21). In conclusion, our results showed that most of the hypoxic tissues in tumor had different levels of HIF-1α expression, and its expression was significantly different with the different treatments; HIF-1α expression had good correlation with tumor hypoxia imaging. Our findings strongly proved that 99 Tc m -MNLS tumor hypoxic imaging is likely to accurately predict the changes of the hypoxic state of the tumor.
As the main point of the study findings, we can say that 99 Tc m -MNLS tumor hypoxic imaging can truly and accurately reflect the changes of tumor hypoxia. 99 Tc m -MNLS tumor hypoxic imaging suitable for detecting the tumor hypoxia and monitoring the changes of tumor hypoxic state after radiotherapy. 99 Tc m -MNLS tumor hypoxic imaging expected to become a non-invasive method for the detection of the extent and scope of tumor hypoxia, and to provide a strong basis for the determination of tumor fractionated radiation time.
Footnotes
Authors' Contributions: Yujing Hu performed the experiments, analyzed the data and wrote the paper. Dayong Wu and Congna Tian contributed to experimental design and paper discussion. Wenyan Zhang and Qiang Wei contributed to the data analyses. Yanzhu Bian conceived and directed the research. All authors read and approved the final manuscript.
Figure 1 .
199 Tc m -2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl dihydrogen phosphate ( 99 Tc m -MNLS) imaging in each group. A -D, Radiotherapy groups [Aimmediately (A), A24 h before (B), A24 h (C), and A48 h (D)]; E -H, Control groups [Cimmediately (E), C24 h before (F), C24 h (G), and C48 h (H)]. Arrow shows the tumor location.
Figure 2 .
2The expression of hypoxia inducible factor-1α (HIF-1α) in each group determined by immunohistochemistry (IHC) (SP method, 400×). A -D, Radiotherapy groups [Aimmediately (A), A24 h before (B), A24 h (C) and A48 h (D)]; E -H, Control groups [Cimmediately (E), C24 h before (F), C24 h (G) and C48 h (H)]. 4 Iran J Radiol. 2019; 16(4):e86127.
3.38 ± 0.23 vs. 2.78 ± 0.29, t = 4.125) (P < 0.01), but there was no significant difference in T/NT ratio between A 24 h and C 24 h (2.78 ± 0.37 vs. 2.98 ±0.16, t = 0.546, P > 0.05), while that in A 48 h group was lower
than that in C 48 h group (2.22 ± 0.43 vs. 3.17 ± 0.76, t = 7.893,
P < 0.05, Table 2).
Table 1 .
1In Vivo Biological Distribution of 99 Tc m -MNLS in Tumor-Bearing Mice (% ID/g, N = 6) aTissues
30 min
1 h
2 h
3 h
4 h
6 h
8 h
Brain
0.004 ± 0.001
0.003 ± 0.001
0.002 ± 0.001
0.002 ± 0.001
0.004 ± 0.001
0.018 ± 0.003
0.036 ± 0.001
Heart
0.037 ± 0.051
0.026 ± 0.009
0.016 ± 0.003
0.014 ± 0.002
0.019 ± 0.006
0.018 ± 0.013
0.009 ± 0.001
Liver
0.503 ± 0.191
0.491 ± 0.051
0.336 ± 0.151
0.289 ± 0.150
0.168 ± 0.138
0.115 ± 0.053
0.038 ± 0.009
Spleen
0.072 ± 0.051
0.044 ± 0.011
0.079 ± 0.021
0.083 ± 0.018
0.025 ± 0.009
0.115 ± 0.064
0.033 ± 0.167
Lung
0.072 ± 0.152
0.070 ± 0.016
0.043 ± 0.008
0.056 ± 0.005
0.062 ± 0.020
0.085 ± 0.010
0.029 ± 0.015
Kidney
3.562 ± 0.410
5.929 ± 1.028
6.071 ± 0.765
2.938 ± 0.931
2.104 ± 0.651
2.885 ± 0.959
0.350 ± 0.074
Stomach
0.037 ± 0.003
0.029 ± 0.009
0.073 ± 0.022
0.089 ± 0.050
0.093 ± 0.019
0.053 ± 0.027
0.034 ± 0.007
Intestines
0.059 ± 0.136
0.120 ± 0.034
0.094 ± 0.029
0.085 ± 0.173
0.039 ± 0.009
0.026 ± 0.022
0.020 ± 0.014
Blood
0.629 ± 0.020
0.504 ± 0.146
0.286 ± 0.051
0.081 ± 0.009
0.070 ± 0.013
0.052 ± 0.093
0.018 ± 0.014
Muscle
0.091 ± 0.008
0.084 ± 0.007
0.067 ± 0.002
0.052 ± 0.001
0.036 ± 0.002
0.029 ± 0.006
0.020 ± 0.001
Tumor
0.243 ± 0.026 *
0.365 ± 0.144 *
0.359 ± 0.033
0.279 ± 0.060 *
0.178 ± 0.075 *
0.125 ± 0.035 *
0.043 ± 0.012 *
Bone
0.278 ± 0.099
0.051 ± 0.009
0.062 ± 0.014
0.081 ± 0.014
0.082 ± 0.005
0.050 ± 0.004
0.033 ± 0.002
Abbreviations: SD, standard deviation; h, hours; 99 Tc m -MNLS, 99 Tc m -2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl dihydrogen phosphate; %ID/g, percentage injected dose
per gram.
a Values are expressed as mean ± SD.
* P < 0.05 vs. 2 hours.
Table 2 .
2T/NT of 99 Tc m -MNLS Hypoxic Imaging and Expression of Hypoxia Inducible Factor-1α in Different Subgroups a99 Tc m -MNLS hypoxic imaging
Hypoxia inducible factor-1α
Radiotherapy group
Control group
P value
Radiotherapy group, %
Control group, %
P value
24 h before
2.17 ± 0.19
2.68 ± 0.12
> 0.05
45.43 ± 2.35
40.52 ± 3.24
> 0.05
Immediately
3.38 ± 0.23
2.78 ± 0.29
< 0.05
87.52 ± 0.84
48.26 ± 1.70
< 0.05
24 h
2.78 ± 0.37
2.98 ± 0.16
> 0.05
49.12 ± 3.78
62.38 ± 0.89
> 0.05
48 h
2.22 ± 0.43
3.17 ± 0.76
< 0.05
21.58 ± 0.89
74.57 ± 2.98
< 0.05
Abbreviations: T/NT, tumor/non-tumor, SD, standard deviation; h, hours; 99 Tc m -MNLS, 99 Tc m -2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl dihydrogen phosphate.
a Values are expressed as mean ± SD.
Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
Iran J Radiol. 2019; 16(4):e86127.
Conflict of Interests
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| 499 | A review of the literature on the effect of hypoxia on in vitro drug sensitivity had suggested that there was consistently more cytotoxicity under hypoxic conditions for the drugs misonidazole and mitomycin C while there was much conflicting data for the drugs Adriamycin and bleomycin. We have examined the effect of oxygen on the cellular response of Ehrlich's ascites tumor cells to the drugs mitomycin C, misonidazole, Adriamycin, and bleomycin. Significant differences were observed when we compared the cytotoxicity of mitomycin C and misonidazole as a function of oxygen concentration. For the drugs Adriamycin and bleomycin no differential effects of oxygen were noted for a 1-h drug exposure with hypoxia while some differences were noted only for bleomycin for an 8-h drug exposure time. Because differences dependent on oxygen concentration were observed for some drugs but not others at the same experimental conditions, and as indicated by a review of the literature, it is suggested that some of the conflicting data in the literature with respect to some of these drugs may be cell-line dependent. Other variables which may also be of importance were the duration of drug exposure time in hypoxia and cell density. The observed oxygen concentration-dependent changes in cell survival for the Ehrlich cells with drugs examined could not be explained on the basis of changes in drug-induced cellular oxygen consumption. | ROS (reactive oxygen species) generated by hypoxia facilitate the vascular inflammatory response, but whether systemic hypoxia influences leucocyte bactericidal activity by modulating circulatory redox status remains unclear. The present study elucidates how various hypoxic interventions influence the bactericidal activity of PMNs (polymorphonuclear leucocytes) following treatment with an antioxidant, vitamin E (D-α-tocopheryl acetate). Forty healthy sedentary men were randomly assigned to vitamin E ( n =20) and placebo ( n =20) groups. At 1 h following oral administration of 1000 i.u. of vitamin E or placebo, each subject in the two groups was randomly exposed to 12%, 15%, 18% and 21% O 2 for 2 h in a normobaric hypoxia chamber. The results demonstrated that exposure to 12% O 2 in the placebo group increased urinary 8-isoprostane and plasma malondialdehyde levels and decreased plasma total antioxidant content and superoxide dismutase activity, but did not alter plasma complement-C3a desArg/C4a desArg/C5a concentrations. Moreover, this hypoxic exposure also increased the chemotaxis of PMNs by exposure to N -formyl-Met-Leu-Phe, the phagocytosis of PMNs to Escherichia coli and the release of PMN oxidant products by E. coli , processes which were accompanied by increased expressions of L-selectin, LFA-1 (lymphocyte function-associated antigen 1), Mac-1, FcγIIIBR, C1qRp and C5aR on PMNs. However, exposure to 12% O 2 in the vitamin E group did not influence expression of the opsonic/complement receptors on PMNs, and the chemotactic, phagocytic or oxidative burst activities of PMN, whereas the circulatory redox status and complement fragment levels were unaltered following this hypoxic exposure and pretreatment with vitamin E. Additionally, the circulatory redox status, complement systems, PMN-mediated bactericidal processes and the opsonic/complement receptors on PMNs were constant following exposure to 15%, 18% or 21% O 2 in the two groups. We conclude that exposure to 12% O 2 promotes the chemotactic, phagocytic and oxidative burst activities of PMNs, possibly by increasing lipid peroxidation and decreasing antioxidative capacity. However, this hypoxic effect on PMN bactericidal activity is ameliorated by pretreatment with vitamin E. | The enhancement of melphalan toxicity was observed by preincubation of V-79- 379A cells in spinner culture with multiple doses of misonidazole (miso) or SR-2508 under hypoxic conditions. Chemosensitization was shown to be a function of sensitizer concentration and duration of exposure to the alkylating agent. A preincubation exposure of cells with 5 mM miso reduced endogenous cell thiols to less than 5% of controls and enhanced melphalan toxicity by a factor of 4.7. Cells preincubated with miso not only had lower levels of nonprotein thiols, but also were shown to have altered levels of intracellular calcium and a lower threshold to oxidative stress as measured by toxicity to cysteamine or H2O2. Preincubated cells, hypoxic cells, and cells receiving moderate hyperthermia (42.5 degrees C for 3 hr) all showed increased sensitivity to either cysteamine or H2O2. The increased killing of preincubated cells by cysteamine was shown to be similar to that of H2O2, and the dramatic reduction of cysteamine toxicity by catalase indicated H2O2 was the major reaction associated with this effect. These results indicate that preincubated cells exhibit a variety of biological effects that may significantly influence their response to further treatment with drugs or radiation, especially where peroxidative and free radical mechanisms are involved. The depletion of endogenous thiols, calcium disturbance, and vulnerability to oxidative stress are factors to be considered when interpreting mechanisms of combined drug action and effects that may potentially be exploited in terms of therapeutic gains. | Rat-liver microsomes and NADPH could reduce Adriamycin, epirubicin and daunorubicin to their free radical forms, which enhanced peroxidation of microsomal lipids less than 2-fold in air but 3- to 5-fold at a pO2 of 4 mm Hg. Mitoxantrone was not reduced by microsomes and had no effect on microsomal peroxidation. Daunorubicin caused more lipid peroxidation than similar concentrations of either Adriamycin or epirubicin, which were equally efficient. In each case peroxidation was iron-dependent and could be catalysed by ferritin. The antioxidants β-carotene and α-tocopherol inhibited lipid peroxidation at low or high pO2. The dose-for-dose difference in the cardiotoxicity of epirubicin compared with Adriamycin is not explained by its effect on microsomal lipid peroxidation. However, the lower incidence of cardiotoxicity with mitoxantrone may be a consequence of its inability to form free radical species and promote lipid peroxidation. | Summary The drugs [1-β-d-arabinofuranosylcytosine (ara-C), hydroxyurea (HU), 5-hydroxy-2-formylpyridine thiosemicarbazone (5-HP), and camptothecin sodium salt (camptothecin)] considered in this paper markedly inhibit DNA synthesis and are maximally cytotoxic to cells in S phase. In these studies, high-specific-activity thymidine- 3 H (HSA-TdR- 3 H) was used as a control compound which killed cells in S but which did not affect the progression of cells into S. The cell-kill kinetics indicated that ara-C, HU, and 5-HP, unlike camptothecin, blocked the L1210 cells from progressing into S in the presence of the drug. We found that L1210 cells that were blocked in G 1 by HU started moving into S immediately after the drug was removed. Therefore, the time interval between two doses of HU that gave maximal cell kill was the same as that for HSA-TdR- 3 H. However, L1210 cells exposed to ara-C and 5-HP took about 2 hr to recover from the effect of the drug and then progress into S. Therefore, the time interval (between two doses of either ara-C or 5-HP) that gave maximal cell kill was longer than that needed for HSA-TdR- 3 H. Camptothecin did not block L1210 cells from moving into S and, therefore, the cell-kill kinetics with camptothecin were the same as those with HSA-TdR- 3 H. The time for maximal recovery of DNA synthesis by L1210 cells after a single exposure to the drugs was determined. The time for maximal recovery of DNA synthesis correlated well with the interval required for maximal cell kill. | Bleomycin cytotoxicity is prevented by superoxide dismutase in vitro. | Abstract The quaternary benzo[c]phenanthridine alkaloids (QBA) produce a plethora of species- and tissue-specific effects but the molecular basis of their biological activities remain mysterious. The objective of the present study was to investigate the cytotoxicity of QBA alkaloids, sanguinarine (SA), chelerythrine (CHE), fagaronine (FA), and the extract from Macleaya cordata in primary cultures of human and porcine hepatocytes. The cellular damage was assessed by the MTT assay, lactate dehydrogenase (LDH) leakage and the determination of intracellular glutathione (GSH) levels. The results are summarised as follows: (i) The alkaloids tested in doses 0.1 and 10 μM did not display statistically significant cytotoxicity for 0–3 h incubation; (ii) SA and CHE showed the dose- and time-dependent toxicity within the range 25–100 μM whereas FA was not toxic; (iii) the LDH leakage into the medium was higher for SA than for CHE, thus revealing a potent potential of SA to disturb cell-membrane integrity; (iv) after 3 h incubation with 100 μM SA/CHE, mitochondrial dehydrogenase activity (MTT assay) and the cellular GSH levels decreased to residual values of about 40% suggesting that mitochondria are unlikely to be a primary target for SA/CHE in the cell; (v) no differences were found in the response to QBA application in human vs porcine hepatocyte. | Background: The aim of this study was to evaluate the protective effects of subacute pre-treatment with chamomile (Matricaria recutita L.) decoction extract (CDE) against stimulated neutrophils ROS production as well as ethanol (EtOH)-induced haematological changes and erythrocytes oxidative stress in rat. Methods: Neutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. Adult male wistar rats were used and divided into six groups of ten each: control, EtOH, EtOH + various doses of CDE (25, 50, and 100 mg/kg, b.w.), and EtOH+ ascorbic acid (AA). Animals were pre-treated with CDE extract during 10 days. Results: We found that CDE inhibited (P ≤ 0.0003) luminol-amplified chemiluminescence of resting neutrophils and N-formyl methionylleucyl-phenylalanine (fMLF) or phorbolmyristate acetate (PMA) stimulated neutrophils, in a dose-dependent manner. CDE had no effect on superoxide anion, but it inhibited (P ≤ 0.0004) H 2 O 2 production in cell free system. In vivo, CDE counteracted (P ≤ 0.0034) the effect of single EtOH administration which induced (P < 0.0001) an increase of white blood cells (WBC) and platelets (PLT) counts. Our results also demonstrated that alcohol administration significantly (P < 0.0001) induced erythrocytes lipoperoxidation increase and depletion of sulfhydryl groups (−SH) content as well as antioxidant enzyme activities as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). More importantly, we found that acute alcohol administration increased (P < 0.0001) erythrocytes and plasma hydrogen peroxide (H 2 O 2 ), free iron, and calcium levels while the CDE pre-treatment reversed increased (P ≤ 0.0051) all these intracellular disturbances. Conclusions: These findings suggest that CDE inhibits neutrophil ROS production and protects against EtOH-induced haematologiacal parameters changes and erythrocytes oxidative stress. The haematoprotection offered by chamomile might involve in part its antioxidant properties as well as its opposite effect on some intracellular mediators such as H 2 O 2 , free iron, and calcium. | 1. The in vitro sulphoxidation of Albendazole (ABZ) by rat intestinal microsomes has been examined. The results revealed intestinal sulphoxidation of ABZ by intestinal microsomes in a NADPH-dependent enzymatic system. The kinetic constants for sulphoxidase activity were Vmax = 46 pmol/min/mg protein and Michaelis constant Km = 6·8 μM.2. The possible effect of inducers (Arochlor 1254 and ABZ pretreatment) and inhibitors (erythromycin, methimazole, carbon monoxide and fenbendazole), was also studied. In rat pretreated with Arochlor 1254, Vmax was 52 pmol/min/mg protein, whereas oral administration of ABZ increased the intestinal sulphoxidation of the drug, Vmax being 103 pmol/min/mg protein.3. Erythromycin did not change the enzymatic biconversion of ABZ, but methimazole and carbon monoxide inhibited the enzyme activity by approximately 60 and 30% respectively. Fenbendazole (a structural analogue of ABZ) was a competitive inhibitor of the sulphoxidation process, characterized by a Ki or 69 μM.4. These data ... |
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