Monthly Archives: March 2017

BioTechniques – Semi-automated Tip Snip cloning of restriction … – BioTechniques.com

Posted: March 17, 2017 at 7:23 am

Semi-automated Tip Snip cloning of restriction fragments into and out of plasmid polylinkers

Emory University School of Medicine, Department of Biochemistry, O. Wayne Rollins Research Center, Atlanta, GA

BioTechniques, Vol. 62, No. 3, March 2017, pp. 99106

Supplementary Material

Abstract

Synthetic biologists rely on semi-synthetic recombinant plasmids, but DNA synthesis is constrained by practical limits on length, accuracy, and sequence composition. Cloned DNA parts can be assembled into longer constructs via subcloning, but conventional methods are labor-intensive. One-pot recombination reactions are more convenient but harder to troubleshoot, and those that depend on PCR to create fragments with compatible ends necessitate re-sequencing. The Tip Snip protocol described here enables the subcloning of an insert from one plasmid polylinker into another without PCR or gel purification steps. Cohesive ends of unwanted restriction fragments are snipped off by additional restriction endonucleases. The resulting short fragments (snippets) are eliminated by hybridization to complementary oligonucleotides (anti-snippets) and subsequent size-selection spin-column chromatography. Unwanted linear donor vectors are ligated to double-stranded oligonucleotides (unlinkers) so that only the desired insert and recipient plasmid form circular DNA capable of transforming bacteria. This new method is compatible with high-throughput processing and automated liquid handling, and because no specialized vectors, reagents, selection schemes, or analytical techniques are required, the barriers to adoption are low.

DNA synthesis costs are decreasing (1), but the assembly and cloning of synthetic DNA remains relatively labor-intensive and expensive. Nucleoside phosphoramidites are chemically synthesized on large scales. Single-stranded oligonucleotides and double-stranded synthetic genes are custom manufactured by machines, so turnover is rapid, throughput is high, and production costs are relatively low. Automation and miniaturization have decreased the per-unit cost of synthesizing gene-length (2 kb) DNAs, but further innovations are required to overcome practical limitations in length, nucleotide composition, accuracy, and yield (1). It is not yet feasible to have every new construct synthesized with its vector de novo, so PCR products and synthetic genes are most often cloned into plasmids and then sequenced.

Cloned parts are often assembled into larger constructs by subcloning, but this classical approach is recalcitrant to automation for three reasons. First, robots that can load agarose gels and purify particular restriction fragments have not yet been invented. Furthermore, DNA purification, restriction digests, and ligation reactions arent reliably efficient, making monitoring and troubleshooting necessary. Finally, the design of cloning experiments is idiosyncratic, so the development of software algorithms that emulate decision making by experienced molecular biologists is non-trivial. The per-unit labor cost of subcloning (~10 h of labor per attempt, not including incubation times) far exceeds those of reagents (e.g., enzymes and purification kits).

METHOD SUMMARY

Tip Snip cloning uses restriction enzymes to shorten unwanted DNA fragments; the unwanted sticky ends are then neutralized by synthetic oligonucleotides. By eliminating the need to gel purify the desired restriction fragments, Tip Snip enables automation of the entire subcloning workflow.

The high cost of molecular cloning has motivated the invention of new methods (2-5). In general, one-pot sequence-specific recombination reactions, such as those catalyzed by recombinases (e.g., Gateway cloning) (6, 7), thermostable polymerases (overlap extension PCR) (8), thermostable ligases (ligase chain reaction) (9), or combinations of exonuclease, polymerase, and ligase (Gibson assembly or ligase-independent cloning (10,11) are the most amenable to high-throughput and automated techniques (12). These protocols are less labor-intensive than traditional cloning workflows with discrete steps but are more difficult to troubleshoot. Another drawback of many seamless assembly techniques is their reliance on PCR or gene synthesis to create fragments with compatible ends. Every part must be re-sequenced each time it is seamlessly combined with another element and re-cloned (Special News Report. Weaver, J. 2015. BioTechniques. 59:II-III.), because the DNA polymerase I homologs used in PCR are three to five orders of magnitude less accurate than those responsible for in vivo plasmid replication and repair. Next-generation sequencing techniques lower per-unit cost, but they cannot be applied to individual plasmids. Thus, many synthetic biologists continue to assemble parts by manual subcloning.

I therefore sought a way to automate the conventional restriction endonuclease/T4 DNA ligasedependent subcloning workflow. Golden Gate assembly, which utilizes type IIS restriction endonucleases (13), and 2ab assembly (14), which utilizes plasmids with two selectable markers separated by a unique restriction site, obviate gel purification but necessitate the employment of specialized vectors incompatible with those of other cloning standards. The three antibiotic assembly (3A) protocol (15) was specifically designed to assemble parts compatible with the seminal BioBrick Assembly Standard (RFC10) used by many synthetic biologists (16). Two donor plasmids carrying parts and a recipient plasmid encoding a counter-selection marker along with a selectable marker different than those of the donors are digested with different pairs of restriction enzymes. All six of the resulting restriction fragments are ligated together and used to transform Escherichia coli. The desired recombinant construct is distinguished from the parental plasmids using an antibiotic and the counter-selection scheme. This technique circumvents gel purification, but sacrifices efficiency for convenience. Three-fragment ligations dont occur as frequently as two-fragment reactions, particularly when three other unwanted fragments with compatible cohesive ends are present. The extraneous DNA also inhibits heat shock transformation of chemically competent E. coli (17), and electroporation is sensitive to salts in ligation reactions so it is less amenable to high-throughput experiments. Here, I describe a set of expedients that in combination facilitate efficient and reliable cloning of DNA into or out of almost any existing plasmid polylinker (multiple cloning site) without the need for PCR amplification or gel purification. Materials and methods

The approach described here builds upon the following classical cloning techniques (18), except as noted. A more detailed step-by-step protocol is included in the Supplementary Material. Plasmids were purified from transformed E. coli using silica spin columns (QIAGEN, Valencia, CA) and hydrated Sephadex G-50 (GE Healthcare Life Sciences, Pittsburgh, PA) in empty spin columns (Epoch Life Science, Missouri City, TX) as directed by their manufacturers. Restriction digests were set up as recommended by the supplier [New England BioLabs (NEB), Ipswich, MA]. Whenever possible 2 g of DNA (6 nM for a 5 kb plasmid) were digested to completion (or nearly so) at 37C overnight with 2040 U of each restriction enzyme (12 nM) in 100 L total reaction volume. Approximately 20 fmol of digested, purified recipient plasmid and 20 fmol of digested insert (and donor plasmid) were reacted with 0.31 Weiss units of T4 DNA ligase in NEB T4 DNA ligase buffer containing 5% polyethylene glycol (molecular weight: 8000) (19). The reactions were temperature cycled in a Bio-Rad (Hercules, CA) MJ mini thermocycler between 10C for 30 s and 30C for 30 s for a total of 418 h (20).

Chemically competent E. coli OmniMax2 cells (Thermo Fisher Scientific, Waltham, MA) were prepared according to Inoue et al. (17). For each transformation, up to 1.25 ng total DNA in ligation reactions were used to transform 25 L of competent cells in the thermocycler. The transformants were spread on lysogeny broth medium (LB) agar plates containing 100 g/mL ampicillin. Some agar plates also contained inducer and a histochemical substrate as described below. Some colonies were adsorbed to a nitrocellulose filter and transferred colony-side up to fresh LB-ampicillin plates supplemented with inducer (1 mM IPTG, 10 g/mL tetracycline, 0.4% L-arabinose or 0.4% rhamnose) and 2 mg/25 mL plate X-gal (for colonies carrying lacZ expression vectors). Additional information the reagents and materials used to culture the bacteria can be found in the Supplementary Material. Results and discussion

The Tip Snip cloning workflow begins with plasmids prepared via alkaline lysis and silica spin-column chromatography. A QIAcube robotic workstation (QIAGEN) can be used to automate this and other purification protocols. To eliminate small molecules that might inhibit restriction endonucleases or broaden their sequence specificity (21), plasmids are further purified via manual gel-filtration spin-column chromatography. Recipient and donor plasmids are digested as usual with restriction enzymes that produce fragments with compatible cohesive ends. Additional restriction enzymes recognizing sites in the polylinker are used to shorten undesired restriction fragments (the stuffer fragment of the recipient plasmid or the donor plasmid of a subcloning experiment) (Figure 1).

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Chemical Cloning To Create Copycat Wines – Forbes

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Chemical Cloning To Create Copycat Wines
Forbes
In her March 7, 2017 Press Democrat story, Peg Melnik talked about a kind of scientific brand named Replica Wine. Shutterstock. Melnik started her story with this quote attributed to Oscar Wilde: "imitation is the sincerest form of flattery." Perhaps ...

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How Many Times Can Facebook Keep Cloning Snapchat? – PSFK (subscription)

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Over the past year, Facebook has been taking on Snapchat by importing its defining features wholesale into the companys own apps

This article titled How many Snapchat clones does it take for Facebook to lose its self-respect? was written by Alex Hern, for theguardian.com on Friday 10th March 2017 12.27 UTC

It must be getting hard to walk into work at Facebook with your head held high. You roll into the campus in your Tesla, waltz over to your desk, and sit down, head full of ideas as to how to make the social network better for users and advertisers alike. Then a notification pings up on Messenger. Its Mark Zuckerberg himself! The boss is speaking to you! What could he have to say?

Clone Snapchat a bit more. Thanks, Mark.

Over the past year, Facebook has shown an almost monomaniacal dedication to taking on Snapchat by importing its defining features wholesale into the companys own apps. Facebook Live has masks now (think Snapchats Lenses). Instagram has geostickers (like Snapchats location-aware stickers.) WhatsApp has Status (think Snapchat Stories). Instagram has Stories (think Snapchat stories).

The latest fruit of Facebooks labours is Messenger Day a way for you to share these photos and videos as they happen by adding to your Messenger Day, where many of your friends can view and reply to them. Its Snapchat Stories. Again.

On top of the Stories clone comes a fully fledged Snapchat clone: Booting into the camera in Messenger now brings up an interface which is Snapchatesque in the same way that The Bootleg Beatles are heavily influenced by the works of Lennon and McCartney. You can take a picture, scribble over it, then send it as a message to your contacts or add it to your Day.

Announcing the feature, Facebooks Tony Leach gave a cringeworthy interview to TechCrunch in which he discusses how Messenger evolved into a Chat Camera. Ive not heard a more obvious attempt to rewrite corporate history since Microsoft refused to acknowledge the iPod in its attempt to branch out into podcasting (The term podcast is a combination of pod (Portable On Demand) and broadcast, it helpfully told Windows Vista users in 2009). The interview spends more time acknowledging AOL instant messenger, a 20-year-old chat program, than it does discussing Snapchat.

This is backsliding even for Facebook. When Instagram cloned Stories, in mid 2016, its chief executive Kevin Systrom did at least acknowledge that the format had been invented by his competitor, but argued that it was bound to become more universal. He told the Verge: Just like when Facebook invented the [News] Feed, and every social product was like, Thats an innovation, how do we adapt that to our network, youre going to see stories pop up in other networks over time, because its one of the best ways to show visual information in chronological order.

Snap Inc CEO, Evan Spiegel (centre), is one of the few who has declined a significant acquisition offer from Facebook. Photograph: Drew Angerer/Getty Images

The rot starts at the top. Even if Mark Zuckerberg isnt directly commanding the creation of Snapchat clones, its clearly something he cares strongly about. In 2012, when Facebook built its first ever clone, Poke, it boasted that the app took only 12 days to create. Practice makes perfect, clearly, because the app flopped. By 2014, Zuckerberg was spinning that Poke was more of a joke than a real app, telling Businessweek that a few people built it in a hackathon thing and then just kind of abandoned it.

The jokes continued, though. Later in 2014, Facebook released a second clone, Slingshot, which also died on its feet. In between, of course, the company tried a more direct approach, making a $3bn acquisition attempt which Snapchat rebuffed.

By October 2016, when I started tracking it, Facebook had made some sort of attempt to usurp Snapchat 10 times. By February, it was 17; the launch of Messenger Day takes it to 18.

And sure, some of those may be a stretch disappearing messages have a strong role in the operations security of an encrypted messaging app, for instance, which is probably why the feature made it into Messengers end-to-end encrypted mode but the overall trend is unmistakeable. Facebook feels no shame at all at the steady downgrade of its product team from groundbreaking designers to photocopier operators.

Theres two ways to look at that change. One is that this is the outcome of Facebooks no-nonsense approach to business, and the very reason why the company is on top of the world. In that reading, Mark Zuckerberg has become the Simpsons parody of Bill Gates, laughing as his goons smash up Homer Simpsons computer equipment to put another competitor out of business. This Facebook plays dirty to stay on top, and doesnt care whether or not you like it.

The other possibility is that Facebook is scared. Its seen potential threats on the horizon before, but its chequebook has always been enough to ward off real danger: thats why it bought Instagram, thats why it bought WhatsApp, and thats why it tried to buy Snapchat.

But it couldnt get the companys fiercely independent co-founder, Evan Spiegel, to sell. And now its in uncharted waters, with a competitor stealing advertising revenue, desirable millennial users, and industry credibility, and with no obvious way to reverse that trend.

Even if Facebook is lashing out in fear, that doesnt mean it wont be successful. A wild animal is at its most dangerous when cornered, after all. But its hard to sit in your open-plan office in Menlo Park feeling like a master of the universe while secretly shitting yourself about whats going on 400 miles south in Venice Beach.

Facebooks time at the top probably isnt up. But its self-respect deficit is going to take years to pay off.

guardian.co.uk Guardian News & Media Limited 2010

Published via the Guardian News Feed plugin for WordPress.

LEAD IMAGE:Instagram Stories, one of Facebooks 18 attempts to beat Snapchat at its own game. Photograph: Instagram

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Evolution – Salon.com

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They don't believe in science, and pander to evangelicals -- as a result, the Republicans remain a party of stupid

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The skeptics fall short for the same reason the creationists can't overturn evolution. Their doubts are unfounded

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A University of California cognitive scientist believes we learned to see things as they are as a means of survival

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It's easy to mock and criticize the Creation Museum. We also need to understand the worldview of those who visit

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Mayhem and chaos at a Kansas theater when a genius takes his gun and permit to the movies, then shoots himself VIDEO

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Exclusive: We talk with sparring partner Richard Dawkins about New Atheism, Twitter & why he sees Ahmed as a fraud

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No one yet knows where the newly discovered Homo naledi fits in the complicated story of hominids on Earth

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Hide your children, hide your scalps

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Research suggests the disease is linked to changes in brain size on the evolutionary road from ape to man

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The justice claims to be an originalist, but his real loyalty is to religion and a phony man in the sky

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Louisianans can now get a 19th Century education to accompany their 19th century economy

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The young scientist was stuck on the concept of species. Then something amazing happened to energize his thoughts

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The Manx, the Munchkin -- without fancy cats, we'd have no memes. Tip your hat to the genius of evolution

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Pigs provided meat, and also solved public health problems in ancient days. Here's how evolution worked

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They hope this attempt will fare better than the last four failed ones.

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Who would've thought a few extra inches could make it easier to survive a frigid winter? VIDEO

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Evolution and the "sexy geek" syndrome: What if humans could re-evolve and be perfect?

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A fascinating new study offers fresh clues about the origins of mankind -- and his home planet

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Who knew evolution could be so orgasmic?

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Tyson, Richard Dawkins and Bill Nye discuss evolution on "StarTalk Live!" VIDEO

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What Your Nose Knows About Human Evolution – Live Science

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They can be bulbous, pert or pointy, but why do noses look so different from one another? It could have something to do with how humans evolved to live in certain climates, a new study suggests.

In the study, the researchers found that wider noses are more commonly found among people living in warm and humid climates, and narrower noses are more commonly found among people in cold and dry climates.

One possible explanation for why nose shapes vary around the world is genetic drift, which is a mechanism of evolution through which the frequency of certain genes "drifts" upward or downward at random, leading to measurable differences between populations that don't often mingle. Genetic drift has a played a predominant role in human evolution, said the study, published today (March 16) in the journal PLOS Genetics.

But for the evolution of some human traits, it's likely that another mechanism, natural selection, also played a role, the researchers wrote. In other words, the evolution of some traits occurred not solely due to a random drift of genes, but also in response to outside factors. For example, human skin color is thought to have evolved in different human populations in response to the amounts of ultraviolet radiation they were exposed to, the study authors wrote. [7 Biggest Mysteries of the Human Body]

To see what mechanism likely influenced nose shape, the researchers used 3D facial imaging to measure the noses of more than 2,600 participants from West Africa, South Asia, East Asia and Northern Europe. The researchers scrutinized the noses, measuring the nostril width, distance between nostrils, nose height, nose-ridge length, nose protrusion and nostril area. In addition, the researchers estimated each participant's ancestry using genetic testing.

The researchers found that two nose measurements nostril width and the width of the nose at its base appeared to be linked to climate. People with wider nostrils were more likely to live in hot, humid climates, and people with narrower nostrils were more likely to live in cold and dry climates, the study said.

The nose's purpose goes beyond smelling and breathing. It also helps warm and moisten the air before it reaches the lungs. The right temperature and humidity levels are important throughout the respiratory tract, because they help the tiny, hair-like cells that line the tract to keep out germs and allergens.

In fact, the nose is so good at regulating air temperature and humidity levels that the air is already 90 of the way to its ideal temperature and moisture level by the time the air reaches the back of the throat, the researchers wrote. [Gasp! 11 Surprising Facts About the Respiratory System]

Air that is already hot and humid doesn't need to change much as it flows through the nostrils. Cool and dry air, on the other hand, needs to be warmed, and moisture must be added. Narrower nostrils could help facilitate this, as they make the air flow in more turbulently and come into greater contact with the warm, moist mucus in the nose, the researchers wrote. Indeed, it was probably more helpful for humans in cold and dry climates to have a narrower nose, senior study Mark Shriver, a professor of anthropology at Pennsylvania State University, said in statement.

The new study's findings appear to support "Thomson's Rule," an idea put forth by the British anatomist Arthur Thomson in the late 1800s, Shriver said. Thomson "said that long and thin noses occurred in dry, cold areas, while short and wide noses occurred in hot, humid areas," Shriver said. People have tested this rule by measuring skulls; however, no one had done the measurements on living people, Shriver added.

He noted that natural selection isn't the only possible explanation for nose differences. Another explanation could be sexual dimorphism, in other words, differences between males and females, the study said. The researchers did note that there were differences between men's noses and women's in their findings, for example, men's noses were larger, on average, than women's noses.

The findings could also have medical implications, particularly as people travel more around the world, the study said. For example, the researchers asked if someone with a narrow nose could have an increased risk for respiratory problems if he or she lived in a hot and humid climate.

In future studies, the researchers hope to also look at people who live at high altitudes, such as people in the Andes, Tibet and Ethiopia, to learn if low atmospheric-oxygen levels also play a role in nose shape, the researchers said.

Originally published on Live Science.

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Evolution Runs Faster on Short Timescales – Quanta Magazine

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In the 1950s, the Finnish biologist Bjrn Kurtn noticed something unusual in the fossilized horses he was studying. When he compared the shapes of the bones of species separated by only a few generations, he could detect lots of small but significant changes. Horse species separated by millions of years, however, showed far fewer differences in their morphology. Subsequent studies over the next half century found similar effects organisms appeared to evolve more quickly when biologists tracked them over shorter timescales.

Then, in the mid-2000s, Simon Ho, an evolutionary biologist at the University of Sydney, encountered a similar phenomenon in the genomes he was analyzing. When he calculated how quickly DNA mutations accumulated in birds and primates over just a few thousand years, Ho found the genomes chock-full of small mutations. This indicated a briskly ticking evolutionary clock. But when he zoomed out and compared DNA sequences separated by millions of years, he found something very different. The evolutionary clock had slowed to a crawl.

Baffled by his results, Ho set to work trying to figure out what was going on. He stumbled upon Kurtns 1959 work and realized that the differences in rates of physical change Kurtn saw also appeared in genetic sequences.

His instincts as an evolutionary biologist told him that the mutation rates he was seeing in the short term were the correct ones. The genomes varied at only a few locations, and each change was as obvious as a splash of paint on a white wall.

But if more splashes of paint appear on a wall, they will gradually conceal some of the original color beneath new layers. Similarly, evolution and natural selection write over the initial mutations that appear over short timescales. Over millions of years, an A in the DNA may become a T, but in the intervening time it may be a C or a G for a while. Ho believes that this mutational saturation is a major cause of what he calls the time-dependent rate phenomenon.

Courtesy of University of Sydney

Simon Ho, an evolutionary biologist at the University of Sydney, found that evolution takes place at varying rates.

Think of it like the stock market, he said. Look at the hourly or daily fluctuations of Standard & Poors 500 index, and it will appear wildly unstable, swinging this way and that. Zoom out, however, and the market appears much more stable as the daily shifts start to average out. In the same way, the forces of natural selection weed out the less advantageous and more deleterious mutations over time.

Hos discovery of the time-dependent rate phenomenon in the genome had major implications for biologists. It meant that many of the dates they used as bookmarks when reading lifes saga everything from the first split between eukaryotes and prokaryotes billions of years ago to the re-emergence of the Ebola virus in 2014 could be wrong. When this work came out, everyone went Oh. Oh, dear, said Rob Lanfear, an evolutionary biologist at the Australian National University in Canberra.

The time-dependent rate phenomenon wasnt fully appreciated at first. For one thing, it is such a large and consequential concept that biologists needed time to wrap their heads around it. But theres a bigger stumbling block: The concept has been all but impossible to use. Biologists have not been able to quantify exactly how much they should change their estimates of when things happened over the course of evolutionary history. Without a concrete way to calculate the shifts in evolutionary rates over time, scientists couldnt compare dates.

Recently, Aris Katzourakis, a paleovirologist at the University of Oxford, has taken the time-dependent rate phenomenon and applied it to the evolution of viruses. In doing so, he has not only pushed back the origin of certain classes of retroviruses to around half a billion years ago long before the first animals moved from the seas to terra firma he has also developed a mathematical model that can be used to account for the time-dependent rate phenomenon, providing biologists with much more accurate dates for evolutionary events.

Other scientists are excited by the prospect. Its like Einsteins theory of relativity, but for viruses, said Sebastin Duchne, a computational evolutionary biologist at the University of Melbourne. The time-dependent rate phenomenon says that the speed of an organisms evolution will depend on the time frame over which the observer is looking at it. And as with relativity, researchers can now calculate by how much.

Viral Fossil Hunting

Katzourakis has spent his career trying to pin down the origin of HIV and other so-called retroviruses, which are made out of single strings of RNA.

When he looked at the mutation rates of HIV, he found that it is among the fastest-evolving viruses ever studied. The speedy mutation rate makes sense: Double-stranded molecules like DNA have molecular proofreaders that can often correct errors made during replication, but HIV and other single-strand RNA viruses dont. Spelling errors occur on top of spelling errors.

Lucy Reading-Ikkanda/Quanta Magazine

Because of this, virologists can directly study only the recent history of viruses like this. Older samples have reached mutation saturation, with so many accumulated spelling errors that scientists cant account for them all. Taking the history of retroviruses back thousands or millions of years would require a different way to measure mutation rates.

Katzourakis turned to another technique. He searched for something akin to viral fossils inside the DNA of their hosts. Retroviruses often insert copies of their genetic material into their hosts cells. Most of the time, the information dies with the host. On rare occasions, however, a retrovirus hits the evolutionary jackpot and slips inside the genome of a sperm or egg cell. Nestled securely in its hosts DNA, the virus gets passed down through the generations.

Katzourakis used these viral relics to study the ancient origin of retroviruses. But when he did so, he got a big surprise. The rate of evolution of these retroviruses over long periods appeared to slow dramatically, nearly matching that of humans and other complex life organisms that have proofreader machinery and thus should change at a much slower pace.

If the viruses were evolving much more slowly than scientists thought, it could imply that the viruses were much older than expected as well. After all, a slowly evolving virus will need more time to change by the same amount as a quickly evolving virus.

So he set out to find an accurate date for the origin of retroviruses. To do this, he turned to a group of the most ancient retroviruses, the so-called foamy viruses, which infect everything from monkeys to cows. This promiscuity enabled Katzourakis to calibrate his evolutionary clock to determine precisely when foamy viruses emerged. If two species shared a foamy-virus sequence, the virus must have infected their common ancestor, before the two species diverged.

It gives us a way to date events in deep evolutionary history thats independent of the sequences themselves, Katzourakis said.

Gillman & Soame

Aris Katzourakis, a paleovirologist at the University of Oxford, dated a class of viruses to the era before the sea-to-land transition.

Researchers in labs around the world had slowly pushed back the date of origin of foamy viruses to 100 million years ago. But Katzourakis found hints that the virus had infected reptiles, amphibians and even fish far earlier than 100 million years ago. To conclusively show that retroviruses were older than the accepted date of 100 million years, however, Katzourakis would need to date the virus itself.

He dived into Hos papers on the time-dependent rate phenomenon, hoping to figure out how to apply it to viruses. He also wanted to create a general model that would allow researchers to input the timescale they were observing and get back details about the organisms evolutionary rate.

Katzourakis and his student Pakorn Aiewsakun tried out four different ways to quantify how quickly the evolutionary rate appeared to change based on timescale. They found that a power law rate-decay model fit their data best and showed that evolutionary rates decrease exponentially as the timescale increases. A subsequent study of 396 different viruses revealed that the evolutionary rate slows at the same rate across almost all genome types and replication strategies. Existing evolutionary clocks, which fail to account for the time-dependent rate phenomenon, inaccurately date ancient viruses as being much younger than they really are.

Katzourakis and Aiewsakun then used the newly developed mathematical framework to recalculate the emergence of foamy viruses. Using their newly developed model, the scientists showed in a paper published in January that foamy viruses emerged somewhere between 460 and 550 million years ago. Independent work by the University of Arizona virologist Michael Worobey, published in Virus Evolution nearly simultaneously, also suggested that these viruses originated earlier than expected. These studies established the oldest date for any known group of viruses, although Katzourakis believes other viral groups may be even more ancient.

The findings have implications far beyond the earning of a trophy for the oldest virus. A convergence on the same date of origin for foamy viruses provides evidence that the time-dependent rate phenomenon isnt just a relic of statistics or the methods researchers use to date species. Katzourakiss model also gives researchers a tool to quantify the effects of the time-dependent rate phenomenon, which will prove key to understanding the factors that drive this phenomenon.

More broadly, the work by Katzourakis and Ho challenges the idea of a steadily ticking evolutionary clock. This changes the way we conceive of molecular evolution, Duchne said. It shows that there is no universal rate of evolution. Even the same organisms have rates that vary over time.

It also means that scientists may need to revise the dates of evolutionary events in the deep past, as they likely underestimated how long ago they truly happened, Katzourakis said. He is trying to understand whether the pruning of mutations by natural selection and mutational saturation is the sole contributor to the time-dependent rate phenomenon, or whether other factors play a role in how and why the phenomenon emerges.

Is it a limitation of our tools, or is there something that weve overlooked? If we can understand this process, it will give us some big evolutionary insights, Katzourakis said.

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‘Teach the controversy’: States introduce bills to teach evolution as debatable theory – RT

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Texas is the eighth state this year to introduce legislation that would protect teachers who present science, including evolution, as a debatable theory. Critics contend these bills are a way to open the door to religion in the classroom.

The Lone Star State joins Alabama, Arkansas, Florida, Indiana, Iowa, Oklahoma and South Dakota as states that have proposed so-called academic freedom or teach the controversy bills, which allow public school teachers to include opposing points of view or beliefs when teaching scientific topics like global warming, evolution and the origins of life.

Louisiana (2008) and Tennessee (2012) have both passed laws that allow teachers to introduce religious materials in science classes as supplementary texts. When Louisianas school board voted to update its science standards on March 7, the revisions passed only after the inclusion of language reminding teachers about the 2008 law, AP reported. The revised standards are set to go into effect for the 2018-2019 academic year.

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The majority of Americans believe that humanity was created or guided by God in its evolution, a 2014 Gallup poll found. Only 19 percent believe humans evolved from a more primitive form of life without divine intervention (Darwinism), while 42 percent believe God created humanity in its current form in the last 10,000 years (creationism), and 31 percent think that humans evolved from lesser advanced life forms, but God guided this process (intelligent design).

Kimberly Villanueva, a middle school teacher in southwest Texas, supports the bill because it would give her the flexibility to answer her students questions and "open [their] minds to scientific possibilities as well."

"I had children last year get up and leave the classroom when we taught plate tectonics and evolution," she told AFP, adding that current law prevents her from answering when her students ask her, Dont you believe in God?

Not all teachers agree that the bill is necessary, however. Pam Wells, a high school science teacher in South Dakota, looked into teaching intelligent design at the request of some parents, but decided against it because the theories werent based in science, she told AP.

One high school senior told her he wouldnt come to class if she opted to teach intelligent design over evolutionary theory. "He said, 'If I wanted to learn about that I'd go to church," Wells recalled.

The sponsor of the South Dakota bill, Republican Senator Jeff Monroe, said it would allow teachers to explain potential flaws in theories and introduce alternative scientific theories without fear of retribution because current state standards for teaching evolution and climate change are one-directional directives that dont allow for analysis, the Sioux Falls Argus Leader reported. Such protections, Monroe added, would benefit students critical thinking skills.

Monroes bill died in committee at the end of February, while Iowas experienced a similar demise in early March.

Opponents say the bills are antiscience, and introduce religion, alternative facts and misleading rhetoric into the classroom.

Although these proposed changes are typically presented by their supporters as giving teachers the chance to discuss genuine scientific controversies, in truth they are (very) thinly veiled attempts to pursue political and religious agendas that have no place in school science lessons for whatever age, the editorial board of Nature wrote.

Regardless of the outcome of these bills, schools will still be open to lawsuits, according to Glenn Branch, deputy director of the National Center for Science Education.

"Allowing the teacher to teach Creationism would risk the possibility of a lawsuit from a parent objecting that it's unconstitutional to teach Creationism, he told AFP. "Stopping the teacher from proceeding would risk the possibility of a lawsuit from the teacher."

Louisiana, where teach the controversy is already in effect, ranks poorly in science testing results compared to other states, AP reported.

The Supreme Court banned the teaching of creationism in science classes in a 1987 decision, ruling that teaching that a supreme being created humankind endorses religion.

In 1997, a US District Court barred the teaching of intelligent design. Another District Court judge ruled that disclaimers stating that evolution is a theory, not a fact violated the First Amendments Establishment Clause in 2005.

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'Teach the controversy': States introduce bills to teach evolution as debatable theory - RT

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Chimps Hold Funeral? – Discovery Institute

Posted: at 7:23 am

A chimp died of a lung infection. Other chimps hovered around the body. One, a female, cleaned the deceaseds teeth with a bit of grass, then sampled the grass herself.

Anthropomorphizing interpretation, please? And make sure it brutally conflates animal behavior with the most refined, sensitive aspects of human culture, if you dont mind.

Certainly. Well, you see they were holding a funeral, complete with mortuary rites. From the article in The Telegraph, Chimp mother filmed cleaning dead body of son in first hint of primate funeral rites:

Female chimp Noel, who lives at the Chimfunshi Wildlife Orphange Trust in Zambia, was seen using a stem of grass to remove debris from the teeth of a young male named Thomas, who she had looked after since the death of his own mother four years earlier.

She was one of a number of chimpswho surrounded the body for around 20 minutes, gently touching and sniffing Thomas despite offers of food to tempt them away.

Not only funeral rituals they also practice adoption:

But it was Noel who appeared to be the most upset, staying on her own to clean the teeth of her adopted son, even when the others had left.

In the Abstract of the original article in Scientific Reports, they note that the observation of this behavior

highlights how crucial information for reconstructing the evolutionary origins of human mortuary practices may be missed by refraining from developing adequate observation techniques to capture non-human animals death responses.

More, from the Discussion section:

Anecdotal evidence needs to be treated cautiously15.

Do you think so? The evidence provided by a single anecdote, they mean.

However, given the availability of high-quality video footage, we present a valid case of unique non-human animal behaviour which could shed light on the evolution of behaviours that are believed to be typically human.

As expected, this is about casting doubt on human uniqueness.

Death responses represent core features of human civilization, with great diversity in mortuary rites found across cultures16. In general, for animals critically depending on group living17, like humans and chimpanzees, responding to death may be a means to reorganize the social unit, especially when so-called brokers die: individuals who play an important role in maintaining group cohesion by connecting sub-groups18,19.

But all the human mortuary practices and mortuary rites we are familiar with stem from beliefs about human life, its dignity, and what comes after it. Its hard to see how in the absence of language, chimps could develop or pass on beliefs of their own about life or death. If what these chimps did was simply to reorganize the social unit following the death of a broker, then calling it a rite would seem to be a case of anthropomorphism.

The lead author of the report is Edwin van Leeuwen at St. Andrews University in Scotland. Speaking with The Telegraph, a colleague interjects a note of sobriety.

Klaus Zuberbuehler, also of St Andrews, said scientists should be cautious in interpreting the behaviour.

Perhaps the chimpanzees are just challenged by the fact that a group member has suddenly become completely motionless, he said.

Thibaud Gruber, of the University of Geneva also told New Scientist that the chimps may have a limited understanding of death.

More than a possibility, the idea that chimps have a limited understanding of death would seem to be a certainty. Given that, the expectation that they would be challenged by a colleagues suddenly becom[ing] completely motionless appears reasonable.

Wikipedia defines anthropomorphism as the attribution of human traits, emotions, and intentions to non-human entities, stating that it is considered to be an innate tendency of human psychology. There is no parallel term for the attribution, by chimps, of chimp traits, etc. to non-chimps. That is because the innate tendency to anthropomorphize is unique to human psychology.

As Ive observed before, attempts to deny the exceptional qualities of human beings, to put us on a plane of equality with animals like chimps, dogs, or cats, share this tendency to self-destruct.

We entertain beliefs that attribute human intentions to chimps. Chimps do not return the favor. That is because contrary to notions widely held in the media and academia, humans are unique in nature. The error of imagining that a chimp holds a funeral for her adopted son is more evidence of that, if any were needed.

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A Ticking Evolutionary Clock – Quanta Magazine

Posted: at 7:23 am

How quickly will a genes rate of evolution slow to a crawl?

Olena Shmahalo/Quanta Magazine

After a string of Insights puzzles related to physics, on the relationship between time and entropy, half-lives, overhang and quantum weirdness, we turn this month to the mysteries of biological evolution. Carrie Arnolds article, Evolution Runs Faster on Short Timescales, explores new research showing that genetic changes that are quite brisk when measured over a few generations seem to slow down considerably when measured over millions of years. One of the researchers who have studied this in genomes is Simon Ho, an evolutionary biologist at the University of Sydney. To quote Arnold:

When [Ho] calculated how quickly DNA mutations accumulated in birds and primates over just a few thousand years, Ho found the genomes chock-full of small mutations. This indicated a briskly ticking evolutionary clock. But when he zoomed out and compared DNA sequences separated by millions of years, he found something very different. The evolutionary clock had slowed to a crawl.

The article goes on to describe how, from a mathematical perspective, evolutionary rates decrease exponentially as the timescale increases.

Can we replicate this in a toy version of a single gene? Lets find out. But first, lets go over the basics for those who need to brush up on them: Heres DNA 101 for puzzle enthusiasts.

A monthly puzzle celebrating the sudden insights and unexpected twists of scientific problem solving. Your guide is Pradeep Mutalik, a medical research scientist at the Yale Center for Medical Informatics and a lifelong puzzle enthusiast.

A gene is a piece of DNA, which is essentially a linear chain of chemical bases that are abbreviated using the letters A, C, G and T. Each of these four letters (bases) appears in random sequence along a given gene, in about equal amounts. Thus the sequence CATGGTACCGAT represents a piece of DNA that is 12 units long. The way DNA works is that each successive three-letter piece of DNA, called a triplet, codes for one of 20 possible units, called amino acids, that make up a protein. Proteins are the bodys workhorses, each one performing different functions thanks to its unique structure and its unique linear sequence of amino acids. Thus in our DNA sequence above, there are four triplets, CAT, GGT, ACC and GAT, each of which codes for a specific amino acid. This piece of DNA, acting through the cell machinery, will form a piece of a specific protein fragment that is four units long.

Now, DNA is generally copied with high fidelity from cell to cell across generations. But, on rare occasions, you can get a point mutation, in which one of the letters of the gene sequence is replaced by another random one, causing the gene to produce a different protein, which may be more or less efficient at doing what it was supposed to. This is basically how evolutionary change happens. We can define the speed at which DNA mutates over time as the evolutionary rate: We can measure it over a given period by counting the number of letters that have changed between the original DNA sequence and the current sequence, divided by the number of years that have passed.

OK, lesson over. Thats all the biology we need for our puzzle.

Question 1:

Imagine a gene that is 108 letters with A, T, G, C in random sequence. Assume that every year, there is a random change one of the letters somewhere on this gene mutates and is replaced by one of the other three. After each year, you compare the current copy of the gene with the original and tally how many letters have changed. After a certain time the evolutionary clock will have slowed to a crawl that is, the number of changed letters will have stopped rising. The evolutionary rate from here on is zero. How many letters of the original gene will have changed at that point? How many years will it take to get to this point? Is the curve exponential?

Question 2:

The above scenario is not very realistic. Every letter in a real-life gene sequence has a different chance of having a mutation that sticks. The letters at some locations in the DNA sequence are preserved, because changes in them are catastrophic; others, at inconsequential locations, can change readily. One general rule is that the third letter of every triplet can change easily. This is because the third position in a triplet is often redundant: The first two positions fix the amino acid the triplet codes for.

Assume that the third letter of each triplet is three times as likely to get mutated as are the first and second. Now try to answer the same questions as in Question 1.

Bonus Question:

I gave this hypothetical piece of DNA 108 letters. What was my reason for choosing that number? Is it because 108 has mystical significance, as a Google search will indicate?

I hope these simple mathematical models give you a feel for how evolutionary rates work. This is, of course, nothing like the complexity of what actually happens in a single real-world gene. First, most genes have many more than 108 letters. Second, even scenario 2 above was an oversimplification: The letters at every location have a different likelihood of being preserved, depending on how important the amino acid they code for is to the function of the final protein. Welcome to biology! Yes, mathematical models can work and can give us some insight, but we must always remember that they are gross oversimplifications. In biology, analytical mathematics can take us only so far, and any attempt to capture the nuance of the real world requires highly sophisticated computer models.

How different this is from the physics we discussed in the last Insights puzzle. In it, some commenters, legitimately voicing one of the influential schools of thought, insisted that mathematics is all there is at the quantum level: Reality either does not exist or cannot be known apart from the models! Whether that is possible is a point worth pondering by all those who make and use mathematical models. For me, it is reassuring to return to the messiness of biology once in a while. Happy puzzling!

Editors note: The reader who submits the most interesting, creative or insightful solution (as judged by the columnist) in the comments section will receive a Quanta Magazine T-shirt. And if youd like to suggest a favorite puzzle for a future Insights column, submit it as a comment below, clearly marked NEW PUZZLE SUGGESTION (it will not appear online, so solutions to the puzzle above should be submitted separately).

Note that we may hold comments for the first day or two to allow for independent contributions by readers.

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Player scouting at the forefront of Major League Soccer rapid … – ESPN FC (blog)

Posted: at 7:23 am

ESPN FC's Alejandro Moreno breaks down the best in MLS with his weekly awards.

Everywhere you look in MLS, the league is evolving at warp speed.

Academies are cranking out players who eventually play for the first team at an increasing rate. Organizations are investing more in sports science and are doing more to look after the welfare of their players off the field. Clubs are also becoming savvier about how they manage the salary budget.

And one area that is undergoing considerable change is player scouting.

Sporting Kansas City manager and technical director Peter Vermes called scouting "one of the last bastions in a club to start to get resources." But now MLS clubs are investing more money in processes, technology and people in a bid to find those players who can turn a team into a title contender.

That teams are investing more in scouting certainly counts as an oddity. To hear Seattle Sounders GM and president of soccer Garth Lagerwey tell it, hitting up ownership for a scouting budget doesn't take much convincing, or at least not as much as it used to.

"I don't care what your philosophy is; you need good players," Lagerwey told ESPN FC in an exclusive interview. "That's not something that is a terribly hard sell to an owner, to say, 'Hey, we want to win, so we need good players, so we should probably spend some money trying to find good players.' Now obviously different owners are willing to commit different levels of money to that, but it's not a hard one to explain."

There have been some catalysts behind the greater emphasis on scouting. Recent additions to the league like New York City FC and Atlanta United, with their vast resources, have increased the competition among teams. Another factor has been the introduction of targeted allocation money. It has meant a higher caliber of player entering the league, and with greater investment comes a need to maximize the odds in terms of making good signings.

"We're in a slightly different market, a slightly different world than just as recently as the last 12-24 months," New England GM Mike Burns told ESPN FC. "I think because of that we're going to have to make a larger investment in scouting for us as well as across the league because you're in a different kind of tier of players you're trying to attract. We're trying to get higher-end guys."

Historically scouting has been very labor intensive. It often required trips abroad and was terribly reactive. The task of scouting also tended to fall on a team's coaching staff, and in some cases still does. Given the responsibilities of an assistant coach during the season, it doesn't take long for the workload to be spread too thin.

"You think you can get it done with your existing staff," Vermes said. "But the bottom line is as this league continues to grow, you have to have areas in your organization that have focus."

Technology has done plenty to change the scouting dynamic. Lagerwey recalled how when he started with Real Salt Lake back in 2007, he would sometimes wait weeks to receive a DVD of a player via regular mail. Usually there would be just one copy, so he and the coaching staff would sit around and watch the DVD together, knowing what they were seeing had to be taken with a degree of skepticism.

"If you're watching one game of a guy, that's inevitably the best game he played in the last six months," Lagerwey said. "That's why the agent is sending you stuff on him."

Now, thanks to services like Match Analysis, Wyscout, Scout7, Opta and InStat, it's possible to watch just about any player, anywhere in the world almost instantaneously. The amount of data is greater too, allowing an analyst to filter video or statistics down to the individual touches, both good and bad. That has led to a significant increase in efficiency in terms of identifying targets and weeding out players who aren't a good fit.

"It used to be go out and scout, and bring 30 names back," Portland Timbers GM and president of soccer Gavin Wilkinson said. "Sending someone out and having them be gone for three weeks no longer makes sense from a business, financial or scouting standpoint. When you're able to have people in the office for eight, nine hours a day, and they're watching three games, or they're able to complete homework on many more players, that's a lot more efficient, a better use of time. I think a lot of it is based on analytics, based on scouting. We still do live scouting, but for us that is one of the final boxes towards signing a player."

Technology alone will not automatically lead to better results, however. Lagerwey noted there is almost too much information available now, so sifting through the noise is important. It also puts a premium on putting processes in place for player identification and tracking, the better to make player scouting a more proactive endeavor.

There still remains a need to have boots on the ground. While technology is a huge help, the eye test still has value, though it is often reserved for the biggest signings.

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"You always want to try to see a guy play live before you sign him, just because it gives you a level of comfort," Lagerwey said. "A big part of that is meeting the person; talking to him, being able to get a measure of him and having a conversation with him. I think that kind of personal stuff is really valuable. Certainly going to a game and watching him live, you can see how he interacts with teammates, you can see what he does outside of the camera, so there's a little bit more information available."

Another way scouting has changed in MLS is that the spectrum of players to be tracked is much broader than it used to be. Scouting in MLS used to focus on foreign and college players. Not anymore.

"As homegrown players have become more and more important and everyone starts to recognize the value of getting these kids in the system, you're scouting the entire country now for youth kids," Lagerwey said. "We're restricted and we can't pull kids out of another MLS team's home territory, but that still leaves big patches of the country to scout and recruit kids."

The emphasis on scouting players takes on added importance for a club like Kansas City. Vermes said SKC's philosophy is to find value in players it recruits while developing its own, as opposed to spending $5 million on one player. The organization is devoting scouting resources to that end.

But finding capable scouts isn't as easy as it sounds. Inside every scout is usually someone who is trying to get into coaching. That can result in considerable turnover in the job, which can lead to a lack of continuity. Finding someone with the breadth of knowledge needed, from youth to domestic pros to international, is difficult as well. That was the challenge for Wilkinson when he was trying to fill the team's director of scouting and recruitment position.

"I think a lot of clubs are still solving this problem," Wilkinson said. "What we did is we went down the road of getting an MLS player that knows the league, knows the philosophy in Portland, knows [coach] Caleb [Porter], knows myself, and he knows the philosophy and expectations of the club."

That player turned out to be recently retired Timbers midfielder Ned Grabavoy, who now oversees all of Portland's scouting efforts.

In terms of staffing dedicated to scouting, the numbers vary wildly. A team like New England still relies heavily on its coaching staff to do scouting, though it has hired a video analyst as well as an additional assistant in Carlos Llamosa this season. Seattle has three dedicated front-office members, led by sporting director Chris Henderson, who handle the scouting duties, splitting up the foreign, domestic and youth buckets. Portland has a more extensive organization, with the number of people -- both internal and external -- dedicated to scouting exceeding double digits. On top of that are the informal networks of contacts that various coaches and staff have developed over the years.

"We have all these tentacles out there that are connected to all these different people that we all know from our years in the game," Vermes said. "The good thing with those connections is that we can double-, triple- or even quadruple-check references on a guy. What's he like off the field, in the locker room? I think those become the more important aspects because a lot of times we can turn to one of our contacts who can really get behind the scenes and find out what a guy is really like."

Kansas City has taken the approach of employing two foreign scouts, one each in Europe and South America. SKC's man in Europe, Rui Marques, is based in Portugal and covers five leagues in Europe. It was through his efforts that Kansas City signed winger Gerso Fernandes and Spanish midfielder Ilie Sanchez.

"That piece has lent itself well because when he's looking at players, they're not far off or they're really close to what we would be looking for ourselves," Vermes said.

By Marques' count, he's seen more than 100 games live since he first started working for the club in summer 2015. He's also watched countless others on video. Marques admits sometimes he has trouble sleeping if he's watched too many games in one day, but that overall he finds the work fulfilling. And throughout the years, he's been able to hone is craft in finding potential signings.

"I think the most difficult task for a scout, it's to evaluate if the player does the best decisions during a match," Marques saidvia email. "It is easy to see if a player has shooting or dribbling skills or if he plays with enough intensity and hardness. I think the best scouts should process in their minds accurate information on a quick way when they are observing a match and within that information must be not only the technical aspects but also the ones that fall into the decision-making category."

If Vermes had his way, he'd prefer to find even more staffers like Marques and spread them throughout the world. Other teams across the spectrum, from Portland to New England, will continue to expand the number of staff dedicated to scouting and player recruitment.

"The rate of foreigners that failed when they came to MLS six, seven years ago, versus where we're at now, I think it's gotten better," Wilkinson said. "There's a lot more information, there's a lot more scouting going on, there's a lot more checks and balances that are going into it. It's just the evolution of the game in this country."

And to compete in MLS, there's no going back.

Jeff Carlisle covers MLS and the U.S. national team for ESPN FC. Follow him on Twitter @JeffreyCarlisle.

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