Friday, February 7, 2020

Are we alone in the Universe? Astrobiology

Two of my astrobiology colleagues: Andrew Steele (l) and Steve Shirey (r), Carnegie 2012

“The probability for the chance of formation of the smallest, simplest form of living organism known is 1 to 10,340,000,000.... The size of this figure is truly staggering, since there are only supposed to be approximately 1080 electrons in the whole universe!” Harold Morowitz, in Energy Flow in Biology (1968).

         In 1996, NASA announced that it had found evidence of life contained in the Martian meteorite Alan Hills 84001 (McKay et al., 1996). They held press conferences and presented evidence for what they termed bacterial cell structures in the meteorites, along with some amino acid profiles. News spread like wildfire among Mars aficionados and skeptics alike. The search for life on Mars stimulated several lander and orbital missions to Mars. At the same time, NASA under the advice of Wesley T. Huntress, Associate Administrator for Space Science, had the idea to form the NASA Astrobiology Institute (NAI), a virtual institute in which scientists in disciplines as far-ranging as astronomy and astrophysics would regularly engage with molecular biologists and geochemists. Objectives central to this effort were to understand how life arose on planet Earth, to determine when it arose, and to devise a set of criteria everyone could agree on that constituted evidence of life.
         This task has been more difficult than it sounds. To this day, NASA and NAI scientists still are discussing how they will determine which chemical or physical line(s) of evidence constitute proof that there is life on another planetary body. I was part of two original NAI teams that took very different approaches to search for life. The Jet Propulsion Lab (JPL) team focused on biosignatures. Biosignatures are fingerprints of life such as isotope fractionation patterns or elemental ratios. Ken Nealson, who specifically moved from University of Wisconsin to JPL, led the effort. Our work was carried out in both modern extreme environments as well as in ancient rocks. Being on the JPL campus, this NAI team used several new techniques that were being specifically designed for space flight. The goal of this group was to develop biosignatures in tandem with new analytical methods in the lab to help us to understand the significance of biosignatures observed in the field. 
(L-R) Wes Huntress, Doug Rumble, Marilyn, Ken Nealson, 2002

         On a geological time scale of hundreds, thousands, or millions of years, biologically-resistant material gradually obtains an isotopic and chemical composition that has little resemblance to original biochemical compounds. If we were to find evidence of life in martian rocks billions of years old, we needed to broaden our thinking. Rather than searching for pristine molecules similar to ones in living organisms, we needed to know what these molecules would look like—their biosignature--after they had been substantially degraded. Working with postdoc Sue Ziegler, we fed a simple mixed culture of microorganisms a single protein, Rubisco, the protein I studied for my PhD work. The bacteria ate the Rubisco protein completely in about 36 hours, turning the Rubisco protein into their own bacterial proteins.
         We compared the chemical isotope biosignature of the amino acids in the Rubisco with the isotope biosignature of the amino acids in the microbes. The biosignatures were completely different. After the bacteria had eaten the Rubisco, some slightly larger heterotrophic organisms ate the bacteria. Bacterial proteins were decomposed and recycled into these larger beasts. In a very short time, hours to days, the original Rubisco protein isotope biosignature had been altered twice. In the real world, almost any biomass made by plants and animals is quickly metabolized by microbes and almost never enters the fossil record. It is a reasonable assumption that if there were living organisms on another planet, a similar hierarchy of organisms would probably have evolved.
         The approach by the Carnegie team, which I also worked with, was completely different. Geophysical Laboratory staff member Bob Hazen and Harold Morowitz (George Mason University) had the idea that we might be able to study some of the most basic reactions fundamental to all organisms.  Bob Hazen is an accomplished writer of popular science books, textbooks, and scientific articles in his discipline. Over the years, I watched him transform from a high-pressure mineralogist to an astrobiologist to a big picture thinker on the nature of carbon in the deep earth. He was never a “lab guy” like the rest of us at the Geophysical Lab. Instead, he partnered with bright people like Larry Finger, George Cody, Robert Downs, and others to anchor the data collecting. Often, we sparred with one another, at unpredictable times. He saw my work as incremental, which is what most scientific advances truly are. I saw his work as broad brush, not getting at the heart of problems. Either way, Hazen sustained having novel scientific ideas which he supported with his encyclopedic ability to read articles, understand them, and write about them with style.
         Harold Morowitz was a real original thinker and intellect. Working at a second tier university in northern Virginia, he struck me as a person who could work anywhere that welcomed complex thoughts. Harold knew the biochemical pathways and enzymes thoroughly, which we appreciated. He visited to discuss the experiments, and sometimes tension arose when hypotheses did not match results and findings. Both he and Bob Hazen were scholars, while the rest of us were soldiers in the laboratory figuring out difficult analytical problems. Fortunately, George Cody and Harold worked on the same plane and were able to match their talents to produce the detailed work on abiotic organic synthesis.
          Hazen and Morowitz’s idea was to use heat and pressure—non-biological hydrothermal chemistry--rather than biological enzymes to see if we could simulate the TCA cycle reactions. The TCA cycle (also known as the Krebs cycle) operates generating energy for cells in just about all known organisms. Some microbes are missing a piece of the cycle, but in general, the TCA cycle is at the heart of metabolism. Geophysical Laboratory staff member George Cody, former Geophysical Laboratory Director Hat Yoder and I, as well as Bob and Harold, postdocs Mark Teece, Jay Brandes, Tim Filley, Jennifer Blank, and Nabil Boctor, discussed how this could be carried out. Bob and his postdocs would seal simple organic reactants into gold tubes—1/4” in diameter and about 4 inches long--that served as reaction vessels.  Then, Hat Yoder would take the gold tubes and subject them to high temperatures (200°C) and high pressures in his laboratory’s apparatus that took up an entire room. He was used to doing experiments at thousands of atmospheres of pressure and at least 500°C. It was a challenge for him to reduce the pressure and temperatures to levels that would not completely burn up the organic starting materials. He would place the small gold tubes into larger metal cylinders that he referred to as “bombs” because if they were made improperly they could explode and had the power to shoot through pretty think steel walls. Fortunately, that did not happen with any of our samples.
We tried to create "life" with heat and temperature, 2002

         George and his postdocs would analyze the products of the reaction. George Cody is one-of-a-kind, often self-absorbed and oblivious to humans, other times thinking about the long-term vision for the Geophysical Laboratory and his colleagues. Cody is most comfortable at the console of his custom built NMR spectrometer. As he evolved from being a coal chemist to an organic geochemist to an astrobiologist to a generalist, his skills in analyzing complex data sets thoroughly distinguish his work. At parties, George often can’t leave his work behind. After a few glasses of red wine, he may regale his friends about hamiltonians (an obscure parameter measured by his NMR spectrometer), while his wife looks patiently on. When we worked together, I could always rely on George to huddle with me and figure out gnarly lab problems, personnel issues, and weird data. Mark Teece and I were to measure the isotopic biosignature in the products; Harold and the rest of us would interpret the results. 
Jay Brandes (l) and George Cody at his NMR, 2001

         Experiments were more complicated than we ever anticipated. Eventually, Cody and team members found a potential reaction that jump started a modification of the biological TCA cycle. This pathway, named the hydrothermal redox pathway, is not used in any extant organisms but may have been the ignition point for primary metabolism during the early evolution of living organisms. One of the keys to getting this pathway to work at all was to choose the correct metals (for example, iron) and their sulfide minerals as catalysts. The potentially critical catalytic role of such metallic catalysts may be a crucial link between geochemistry and biochemistry at the point of life’s emergence. Integrating all of this, they discovered that they were within one reaction of demonstrating a purely geochemical carbon fixation pathway that closely mimics the TCA pathways, but they were unable, ever, to figure out how to do the last remaining reaction non-biologically.
         Origin of life studies were being carried out across the NAI and internationally at this time (2000s). The RNA world hypothesis, promoted by Jack Shostack at MIT, had been a favored pathway for jump-starting life. RNA (ribonucleic acid, one of the molecules carrying the genetic code) can catalyze simple reactions, directs the construction of more complicated molecules like proteins, and holds heritable information. The problem with this model was figuring out how to make RNA molecules in the first place. One hypothesis was that meteorites that bombarded early Earth brought these molecules with them.
         Zita Martins and the organic geochemists at Goddard Space Flight Center were searching for the building blocks of DNA and RNA, nucleobases, in organic extracts of the Murchison meteorite, one of the most famous carbonaceous chondrites of all time. Zita was working with Danny Glavin and Jason Dworkin at Goddard, and her colleagues and PhD advisor in the Netherlands. Using a sophisticated type of mass spectrometry, she could detect nucleobases in some of the Murchison extracts. She was able to zero in on these unusual and complicated structures, essentially picking the “needles out of the haystack” of other compounds. We began a collaboration to measure the stable isotope biosignature patterns in these particular molecules. If they really came from outer space, these compounds should have an isotope biosignature pattern similar to other meteorite organic molecules. Alternatively, if these were terrestrial contaminants, they should resemble isotope biosignature patterns of biological molecules.
         Zita arrived with extracts in hand at the Geophysical Laboratory, and we were confident that we could readily measure the carbon isotope patterns based on the results she measured at Goddard Space Flight Center. Unfortunately, the analytical system I was using, a combustion-GC-IRMS system, combusts all carbon compounds in the chromatogram, which was loaded with a multitude of other compounds, mostly organic acids, as well as with trace amounts of nucleobases (Martins et al., 2008).
         We were able to devise a clever analytical scheme for our instrument that separated the organic acids from the nucleobases without any serious overlap. The carbon isotope biosignature patterns of the organic acids had enriched levels of 13C in them, similar to measurements others had made on these same types of compounds in meteorites. We were thrilled when we were able to measure a 13C-enriched carbon isotope biosignature for uracil, a component in RNA, in the Murchison sample. The two isotope biosignatures were consistent with an extraterrestrial origin for both compounds. We also analyzed uracil from the soil surrounding the location in Australia where the meteorite was found. The soil uracil had an isotope biosignature similar to terrestrial carbon—not at all like the meteorite uracil. This work supports one of the many theories about the origins of life. One of these theories holds that meteorites delivered organic molecules to Earth during its formation and the period when Earth was continually bombarded by incoming asteroids. Although it was known that amino acids (e.g.,  Engel et al., 1980; Martins et al., 2007) were common monomers in some meteorites, no one had ever found nucleobases.
         Eventually, the stable isotope biogeochemistry studies of the Carnegie team shifted in emphasis. In phase two of the NAI project at the Geophysical Laboratory, we began to study the isotopic compositions of ancient sedimentary rocks and stromatolites and began an ambitious field campaign in Svalbard, an archipelago high above the Arctic Circle in a project termed AMASE: Arctic Mars Analogue Svalbard Expedition. Both the NAI and AMASE endeavors required field trips to far flung places to collect samples from ecosystems of interest to astrobiologists.

Andrew Steele AKA Steelie

            With Wes Huntress as our new Director in 1999, we began a search to hire a replacement for Ed Hare—someone who would be a full time astrobiologist. Carnegie and the Geophysical Laboratory hire people in very different ways than universities. It is the sole discretion of the Director of the department to choose and negotiate with a new staff member. For the astrobiology position, we advertised widely and received a number of applications from interesting, qualified individuals. A short list was struck and about five people each spent a couple of days visiting the campus, giving a seminar, and trying to impress the scientific staff.
            At the time, I was involved in using a new time-of-flight mass spectrometer to identify unknown compounds in complex mixtures. The instrument, the Protein Chip Reader, could measure the coupling of an antibody with its antigen very precisely. I had heard about a young man at Johnson Space Flight Center who was developing a similar system—except miniaturized—for flying on an upcoming Mars mission. He also heard of what I was doing and wrote me an email asking to visit the Laboratory. I was excited by the prospect, asked for his CV, and invited him to come for a seminar. Unbeknownst to him, his CV was circulated to the astrobiology hiring committee. We considered him a potentially viable candidate. Andrew Steele was officially on our radar screen.
            Andrew was, by then, working in England, so flew over to the States the weekend before his seminar to get adjusted to the time change. I met him briefly before his Monday seminar, telling him, “Hey! Do you know we have a staff position open for an Astrobiologist?” He did not. The news sent him into a bit of a panic, because Steele is usually informal in his mannerisms, dress, and speaking style. Apparently, he purchased a new outfit, updated his talk, and practiced it again and again before arriving at the Lab for his “visit” early Monday morning, which morphed into an impromptu interview. His combination of microbiology, meteorite geochemistry, technology, and Mars science was a perfect fit for what we were looking for. An offer quickly followed.
Steelie at the 9:30 Club, Washington DC

            Andrew—Steelie, as he is known to almost everyone, looks the part of a 1990s British rocker. In fact, he is an accomplished guitarist and composer of rock music, playing and singing in a Takoma Park band. His light brown hair reaches to the middle of his back, or is frequently up in a man-bun or back in a ragged ponytail. Wearing jeans purchased at a boutique shop in London and a t-shirt with science logos, Steelie can light up a room with his outward enthusiasm. He wears his personality and feelings on his sleeve, however. When he’s in a bad mood, it shows. When he’s deep in thought, he paces, looking at the ground, muttering to himself about thermodynamics and Mars.

            I was barely 13 years old when Steelie was born in January 1965. When we traveled to conferences, NASA meetings, and fieldwork, we looked like an unusual pair. Once—just once—in an airport rental car lot, a stranger said to him, “Your mother is waving at you over there.” That comment resulted in endless teasing. I was not old enough to be his mother, but earned the nickname of “Ma.” While he liked to say he thought I was “matronly” when he first met me, I enjoyed saying about him, “Yeah, he’s my son, living in the basement, doesn't have a girlfriend or a job, plays on an old Atari video game all day.” The razzing continues to this day.
            Steelie hit the ground running at the Lab and built a strong team of young postdocs and students who adored him and his unconventional style. People came from around the world to work with him. He set up his first lab in Ed Hare and John Frantz’s old labs, shoehorning in autoclaves, microbial culture apparatus, DNA identification instruments, and sophisticated microscopes. He was known for working odd hours. I’d see him slink by my office around 11 am, backpack slung over his shoulder, often laughing. He worked until late at night, sometimes regaling his colleagues with emails at midnight. Steelie had never been responsible for lab personnel before. Sometimes he loved the job, other times he found it a bother.
            Often, he was late for lab, committee, and informal meetings that he himself had set up. Finally one day, fed up with this, his lab group and I “decorated” his office with thousands of Styrofoam peanuts. When he saw the mess we created he was furious and let his lab mates know, in no uncertain terms, he was angry with their childish behavior. I let him know that I was the mastermind of the prank, and that if he wanted grown up behavior, he should be on time like a professional adult. We glowered a bit, then burst out laughing. I can’t say he completely changed his ways, but he grew more “adult like” and commanded the respect of his peers and lab group.
Styroform peanut crew, Steelie's lab

            Steelie’s first field trip was with my lab group who were investigating the effects of chicken waste (i.e., chicken &%*t) on the ecosystem. As part of this trip, we used seine nets to sample small fish from rivers that had been potentially polluted by chicken waste. Steelie came dressed in shorts wearing sandals. On his first attempt at fish seining, he lost one sandal in the mud. The remainder of the day he wore one shoe. He’d also forgotten to bring his wallet and drivers license, something we learned was more common than not. Consequently, when we went out for beers at the end of the day, the waitress refused to serve him. 
Steelie missing his shoe, 1st field trip

            Steelie’s next field trip was the 2003 expedition on AMASE. In Longyearbyen, he purchased a pair of fancy red hiking boots, which gave him huge blisters when he climbed Sverrefjell volcano for the first time. I joined the AMASE team in 2004, and by this time, he became skilled at organizing Artic fieldwork and finding signs of life on seemingly barren rocks. Never ever one to give up, Steelie went on to become Chief Scientist and an accomplished Arctic explorer over the years.
Steelie doing science in the Arctic

         Steelie is one of my Science Brothers. Now that we live and work thousands of miles away from each other, when we call the other answers the phone “As I live and breathe!” I watched over his daughter when his second child was born. He mentored my son Evan when Chris and I moved to California. Steelie was one of the first people I told about my ALS diagnosis. I was one of the first he told about his mother’s passing. I may be more matronly than ever. His hair and beard are tinged with gray, but we’ve got a firm hold on life and science. 
Steelie and Marilyn, Merced, 2014

A ride on the Vomit Comet

            We’ve all watched those space movies—Sandra Bullock floating around trying to repair a space ship, her hair twirling around her head, waving herself around the universe. It’s as fun as it looks, which I found out in 2004 on a trip on NASA’s Vomit Comet. In order to train astronauts for the feeling of zero gravity, NASA has a special plane taking off from Johnson Space Flight Center near Houston, Texas. In order to achieve zero gravity, the aircraft, a large C-130 cargo jet aircraft, follows a parabolic flight plan going up in an arc followed by descending in a similar arc. During the upward climb, gravity is about double Earth’s gravity; on the downward trajectory, gravity goes to zero.
            My opportunity to fly on the Vomit Comet was made possible by working with Jake Maule, a postdoc working with Andrew Steele. Jake, now a physician at Duke University Hospital, is a trim, sharp-looking Brit who had dreams of joining the astronaut core. His PhD is in medicine, so he was looking to use that training combined with an astrobiology theme to be attractive to the very competitive NASA program for selecting astronauts. Jake and I were both interested in immunology at that time. I had purchased an instrument capable of making the types of measurements that can detect complex diseases like HIV-AIDS. The instrument, an ELISA reader (enzyme-linked immuno-sorbent assay) measures the amount and strength that antibodies have in binding to the molecules, antigens, they are trying to remove from harming our bodies. 
Jake Maule, Arctic, 2005

            Jake’s idea was to use the ELISA reader to find out whether and how antibodies and antigens hook up together without the benefit of gravity. He asked to borrow my instrument. I told him, “OK, but you have to take me with you!” He thought about it briefly, and agreed. We flew down to Johnson Space Flight Center for 2 days of training. After a morning of lectures, we went into a space simulation chamber that was evacuated leaving almost no air, but all was fine because we were wearing oxygen masks. The chamber was then filled with nitrogen gas—which does not support life—and we were asked to remove our masks. It took me only 20 seconds to feel the effects—I was unable to count to five! My mask went right back on.
            Jake and I practiced our experiments in a lab on the ground. We planned to fly 40 cycles alternating between zero gravity and two-times gravity (2-G) during our 3 hour flight. Each cycle lasted 20 seconds—barely enough time to complete the manipulations needed. The morning of our flight we were given two medications to help—Dexedrine and scopolamine-one to keep you awake and the other to keep you from getting airsick. Donning NASA flight suits, 15 scientists, a flight supervisor, and the flight surgeon entered the plane. Excitedly, we set up our experiments. Ours was inside of a newborn baby’s Isolette, a plastic box with armholes for two people to attend to a premature infant, which kept our supplies from floating around the aircraft.
            We were seated for takeoff, then at 10,000 feet we moved to our workstations, where our feet were placed under straps so we wouldn’t float away. When we were about to enter zero-G, special lights flashed on. Then, as we switched to 2-G, the flight supervisor shouted, “Feet down! Comin’ up!” We heard that phrase more than 40 times.
            The first zero-G experience made your stomach do a flip-flop. Your hair raises up, your equipment floats around. Wow! Then, all too soon, you feel 2-G making you twice your body weight. A slight movement of your head and you felt nauseous, even going so far as to cause vomiting. On the second cycle, we began the manipulations. My skills working at sea on ships tossed around by big waves trained me for this work. Opposite to me, Jake was turning a bit green. The flight surgeon floated by, offered him a barf bag, wrapped it up, and then went on to the next scientist who needed some help. I’m proud to say my stomach remained in check.
            After 40 cycles of zero G, Jake and I unplugged and floated for the next couple of parabolas. The feeling of weightlessness, even for 20 seconds, is something my body has never forgotten. The ability to float in air, even fly, is something we only dream about but never experience. The trip on the Vomit Comet let my soul soar!
            Because I was a trooper on the flight, the pilots invited me to sit in the jump seat just behind them when we were returning to Johnson. What an eye opener!  First, I saw how close we were to another airplane and how the pilots handled that. Then, a warning light came on in the panel of instruments on the plane’s dashboard. We were all connected via headsets, so I could hear them discussing the meaning of the orange light. Apparently, it had to do with the functioning of one of the two jet engines. After we safely landed, the plane was taken into a hanger for maintenance to figure out what was happening with that engine. Turns out that it needed to be completely overhauled. Our 2nd flight was cancelled and the Vomit Comet was out of commission for months.
            Our experiments were successful. We determined that antibodies and antigens had no problems working in the absence of gravity. Although humans evolved with the benefits of a gravitational field, our biochemical systems could adapt to spaceflight.

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