Could High-Energy Radiation Have Played an Important Role in Getting Earth Ready For Life?

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A version of this article first appeared in Astrobiology Magazine, www.astrobio.net.

The fossil remains of a natural nuclear reactor in Oklo, Gabon.  It entered a fission state some 2 billion years ago, and so would not have been involved in any origin of life scenario.  But is a proof of concept that these natural reactors have existed and some were widespread on earth Earth.  It is but one possible source of high energy particles on early Earth. The yellow rock is uranium oxide. (Robert D. Loss, Curtin University, Australia)

Life on early Earth seems to have begun with a paradox: while life needs water as a solvent, the essential chemical backbones of early life-forming molecules fall apart in water. Our universal solvent, it turns out, can be extremely corrosive.

Some have pointed to this paradox as a sign that life, or the precursor of life, originated elsewhere and was delivered here via comets or meteorites. Others have looked for solvents that could have the necessary qualities of water without that bond-breaking corrosiveness.

In recent years the solvent often put forward as the eligible alternative to water is formamide, a clear and moderately irritating liquid consisting of hydrogen, carbon, nitrogen and oxygen. Unlike water, it does not break down the long-chain molecules needed to form the nucleic acids and proteins that make up life’s key initial instruction manual, RNA. Meanwhile it also converts via other useful reactions into key compounds needed to make nucleic acids in the first place.

Although formamide is common in star-forming regions of space, scientists have struggled to find pathways for it to be prevalent, or even locally concentrated, on early Earth. In fact, it is hardly present on Earth today except as a synthetic chemical for companies.

New research presented by Zachary Adam, an earth scientist at Harvard University, and Masashi Aono, a complex systems scientist at Earth-Life Science Institute (ELSI) at Tokyo Institute of Technology, has produced formamide by way of a surprising and reproducible pathway: bombardment with radioactive particles.

 

In a room fitted for cobalt-60 testing on the campus of the Tokyo Institute of Technology, a team of researchers gather around the (still covered) cobalt-60 and vials of the chemicals they were testing. The ELSI scientists are (from left) Masashi Aono,  James Cleaves, Zachary Adam and Riquin Yi.  (Isao Yoda)

The two and their colleagues exposed a mixture of two chemicals known to have existed on early Earth (hydrogen cyanide and aqueous acetonitrile) to the high-energy particles emitted from a cylinder of cobalt-60, an artificially produced radioactive isotope commonly used in cancer therapy. The result, they report, was the production of substantial amounts of formamide more quickly than earlier attempts by researchers using theoretical models and in laboratory settings.

It remains unclear whether early Earth had enough radioactive material in the right places to produce the chemical reactions that led to the formation of formamide. And even if the conditions were right, scientists cannot yet conclude that formamide played an important role in the origin of life.

Still, the new research furthers the evidence of the possible role of alternative solvents and presents a differing picture of the basis of life. Furthermore, it is suggestive of processes that might be at work on other exoplanets as well – where solvents other than water could, with energy supplied by radioactive sources, provide the necessary setting for simple compounds to be transformed into far more complex building blocks.

Formamide is a clear liquid which is miscible with water and has an ammonia-like odor.

“Imagine that water-based life was preceded by completely unique networks of interacting molecules that approximated, but were distinct from and followed different chemical rules, than life as we know it,” said Adam.

Their work was presented at recent gatherings of the International Society for the Study of the Origin of Life, and the Astrobiology Science Conference.

The team of Adam and Aono are hardly the first to put forward the formamide hypothesis as a solution to the water paradox, and they are also not the first to posit a role for high-energy, radioactive particles in the origin of life.

An Italian team led by Rafaelle Saladino of Tuscia University recently proposed formamide as a chemical that would supply necessary elements for life and would avoid the ‘water paradox.’ Since the time that Marie Curie described the phenomenon of radioactivity, scientists have proposed innumerable ways that the emission of particle-shedding atomic nuclei might have played roles, either large or small, in initiating life on Earth.

Merging the science of formamide and radioactivity, as Adam and Aono have done, is a potentially significant step forward, though one that needs deeper study.

“If we have formamide as a solvent, those precursor molecules can be kept stable, a kind of cradle to preserve very interesting products,” said Aono, who has moved to Tokyo-based Keio University while remaining a fellow at ELSI.

Aono and technician Isao Yoda in the radiation room with the cobalt-60 safely tucked away. (Nerissa Escanlar.)

The experiment with cobalt-60 did not begin as a search for a way to concentrate the production of formamide. Rather, Adam was looking more generally into the effects of gamma rays on a variety of molecules and solvents, while Aono was exploring radioactive sources for a role in the origin of life.

The two came together somewhat serendipitously at ELSI, an origins-of-life research center created by the Japanese government. ELSI was designed to be a place for scientists from around the world and from many different disciplines to tackle some of the notoriously difficult issues in origins of life research. At ELSI, Adam, who had been unable to secure sites to conduct laboratory tests in the United States, learned from Aono about a sparingly-used (and free) cobalt-60 lab; they promptly began collaborating.

It is well known that the early Earth was bombarded by high-energy cosmic particles and gamma rays. So is the fact that numerous elements (aluminum-26, iron-60, iodine-129) have existed as radioactive isotopes that can emit radiation for minutes to millennium, and that these isotopes were more common on early Earth than today. Indeed, the three listed above are now extinct on Earth, or nearly extinct, in their natural forms

Less known is the presence of “natural nuclear reactors” as sites where a high concentration of uranium in the presence of water has led to self-sustaining nuclear fission. Only one such spot has been found —in the Oklo region of the African nation of Gabon — where spent radioactive material was identified at 16 sites separate sites. Scientists ultimately concluded widespread natural nuclear reactions occurred in the region some 2 billion years ago.

That time frame would mean that the site would have been active well after life had begun on Earth, but it is a potential proof of concept of what could have existed elsewhere long before

Adam and Aono remain agnostic about where the formamide-producing radioactive particles came from. But they are convinced that it is entirely possible that such reactions took place and helped produce an environment where each of the backbone precursors of RNA could readily be found in close quarters.

Current scientific thinking about how formamide appeared on Earth focuses on limited arrival via asteroid impacts or through the concentration of the chemical in evaporated water-formamide mixtures in desert-like conditions. Adam acknowledges that the prevailing scientific consensus points to low amounts of formamide on early Earth.

“We are not trying to argue to the contrary,” he said, “but we are trying to say that it may not matter.”

If you have a unique place (or places) on the Earth creating significant amounts of formamide over a long period of time through radiolysis, then an opportunity exists for the onset of some unique chemistry that can support the production of essential precursor compounds for life, Adam said.

“So, the argument then shifts to— how likely was it that this unique place existed? We only need one special location on the entire planet to meet these circumstances,” he said.

Zachary Adam, an earth scientist in the lab of Andrew Knoll at Harvard University. (Nerissa Escanlar)

After that, the system set into motion would have the ability to bring together the chemical building blocks of life.

“That’s the possibility that we look forward to investigating in the coming years,” Adam said.

James Cleaves, an organic chemist also at ELSI and a co-author of the cobalt-60 paper, said while production of formamide from much simpler compounds represents progress, “there are no silver bullets in origin of life work. We collect facts like these, and then see where they lead.”

Another member of the cobalt-60 team is Albert Fahrenbach, a former postdoc in the lab of Harvard University’s Nobel laureate Jack Szostak and now an associate principal investigator at ELSI.

An organic chemist, Fahrenbach was a late-comer to the project, brought in because Cleaves thought the project could use his expertise.

“Connecting the origins of life, or precursors chemicals, with radiolysis (or radioactivty) was an active field back in the 70s and 80s,” he said. “Then it pretty much died out and went out of fashion.”

Fahrenbach said he remains uncertain about any possible role for radiolysis in the origin of life story. But the experiment did intrigue him greatly, it led him to experiment with some of the chemicals formed by the gamma ray blasts, and he says the results have been productive.

“Without this experiment, I would definitely not be going down some very interesting paths,” he said

 

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Nobel Laureate Jack Szostak: Exoplanets Gave The Origin of Life Field a Huge Boost

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Jack Szostak, Nobel laureate and pioneering researcher in the origin-of-life field, was the featured speaker at a workshop this week at the Earth-Life Science Institute (ELSI) in Tokyo.  One goal of his Harvard lab is to answer this once seemingly impossible question:  was the origin of life on Earth essentially straight-forward and “easy,” or was it enormously “hard” and consequently rare in the universe. (Nerissa Escanlar)

Sometimes tectonic shifts in scientific disciplines occur because of discoveries and advances in the field.  But sometimes they occur for reasons entirely outside the field itself.  Such appears to be case with origins-of-life studies.

Nobel laureate Jack Szostak was recently in Tokyo to participate in a workshop at the Earth-Life Science Institute (ELSI) at the Tokyo Institute of Technology on “Reconstructing the Phenomenon of Life To Retrace the Emergence of Life.”

The talks were technical and often cutting-edge, but the backstory that Szostak tells of why he and so many other top scientists are now in the origins of life field was especially intriguing and illuminating in terms of how science progresses.

Those ground-shifting discoveries did not involve traditional origin-of-life questions of chemical transformations and pathways.  They involved exoplanets.

“Because of the discovery of all those exoplanets, astronomy has been transformed along with many other fields,” Szostak said after the workshop.

“We now know there’s a large range of planetary environments out there, and that has stimulated a huge amount of interest in where else in the universe might there be life.  Is it just here?  We know for sure that lots of environments could support life and we also would like to know:  do they?

“This has stimulated much more laboratory-based work to try to address the origins question.  What’s really important is for us to know whether the transition from chemistry to biology is easy and can happen frequently and anywhere, or are there one or many difficult steps that make life potentially very rare?”

In other words, the explosion in exoplanet science has led directly to an invigorated scientific effort to better understand that road from a pre-biotic Earth to a biological Earth — with chemistry that allows compounds to replicate, to change, to surround themselves in cell walls, and to grow ever more complex.

With today’s increased pace of research, Szostak said, the chances of finding some solid answers have been growing.  In fact, he’s quite optimistic that an answer will ultimately be forthcoming to the question of how life began on Earth.

“The field is making real progress in understanding the pathway from pre-biotic chemistry to the earliest life,” Szostak told.  “We think this is a difficult but solvable problem.”

And any solution would inevitably shed light on both the potential make-up and prevalence of extraterrestrial life.

This artist’s concept depicts select planetary discoveries made by NASA’s Kepler space telescope.  With more than 4,000 confirmed exoplanets and estimates now that there are billions upon billions more, the question of whether some are inhabited has taken on a new urgency requiring the expertise of scientists from a wide range of fields.
(NASA/W. Stenzel)

Whether it’s ultimately solvable or not, that pathway from non-life to life would appear to be nothing if not winding and complex.  And since it involves trying to understand something that happened some 4 billion years ago, the field has had its share of fits and starts.

It is no trivial fact that probably the biggest advance in modern origin-of-life science — the renown Miller-Urey experiment that produced important-for-life amino acids out of a sparked test tube filled with  gases then believed to be prevalent on early Earth — took place more than 60 years ago.

Much has changed since then, including an understanding that the gases used by Miller and Urey most likely did not reflect the early Earth atmosphere.  But no breakthrough has been so dramatic and paradigm shifting since Miller-Urey.  Scientists have toiled instead in the challenging terrain of how and why a vast array of chemicals associated with life just might be the ones crucial to the enterprise.

But what’s new, Szostak said, is that the chemicals central to the pathway are much better understood today. So, too, are the mechanisms that help turn non-living compounds into self-replicating complex compounds, the process through which protective yet fragile cell walls can be formed, and the earliest dynamics involved in the essential task of collecting energy for a self-replicating chemical system to survive.

The simple protocells that may have enabled life to develop four billion years ago consist of only genetic material surrounded by a fatty acid membrane. This pared down version of a cell—which has not yet been completely recreated in a laboratory—is thought to have been able to grow, replicate, and evolve. (Howard Hughes Medical Institute)

This search for a pathway is a major international undertaking; a collective effort involving many labs where obstacles to understanding the origin-of-life process are being overcome one by one.

Here’s an example from Szostak:  The early RNA replicators needed the element magnesium to do their copying.  Yet magnesium destroyed the cell membranes needed to protect the RNA.

A possible solution was to find potential acids to bond with magnesium and protect the membranes, while still allowing the element to be available for RNA chemistry.  His team found that citric acid, or citrate, worked well when added to the cells.  Problem solved, in the lab at least.

The Szostak lab at Harvard University and the Howard Hughes Medical Institute has focused on creating “protocells” that are engineered by researchers yet can help explain how origin-of-life processes may have taken place on the early Earth.

Their focus, Szostak said, is on “what happens when we have the right molecules and how do they get together to form a cell that can grow and divide.”

It remains a work in progress, but Szostak said much has been accomplished. Protocells have been engineered with the ability to replicate, to divide, to metabolize food for energy and to form and maintain a protective membrane.

The perhaps ultimate goal is to develop a protocell with with the potential for Darwinian evolution.  Were that to be achieved, then an essentially full system would have been created.

How did something alive emerge from a non-living world? It’s a question as old as humanity and seems to pose more questions with every answer.  But Szostak (and some others) are convinced that the problem will in time prove to be solvable. Here blue-green algae in Morning Glory Pool, Yellowstone National Park, Wyoming.

Just as the discovery of a menagerie of exoplanets jump-started the origin of life field, it also changed forever its way of doing business.

No longer was the field the singular realm of chemists, but began to take in geochemists, planetary scientists, evolutionary biologists, atmospheric scientists and even astronomers (one of whom works in Szostak’s lab.)

“A lot of labs are focused on different points in the process,” he said.  “And because origins are now viewed as a process, that means you need to know how planets are formed and what happens on the planetary surface and in the atmospheres when they’re young.

“Then there’s the question of essential volatiles (such as nitrogen, water, carbon dioxide, ammonia, hydrogen, methane and sulfur dioxide); when do they come in and are they too much or not enough.”

These were definitely not issues of importance to Stanley Miller and Harold Urey when they sought to make building blocks of life from some common gases and an electrical charge.

But seeing the origin of life question as a long pathway as opposed to a singular event leaves some researchers cold.   With so many steps needed, and with the precisely right catalysts and purified compounds often essential to allow the next step take place, they argue that these pathways produced in a chemistry lab are unlikely to have anything to do with what actually happened on Earth.

Szostak disagrees, strongly.  “That just not true.  The laws of chemistry haven’t changed since early Earth, and what we’re trying to understand is the fundamental chemistry of these compounds associated with life so we can work out plausible pathways.”

If and when a plausible chemical pathway is established, Szostak said,  it would then be time to turn the scientific process around and see if there is a possible model for the presence of the needed pathway ingredients on early Earth.

And that involves the knowledge of geochemists, researchers expert in photochemistry and planetary scientists who have insight into what conditions were like at a particular time.

Szostak and David Deamer, an evolutionary biologist at the University of California, Santa Cruz, at the ELSI origins workshop.  Deamer supports the view that life on Earth may well have begun in and around hydrothermal springs on land.  That’s where essential compounds could concentrate, where energy was present and organic compounds on interstellar dust could have landed, as they do today. (Nerissa Escanlar)

Given the work that Szostak, his group and others have done to understand possible pathways that lead from simple starting materials to life, the inevitable question is whether there was but one pathway or many.

Szostak is of the school that there may well have been numerous pathways that resulted in life, although only one seems to have won out.  He bases his view, in part at least, on a common experience in his lab.  He and his colleagues can bang their collective heads together for what seems forever on a hard problem only to later find there was not one or two but potentially many answers to it.

An intriguing implication of this “many pathways” hypothesis is that it would seemingly increase the possibility of life starting beyond Earth.  The underlying logic of Szostak’s approach is to find how chemicals can interact to form life-like and then more complex living systems within particular environments.  And those varied environments could be on early Earth or on a planet or moon far away.

“All of this looked very, very hard at the start, trying to identify the pathways that could lead to life.  And sure, there are gaps remaining in our understanding.  But we’ve solved a lot of problems and the remaining big problems are a rather small number.  So I’m optimistic we’ll find the way.”

“And when we get discouraged about our progress I think, you know, life did get started here.  And actually it must quite simple.  We’re just not smart enough to see the answer right away.

“But in the end it generally turns out to be simple and you wonder 20 years later, why didn’t we think of that before?”

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Messy Chemistry, Evolving Rocks, and the Origin of Life

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Ribosomes are life’s oldest and most universal assembly of molecules. Today’s ribosome converts genetic information (RNA) into proteins that carry out various functions in an organism. A growing number of scientists are exploring how earliest components of life such as the ribosome came to be. They’re making surprising progress, but the going remains tough.

Noted synthetic life researcher Steven Benner of Foundation for Applied Molecular Evolution is fond of pointing out that gooey tars are the end product of too many experiments in his field.  His widely-held view is that the tars, made out of chemicals known to be important in the origin of life, are nonetheless a dead end to be avoided when trying to work out how life began.

But in the changing world of origins of life research, others are asking whether those messy tars might not be a breeding ground for the origin of life, rather than an obstacle to it.

One of those is chemist and astrobiologist Irena Mamajanov of the Earth-Life Science Institute (ELSI)  in Tokyo.  As she recently explained during an institute symposium, scientists know that tar-like substances were present on early Earth, and that she and her colleagues are now aggressively studying their potential role in the prebiotic chemical transformations that ultimately allowed life to emerge out of non-life.

“We call what we do messy chemistry, and we think it can help shed light on some important processes that make life possible.”

Irena Mamajanov of the Earth-Life Science Institute (ELSI) in Tokyo was the science lead for a just completed symposium on emerging approaches to the origin of life question. (Credit: Nerissa Escanlar)

It stands to reason that the gunky tar played a role, she said, because tars allow some essential processes to occur:  They can concentrate compounds, it can encapsulate them, and they could provide a kind of primitive (messy) scaffolding that could eventually evolve into the essential backbones of a living entity.

“Scientists in the field have tended to think of the origin of life as a process going from simple to more complex, but we think it may have gone from very complex — messy — to more structured.”

Mamajanov is part of an unusual Japanese and international group gathered at (ELSI), a relatively new site on the campus of the Tokyo Institute of Technology. It is dedicated to origin of life and origin of Earth study, with a mandate to be interdisciplinary and to think big and outside the box.

ELSI just completed its fifth annual symposium, and it brought together researchers from a wide range of fields to share their research on what might have led to the emergence of life.  And being so interdisciplinary, the ELSI gathering was anything but straight and narrow itself.

There was talk of the “evolution” of prebiotic compounds; of how the same universal 30 to 50 genes can be found in all living things from bacteria to us; of the possibility that the genomes of currently alive microbes surviving in extreme environments provide a window into the very earliest life; and even that evolutionary biology suggests that life on other Earth-like planets may well have evolved to form rather familiar creatures.

Except for that last subject, the focus was very much on ways to identify the last universal common ancestor (LUCA), and what about Earth made life possible and what about life changed Earth.

Artist rendering of early Earth on a calm day.  Scientists are trying to understand the many and complex geochemical processes that led to the emergence of life from non-life.

Scientific interest in the origin of life on Earth (and potentially elsewhere) tends to wax and wane, in large part because the problem is so endlessly complex.  It’s one of the biggest questions in science, but some say that it will never be fully answered.

But there has been a relatively recent upsurge in attention being paid and in funding for origin of life researchers.

The Japanese government gave $100 million to build a home for ELSI and support it for ten years, the Simons Foundation has donated another $100 million for an origins of life institute at Harvard, the Templeton Foundation has made numerous origin of life grants and, as it has for years, the NASA Astrobiology Institute has funded researchers.  Some of the findings and theories are most intriguing and represent a break of sorts from the past.

For some decades now, the origins of life field has been pretty sharply divided.  One group holds that life began when metabolism (a small set of reactions able to harness and transform energy ) arose spontaneously; others maintain that it was the ability of a chemical system to replicate itself (the RNA world) that was the turning point.  Metabolism First versus the RNA First, plus some lower-profile theories.

In keeping with its goal of bringing scientists and disciplines together and to avoid as much origin-of-life dogma as possible, Mamajanov sees their “messy chemistry” approach as a third way and a more non-confrontational approach.  It’s not a model for how life began per se, but one of many new approaches designed to shed light and collect data about those myriad processes.

“This division in the field is hurting science because people are not talking to each other ,” she said.  “By design we’re not in one camp or another.”

Loren Williams of Georgia tech

Another speaker who exemplified that approach was Loren Williams of Georgia Tech, a biochemist whose lab studies the genetic makeup of those universal 30 to 50 ribosomes (a complex molecule made of RNA molecules and proteins that form a factory for protein synthesis in cells.)  He was principal investigator for the NASA Astrobiology Institute’s Georgia Tech Center for Ribosome Adaptation and Evolution from 2009-2014.

His goal is to collect hard data on these most common genes, with the inference that they are the oldest and closest to LUCA.

“What becomes quickly clear is that the models of the origin of life don’t fit the data,” he said. “What the RNA model predicts, for instance, is totally disconnected from this data.  So what happens with this disconnect?  The modelers throw away the data.  They say it doesn’t relate.  Instead, I ignore the models.”

A primary conclusion of his work is that early molecules — rather like many symbiotic relationships in nature today — need each other to survive.  He gave the current day example of the fig wasp, which spends its larval stage in a fig, then serves as a pollinator for the tree, and then survives on the fruit that appears.

He sees a parallel “mutualism” in the ribosomes he studies.  “RNA is made by protein; all protein is made by RNA,” he said.  It’s such a powerful concept for him that he wonders if  “mutualism” doesn’t define a living system from the non-living.

 

These stromatolites, wavelike patterns created by bacteria embedded in sediment, are 3.7 billion years old and may represent the oldest life on the planet. Photo by Allen Nutman

 

Stromatolites, sedimentary structures produced by microorganisms,  today at Shark Bay, Australia. Remarkably, the life form has survived through billions of years of radical transformation on Earth, catastrophes and ever-changing ecological dynamics.

 

A consistent theme of the conference was that life emerged from the geochemistry present in early Earth.  It’s an unavoidable truth that leads down some intriguing pathways.

As planetary scientist Marc Hirschmann of the University of Minnesota reported at the gathering, the Earth actually has far less carbon, oxygen, nitrogen and other elements essential for life than the sun, than most asteroids, than even interstellar space.

Since Earth was initially formed with the same galactic chemistry as those other bodies and arenas, Hirschmann said, the story of how the Earth was formed is one of losing substantial amounts of those elements rather than, as is commonly thought, by gaining them.

The logic of this dynamic raises the question of how much of those elements does a planet have to lose, or can lose, to be considered habitable.  And that in turn requires examination of how the Earth lost so much of its primordial inheritance — most likely from the impact that formed the moon,  the resulting destruction of the early Earth atmosphere, and the later movement of the elements into the depths of the planet via plate tectonics. It’s all now considered part of the origins story.

And as argued by Charley Lineweaver, a cosmologist with the Planetary Science Institute and the Australian National University, it has become increasingly difficult to contend that life on other planets is anything but abundant, especially now that we know that virtually all stars have planets orbiting them and that many billions of those planets will be the size of Earth.

Other planets will have similar geochemical regimes and some will have undergone events that make their distribution of elements favorable for life.  And as described by Eric Smith, an expert in complex systems at ELSI and the Santa Fe Institute, the logic of physics says that if life can emerge then it will.

Any particular planetary life may not evolve beyond single cell lifeforms for a variety of reasons, but it will have emerged.  The concept of the “origin of life” has taken on some very new meanings.

ELSI was created in 2012 after its founders won a World Premier International Research Center Initiative grant from the Japanese government. The WPI grant is awarded to institutes with a research vision to become globally competitive centers that can attract the best scientists from around the world to come work in Japan.

The nature and aims of ELSI and its companion group the ELSI Origins Network (EON) strike me as part of the story.  They break many molds.

The creators of ELSI, both Japanese and from elsewhere, say that the institute is highly unusual for its welcome of non-Japanese faculty and students.  They stay for years or months or even weeks as visitors.

While ELSI is an government-funded institute with buildings, professors, researchers and a mission (to greatly enhance origin of life study in Japan), EON is a far-flung collection of top international origins scientists of many disciplines.  Their home bases are places like Princeton’s Institute for Advanced Study, Harvard, Columbia, Dartmouth, Caltech and the University of Minnesota, among others in the U.S., Europe and Asia.  NASA officials also play a supporting, but not financial, role.

ELSI postdocs and other students live in Tokyo, while the EON fellows spend six months at ELSI and six months at home institutions.  All of this is in the pursuit of scientific collaboration, exposing young scientists in one field related to origins to those in another, and generally adding to global knowledge  about the sprawling subject of origins of life.

Jim Cleaves, of ELSI and the Institute for Advanced Study,  is the director of EON and an ambassador of sorts for its unusual mission.  He, and others at the ELSI symposium, are eager to share their science and want young scientists interested in the origins of life to know there are many opportunities with ELSI and EON for research, study and visitorships on the Tokyo campus.

 

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