Scientists prove parasite mimics key plant peptide to feed off roots By Roger Meissen | Bond LSC
A nematode (the oblong object on the left) activates the vascular stem cell pathway in the developing nematode feeding site (syncytium) on a plant root. | photo by Xiaoli Guo, MU post-doctoral research associate
When it comes to nematodes, unraveling the root of the issue is complicated.
These tiny parasites siphon off the nutrients from the roots of important crops like soybeans, and scientists keep uncovering more about how they accomplish this task.
Research from the lab of Bond LSC’s Melissa Mitchum recently pinpointed a new way nematodes take over root cells.
Melissa Mitchum | photo by Roger Meissen, Bond LSC
“In a normal plant, the plant sends different chemical signals to form different types of structures for a plant. One of those structures is the xylem for nutrient flow,” said Mitchum, an associate professor in the Division of Plant Sciences at MU. “Plant researchers discovered a peptide signal for vascular stem cells several years ago, but this is the first time anyone has proven that a nematode is also secreting chemical mimics to keep these stem cells from changing into the plant structures they normally would.”
Stem cells? Xylem? Chemical mimics?
Let’s unpack what’s going on.
First, all plants contain stem cells. These are cells with unbridled potential and are at the growth centers in a plant. Think the tips of shoots and roots. With the right urging, plant stem cells can turn into many different types of cells.
That influence often comes in the form of chemicals. These chemicals are typically made inside the plant and when stem cells are exposed to them at the right time, they turn certain genes either on or off that in turn start a transformation of these cells into more specialized organs.
Want a leaf? Expose a stem cell to a particular combination of chemicals. Need a root? Flood it with a different concoction of peptides. The xylem — the dead cells that pipe water and nutrients up and down the plant — requires a particular type of peptide that connects with just the right receptor to start the process.
But for a nematode, the plan is to hijack the plant’s plan and make plant cells feed it. This microscopic worm attaches itself to a root and uses a needle-like mouthpiece to inject spit into a single root cell. That spit contains chemical signals of its own engineered to look like plant signals. In this case, these chemicals — B-type CLE peptides — and their purpose are just being discovered by Mitchum’s lab.
“Now a nematode doesn’t want to turn its feeding site into xylem because these are dead cells it can’t use, so they may be tapping into part of the pathway required to maintain the stems cells while suppressing xylem differentiation to form a structure that serves as a nutrient sink,” Mitchum said. “To me that’s really cool.”
This means these cells are free to serve the nematode. Many of their cell walls dissolve to create a large nutrient storage container for the nematode and some create finger-like cell wall ingrowths that increase the take up of food being piped through the roots. For a nematode, that’s a lifetime of meals for it while it sits immobile, just eating.
But how did scientists figure out and test that this nematode’s chemical was the cause?
Using next generation sequencing technologies that were previously unavailable, Michael Gardner, a graduate research assistant, and Jianying Wang, a senior research associate in Mitchum’s lab, compared the pieces of the plant and nematode genome and found nearly identical peptides in both — B-type CLE peptides.
“Everything is faster, more sensitive and we can detect things that had gone undetected through these technological advances that didn’t exist 10 years ago,” Mitchum said.
To test their theory, Xiaoli Guo, postdoctoral researcher and first author of the study in Mitchum’s lab synthesized the B-type CLE nematode peptide and applied it to vascular stem cells of the model plant Arabidopsis. They found that the nematode peptides triggered a growth response in much the same way as the plants own peptides affected development.
They used mutant Arabidopsis plants engineered to not be affected as much by this peptide to confirm their findings.
“We knocked out genes in the plant to turn off this pathway, and that caused the nematode’s feeding cell to be compromised. That’s why you see reduced development of the nematode on the plants.”
This all matters because these tiny nematodes cost U.S. farmers billions every year in lost yields from soybeans, and similar nematodes affect sugar beets, potatoes, corn and other crops.
While this discovery is just a piece of a puzzle, these pieces hopefully will come together to build better crops.
“You have to know what is happening before you can intervene,” Mitchum said. “Now our biggest hurdle is to figure out how to not compromise plant growth while blocking only the nematode’s version of this peptide.”
Nga Nguyen hopes to apply her research to increase nutrient contents in crop plants
Nga Nguyen, a doctoral candidate in MU’s Division of Plant Sciences, observes samples of a model plant species, Arabidopsis thaliana, in the Mendoza-Cózatl lab at Bond Life Sciences Center on Feb. 7, 2017. | photo by Eleanor C. Hasenbeck, Bond LSC
By Eleanor C. Hasenbeck | Bond LSC
Plants smell better than animals, at least to Nga Nguyen. That’s one reason why she decided to study them.
“In my undergrad, I studied horticulture,” Nguyen said. “For that you don’t really learn the inside mechanisms of plants, so I decided besides knowing the cultivation techniques, I’d like to also learn about the molecular biology.”
As a fifth year doctoral candidate in the Mendoza-Cózatl lab at Bond Life Sciences Center, she hopes to combine her undergraduate background with her present research in the microbiology of plants to improve the crops of the future.
Nguyen studies how transporter proteins move micronutrients like iron through plants. By understanding how plants move these nutrients in model plants, researchers hope to apply the same understanding and techniques to crops like soy and common beans. Increasing the micronutrient content of these crops could be a useful tool in combatting nutrient deficiencies in areas where people don’t have access to meat and dairy.
But Nguyen says the benefits of studying plants don’t end there. “I hope people pay attention to plant research and study,” Nguyen said. “If you think about it, it’s not just our food, but our clothing and the materials we use.”
Emily Million, a prospective biochemistry graduate student from Truman State University and Kevin Muñoz-Forti of University of Puerto Rico’s Pontifical Catholic University talk at the Graduate Life Sciences Joint Recruitment Weekend on February 4 after looking at posters about many different research programs and projects. | Roger Meissen, Bond LSC
By Jinghong Chen | Bond Life Sciences Center
Nick Dietz was not certain where to start his research journey this time last year.
But the atmosphere during a recruitment weekend nearly a year ago convinced him to pick MU over three other offers of admission. He is now a first-year plant sciences Ph.D. graduate student and life sciences fellow at MU.
“It is crucially important for [prospective] graduate students to feel they are going to feel like home, and Mizzou just knocked out that part with the recruitment weekend,” said Dietz.
The Graduate Life Sciences Joint Recruitment Weekend, an annual event since 2010, builds a two-way street between MU faculties and prospective graduate students and helps them to determine whether MU is the place for them to continue their education.
This year, about 35 prospective students with different academic backgrounds participated in the recruitment event.
“Up to this point, the departments only know these [prospective] students on paper,” said Debbie Allen, coordinator of Graduate Initiatives. “But this is an chance for the faculty and staff to meet them in person to get a feel that whether they are going to be a good fit for our program.”
Conversely, the prospective students also gain deeper understanding of MU via tours around the campus and the laboratories, one-on-one interviews with potential advisors and interdisciplinary poster sessions. The event combines recruiting efforts from the division of Biochemistry, Plant Sciences, Molecular Pathogenesis and Therapeutics graduate program, Genetics Area program, MU Information Institute, the Interdisciplinary Plant Group and Life Sciences Fellowship Program.
More than 100 faculty, graduate students and post-doctoral fellows joined the recruitment weekend. They play a valuable role in interacting with the prospective students, as they are the ones who are in the midst of MU life.
Nick Dietz, a freshman plant sciences graduate student, volunteered as a student ambassador during the the annual Graduate Life Sciences Joint Recruitment Weekend Saturday, Feb. 4. Dietz said last year’s event clinched his decision to attend MU and made him want to help prospective students make their decisions on where to attend. | photo by Roger Meissen, Bond LSC
Dietz joined that effort as a student ambassador. He toured Matthew Murphy, an Illinois College graduate, around the campus and shuttled him to different interviews.
Murphy drove from St. Louis for the recruitment weekend. With a major in biology and a minor in mathematics, he wishes to submerge himself into plant sciences.
During his gap year at the Donald Danforth Plant Science Center after graduation, Murphy learned about the division of Plant Sciences, which is one of the MU’s strongest programs. That eventually got him pumped up to apply for MU.
The recruitment weekend energized him further.
“Every graduate student I have talked to is really helpful and honest,” said Murphy. “They are all saying… how thankful they are to pick Mizzou.”
Lloyd Sumner, professor of biochemistry and director of MU’s Metabolomics Center in Bond LSC, talks with a prospective graduate student Saturday, Feb. 4, during the annual Graduate Life Sciences Joint Recruitment Weekend. | photo by Roger Meissen, Bond LSC
Lloyd Sumner, an MU professor of biochemistry, is expecting new students to join his lab. He had lunch and one-on-one meetings with the 11 prospective students invited by the biochemistry department, and toured them around his lab to showcase the instrumental resources.
“These are educated young adults with often very grand ideas. It is inspiring to visit with them and to be part of their future goals and careers,” Sumner said.
After six months rotating between different labs, Dietz has not yet decided which research route he will take yet. Nevertheless, he remains certain of one thing: he is enjoying the life here.
“It is a really warm atmosphere,” said Dietz. “I don’t feel I am being used as a labor. Professors actually want me to do well and get a good education.”
MU Center for Agroforestry symposium talks medicinal plants
Rob Riedel from Wild Ozark Ginseng Farm introduces their products at the agroforestry symposium on Jan. 26th, 2017 | photo by Jinghong Chen, Bond LSC
By Jinghong Chen | Bond LSC
Researchers, landowners and entrepreneurs converged at Bond Life Sciences Center to discuss current developments and topics in medicinal plants and agroforestry at the eighth UMCA Agroforestry Symposium. This daylong annual event, hosted by the Center for Agroforestry, took place on Thursday, Jan. 26.
People have been using medicinal plants as natural remedies and medicines for thousands of years all over the world. The global market of medicinal plants industry is huge.
“It is going to approach nearly $115 billion by 2020,” said Dr. Shibu Jose, director of MU Center for Agroforestry.
The university practices research projects on how to grow medicinal plants in a sustainable manner and how to harvest and process them, according to Dr. Jose.
Keynote speaker Tom Newmark talks about medicinal plants at the agroforestry symposium on Jan. 26th, 2017 | photo by Jinghong Chen, Bond LSC
Tim Newmark of the American Botanical Council said climate change and the loss of soil are two main threats to herb plants. His keynote speech is on how to use regenerative practices in medicinal plants and agroforestry to positively impact the environment. A recent White House report wrote that without cooperated actions, the United of States will run out of the topsoil by the end of this century.
“We are eating our environment,” said Newmark.
Four main destructive forces leading to the dramatic loss of soil are excessive tilling, monoculture, synthetic nitrogen fertilization and pesticides.
Newmark did a side-by-side test in his farm in Costa Rica during the worst drought in the country. He implanted cassava in two fields under identical conditions and applied the best practice of conventional agrochemical agriculture and regenerative practice, respectively.
When the drought happened with six weeks of no rain in the rainforest, only the crop in conventional field was a complete failure.
Newmark said the next trend in the plants industry is agriculture focusing on regenerative plant soil.
Seven other speakers also presented on medicinal plants and included:
Dr. Jim Chamberlain, from US Forest Service, on forest management and medicinal plants
Dr. Susan Leopold, from United Plant Savers, on the conservation of medicinal plants
Dr. Jed W. Fahey, from Johns Hopkins University, on researches on moringa oleifera
Dr. Lloyd Sumner, from the University of Missouri, on the metabolomics opportunities and application in pecan
Dr. Chung-Ho Lin, from the University of Missouri, on how to identify value-added compounds from waste plant materials
Dr. Bill Folk, from University of Missouri, on International partnerships in medicinal plants
Steven Foster, an author and photographer, on field guide on medicinal plants and herbs
The agroforestry symposium is held annually with different themes. It has focused on climate change and pollinators, previously.
It feels good to get recognition, especially when it comes from the White House.
This week D Cornelison, a Bond Life Sciences Center researcher and associate professor of biological sciences found out she will receive a Presidential Early Career Award for Scientists and Engineers (PECASE). The award is the highest honor bestowed by the United States government on science and engineering professionals in the early stages of their independent research careers. She joins 102 researchers this year selected by the White House to receive this prestigious award.
This is a first for Missouri as a state as well as MU, making her the only scientist based in Missouri to ever be selected. Cornelison was nominated by her program officer at the National Institutes of Health, which funds her work on satellite cells.
Read more here from Melody Kroll on the Division of Biological Sciences website.
Bond LSC scientist works with MU eye surgeon to help people suffering from autoimmune-disease Sjögren’s syndrome
Dr. Carisa Petris stands in the McQuinn atrium of Bond Life Science Center. She and Bond LSC researcher Gary Weisman are using funding from a $100,000 Bond LSC grant to study the mechanisms of an auto-immune disease in the lacrimal glands of the eyes. They are hoping treatments for the disease in mice they study could be applied to humans. | photo by Phillip Sitter, Bond LSC
By Phillip Sitter | Bond LSC
They may not get much respect, but tears and spit are the products of a delicate secretive system that people would pay their respects to in mourning if they discovered that system was dying.
Gary Weisman and Dr. Carisa Petris are working together to help heal the damage caused by such a chronic lack of tears and saliva. The pair recently received a $100,000 Bond Life Sciences Center Grant for Innovative Collaborative Research to allow Bond LSC’s Weisman to partner with Petris, an eye surgeon working at MU Hospital.
They want to study the mechanism by which the auto-immune disease Sjögren’s syndrome cripples the glands of the eyes in mice. By comparing that mechanism to how it works in human eyes, they hope to examine if effective treatments for the mice could in turn help people.
“Dr. Weisman has characterized [Sjögren’s syndrome] in the salivary glands, and then there are similar glands in the eye called the lacrimal glands, and those are the tissues that we’re going to study,” she said of their collaboration.
Much of the grant money will go toward the costs of obtaining and housing new knockout mice for the study. These mice have a disabled, or knocked out, gene that causes them to express a certain trait like the dry eyes and development of Sjögren’s in this case.
“It takes a few weeks to a couple months for the disease to fully manifest itself, so we’ll house those mice for that time, and then of course, we’ll be treating them with the drug, and not with the drug, some for harvesting just the lacrimal glands and [studying] the surface of the eye,” Petris said.
Even though Sjögren’s syndrome and inflammation research are big topics, there’s just no good solution to the problems yet.
“There are a few [eye] drops that are used for Sjögren’s now, and they’re at best helpful, but they don’t cure the disease, so that would be the ultimate goal. They help decrease the inflammation that goes along with it and increase the tear production. The drops are also limited in their longevity too — you can only use them a certain length of time before they tend to not work so well anymore,” Petris said.
Petris referred to one drug that shows promise. The drug or another like it would interrupt the autoimmune response that causes the damaging inflammation that leads to Sjögren’s. It has already shown good results for reducing the symptom of dry mouth in mice, so Petris said she and Weisman will add it to some of the eyes of their mice and see if has any similar effect it reducing dryness there.
Dr. Peter Ostrum spoke at Bond LSC in celebration of World One Health Day
Dr. Peter Ostrum, who once played the character of Charlie Bucket in 1971’s “Willy Wonka and the Chocolate Factory” —also starring the late Gene Wilder — smiles after giving a lecture to an audience at Monsanto Auditorium in Bond LSC. After “Willy Wonka,” Ostrum did not pursue acting further, and went into a career in veterinary medicine. | photo by Phillip Sitter, Bond LSC
By Phillip Sitter |Bond LSC
The character of Charlie Bucket found his golden ticket to a happy life wrapped in a Willy Wonka chocolate bar. Peter Ostrum, who at the time was just a child actor playing Charlie, later found his in horse pastures.
After playing Charlie in 1971’s “Willy Wonka and the Chocolate Factory” alongside the late Gene Wilder starring in the titular role, Ostrum didn’t pursue acting any further. He spoke about life as a veterinarian Nov. 3 at Monsanto Auditorium in Bond Life Sciences Center.
“People are always curious about what happened to Charlie. Why wasn’t he in any other films? Did he survive Hollywood? I’m relieved to tell you that my life didn’t end up as a trainwreck,” Ostrum said, getting some laughs from the crowd gathered to listen to him speak.
“The film industry just wasn’t for me,” he explained, although he did enjoy working alongside Wilder and co-star Jack Albertson, who played Grandpa Joe. Ostrum said that every day on lunch break during filming in Munich, Germany, Wilder would share a chocolate bar with him.
Back at home in Ohio, Ostrum worked at a stable, and had several positive interactions with veterinarians. He admired the profession, and working with horses specifically. He even went on to be a groomer for the Japanese three-day equestrian event team at the 1976 Summer Olympics in Montreal.
He wanted to become an equine veterinarian after a year working at an equine veterinary clinic. However, Ostrum discovered that dairy cow care fell more in line with his dreams, and after getting his veterinary degree at Cornell, he’s been doing that ever since — in upstate New York where he is also a husband and father of two children.
Ostrum described how agriculture and veterinary medicine have changed over recent years, with changing numbers and sizes of farms, the rising power of animal welfare groups and an increased desire from consumers to know where their food comes from. People want to know whether animals are treated humanely and whether farms are negatively affecting the environment, he said.
All of these changes and others require increased transparency, education and community outreach efforts by everyone working in agriculture, Ostrum said. In candidates for veterinary associates, he said that he looks for “the intangible skills at the heart of who people are” — their character and their ability to connect with clients and patients.
Ostrum also mentioned the importance of mental health awareness among veterinarians and other health professionals. “We can’t help others if we can’t help and support ourselves,” he said.
Efforts to understand the genome of one plant through its many genetic varieties could lead to nutritional improvements in the staple crops billions of people depend on
By Phillip Sitter | Bond LSC
Ruthie Angelovici stands next to some Arabidopsis thaliana samples in the basement of Bond LSC. She is leading projects to study the relationships between genotypic and phenotypic variation in Arabidopsis and how this affects the amino acid content of the plants, and the resistance of their seeds to drought conditions. | Phillip Sitter, Bond LSC
It’s hard to avoid corn, rice or soybeans in your diet, and you’ve probably eaten or drank something today with at least one ingredient from them.
Unfortunately for the billions of people worldwide who depend on these crops as a staple, they aren’t actually all that nutritious. Specifically, they lack sufficient quantities of amino acids.
Twenty amino acids are required to build any protein, and within that about ten are considered essential, Bond Life Sciences researcher Ruthie Angelovici said. “Without amino acids, you can’t live.”
Amino acids might seem minor, but important parts and processes in our bodies from our muscles to enzymes are built from or work through them. That’s why Angelovici wants to enhance their availability in key foodcrops.
In the case of amino acids, “What we’re trying to understand is the basic question of how those accumulate in seeds, and then from that basic concept we’re going to try to improve that in grain,” Angelovici said.
The evolution of poor nutrition
No one really knows why so many of our most important crops that essentially sustain humanity lack sufficient essential amino acids.
Maybe plants don’t synthesize amino acids because the cost in energy for the plant is too high, or because higher levels of amino acids might make them more vulnerable to attacks from hungry insects. Maybe if plants produced higher levels of amino acids, the taste would be too strong for human palates, and so our ancestors long ago selectively bred those traits out of crop populations. Or, maybe in ancient farmers’ pursuits of other traits in their crops, like higher quantities of starch, humanity accidentally boosted one nutritional trait at another’s expense. There are a lot of unknowns when it comes to these theories, Angelovici said.
What is clear — and something Angelovici said she cannot stress enough — is how powerful a genetic tool she and her fellow researchers at Bond LSC have in the form of a collection of a vast amount of genetic variation of Arabidopsis thaliana.
“Arabidopsis thaliana is a model [plant] system that a lot of plant scientists use, although it is not a crop, or anything like that, but it’s a great model plant to start with, and then everything we learn from it, we can try and figure out if it’s the same in maize, rice, soybean, and translate it,” Angelovici explained.
Part of the mustard family, Arabidopsis grows quickly so researchers can study four or five generations in one year. As an added bonus, this huge genetic variety but can be grown in just one room instead of large fields. For Angelovici, that room is in Bond LSC’s basement and the basement of greenhouses nearby.
“We are growing right now 1,200 ecotypes of this Arabidopsis thaliana. So, what is an ecotype? It’s basically from the same species, but they have a slightly different genotypes. So, we’re looking at a vast genetic variation that represents genetic variation of this species across the world. Each ecotype comes from a different place,” she said.
For those of you wondering, a genotype is the specific sequence of information in an organism’s genetic code — its genetic identity. A phenotype is an observable physical trait controlled by the genetic sequence. For phenotype, think in terms of color, size, shape — just like in different breeds of dogs and cats, for example.
Even the smallest differences in genetics can produce the range of traits we observe, like the size difference between a Chihuahua and a St. Bernard — even though all the breeds are the same species. The same thing applies to plant species, too.
Angelovici said researchers can use all the genetic variation in their extensive Arapidopsis collection understand questions of how observable traits relate to genes, and vice versa.
Once that connection is established, “we basically have an address on the genome, and then we can go after the gene itself, understanding the function of the gene, and how that is affecting our variation of the phenotype, basically to help us understand the mechanism,” Angelovici explained.
“And if you understand the mechanism, we might be able to improve it, change it, either through genetic engineering or breeding. Basically, mining what Mother Nature has already done throughout many generations, and trying to figure out if we can utilize that in crops,” she added.
“We can measure the level of amino acid, but does the plant really care about the absolute level of amino acid, or relative level, and how they correlate with one another? It appears that these relationships are really important.”
All this algorithmic analysis can eventually improve results.
“When we get a candidate gene that we think affects one of the traits that we are interested in, we either knock it out or over-express it, and go back to the phenotype and figure out if it changes, and how,” Angelovici said.
“Along the way, we also try to understand if the phenotype is correlating with something that is larger, for example the plant’s growth, its development or the development of seeds.”
A plant under stress
An understanding of seed development might be especially important in understanding how drought affects the nutritional quality of future generations of water-stressed plants.
“Surprisingly, those are processes that are not well-understood — how the seed itself is adapting to water stress. A lot of people are working on water stress and drought at the plant level, in the yield [of a crop], but we’re trying to really understand what is happening on the level of the seeds, on the bio-chemical level, and then how that affects the next generation,” Angelovici explained.
If she and her fellow researchers find a super-resilient seed, they could learn to transfer its resiliency to drought to future generations of seeds.
Something they’ve seen already is that if you really water-stress a plant, while it may produce less seeds, seeds that it does produce are bigger.
“Right now the question is, are they bigger because they are trying to adapt for their harsher environment, or are they just trying to survive?” she said. Is the parent developing its offspring in a certain way to ensure the best possibility of success of that offspring, or just so it can survive to reproduce another day?
“We can only provide the data,” Angelovici said of her work in trying to answer questions like these, in order to improve the quality of human life by understanding and improving the quality of our food.
“This is the mechanism, and that is a tool we can provide,” Angelovici said of what the research can offer to people like farmers and other plant breeders. “Knowledge is power. What we do with this power is up to a lot of people.”
Ruthie Angelovici is an assistant professor in the Division of Biological Sciences, and is a researcher at Bond Life Sciences Center. She received her degrees in plant science from institutions in Israel — her B.S. and M.S. from Tel Aviv University, and her Ph.D. from the Weizmann Institute of Science in Rehovot. She was a postdoctoral fellow at the Weizmann Institute and at Michigan State University, and has been at MU since fall of 2015.
This file photo shows at least one other ecotype of Arabidopsis thaliana in a greenhouse in Bond LSC. Even small variations in the species’s genome can create the large number of observable varieties, sometimes with distinct sizes and shapes, other times with genetic differences that can only be observed on the microscopic level. | Roger Meissen, Bond LSC
Inter-departmental MU team aims to improve enzyme use and recovery for spectrum of industrial, medical and military applications
By Phillip Sitter | Bond LSC
A mostly-finished cylindrical bio-reactor site sits in a 3D printer after the printing has stopped. With a 3D printer in-house, Chung-Ho Lin said that the inter-departmental team he is part of can generate four or five different prototypes a day to test in their bio-reactor model, instead of having to order from different fabrication companies. A basic printer like this used to cost $8,000, but within the last year prices dropped to only about $1,000. Lin is a research assistant professor at MU’s Center for Agroforestry, and the team and project are coordinated by Hsinyeh Hsieh, a veterinary pathobiology research scientist in George Stewart’s Bond LSC lab, where the team also does most of its work. | Phillip Sitter, Bond LSC
As Sagar Gupta watched a 3-D printer on a lab countertop construct a jumbo pencil eraser-sized, white plastic cylinder of what looked like a shell holding inter-woven letter Xs, he remarked that the only limitation to what you can print is the size of the printer.
“The timing is perfect, otherwise we wouldn’t have been able to afford it,” Chung-Ho Lin said of the availability of cheaper 3-D printers within the past couple years.
The two men were acutely aware, as the printer continued its methodical manufacture, that they may be architects of the first steps in a bio-chemical revolution.
It’s a revolution that could be hugely profitable financially and may help to save lives on battlefields, clean up some kinds of pollution and enable humans to venture further into space for a cheaper cost, among other things.
To understand how this cross-disciplinary team working in George Stewart’s lab at the Bond Life Sciences Center got there, we have to back up a little bit.
Sagar Gupta holds a vial of carbon solution. Most of the team’s prototype designs for bio-reaction sites are made of carbon, and some are even bio-degradable. | Phillip Sitter, Bond LSC
From a bottleneck to a bioreactor
Their work began three years ago with a project to develop technology to reduce the cost of converting cellulose into glucose for biofuels — essentially the process by which raw plant fiber from wood or leaves is turned into a sugar that can be more efficiently burned to produce energy.
“That has been the bottleneck for the biofuel industry,” said Lin.
The team — consisting of Lin, a research assistant professor at MU’s Center for Agroforestry; Stewart, Hsinyeh Hsieh and several undergraduate and recently graduated students including Gupta — already developed E. coli bacteria that can mass-produce engineered enzymes to convert cellulose into glucose.
These enzymes speed up the reactions and reduce the cost because they have linkers attached to them — protein hooks that let them be recovered after a single use as catalysts in biological reactions, rather than having to throw them out. Hsieh said she developed this with Stewart’s input, and the assistance of a recently graduated student, Che-Min Su.
However, the team needed a platform for the linkers to hook onto — something they could continuously use to reel in their catch.
The answer in their search for the correct platform arrived when affordable 3-D printing technology came onto the market. With their own 3-D printer in-house, they custom-designed different platforms for their experiments and completely bypassed having to shop around with different fabrication companies.
All of the ingredients were there with that plastic cylinder Gupta and Lin watched print. The team now had a cheap way to mass produce and repeatedly recover enzymes. With this capability, they could produce a more efficient bioreactor — a controlled, isolated system in which desired reactions can take place with higher outputs of quantity and quality of a desired product.
It’s much like the more familiar concept of a nuclear reactor, which controls and isolates a nuclear chain reaction to harvest the most energy possible. The catalysts in that reaction are radioactive particles that give off heat as they decay. In a physical reaction, the heat released boils liquid water into gaseous steam, and the steam turns a turbine generator that makes electricity.
But in the team’s bioreactors, catalysts are enzymes that chemically react with cellulose and transform it into glucose instead of electricity. The glucose can be fermented further into butanol that can ultimately be used for liquid fuels to power vehicles.
A bio-reactor column stands packed with carbon fibers submerged in enzymes. If the column were hooked up a continous flow system, substrate would be pumped through it to spur bio-chemical reactions on the surface of the carbon fibers, or whatever other type of site is packed inside. | Phillip Sitter, Bond LSC
Money and blood
While only at a bench-top, proof-of-concept scale, the team’s first bioreactor has lasted more than four months. With prospects to increase its size, they “could be saving at least $10 to $12 million per year on an industrial scale,” said Gupta. Gupta graduated in May from MU with an MBA, and now works for Lin.
That estimate is just for one individual bioreactor. Begin to multiply it, and the cost-savings add up very quick.
“Nowadays, probably a majority of pharmaceutical companies have already switched their manufacturing process into the enzymatic process. One thing nice about the enzymatic process is that it can eliminate [the need for] a lot of hazardous chemicals. They also tend to have a better yield,” Lin explained.
Lin added that there is a bonus of complexity within this kind of 3-D platform system. Individual enzymes have different linkers, and this allows for multiple enzymes to catalyze reactions and be recovered on the platform at the same time. This is especially cost-saving because the conversion of cellulose into glucose requires three different kinds of enzymes.
“Because of this high specificity, we don’t need any enzymatic purification process,” he said.
Once the enzymes hooked to a platform start to naturally decay, the team can simply remove the decayed enzymes by a hot water bath and soak it in a new batch of enzymes, just like swapping out an empty printer cartridge for a full one with fresh ink.
While their primary focus is on biofuels, they are very aware that more efficient and cheaper bioreactors could have huge implications for a broad spectrum of industries.
One use they are developing could effectively transform one blood type into another using enzymes.
“This is not a completely new technology, but in the past, I would say back in the 90’s, some people tried some clinical trials and they ran into a problem, because a lot of times after the conversion, [loose] enzymes would get into the recipients’ bloodstream and cause an auto-immune reaction,” said Lin.
However, by being able to immobilize enzymes with their linkers on this 3-D device, they should be able to get around that problem, he said.
“I think there’s great potential for the soldier on the battlefield,” Lin cited as an application for the technology. A field doctor or medic wouldn’t have to worry about waiting on a certain type of blood for a transfusion, because they could convert another batch of blood into a universal-donor type.
Another team member, Hien Huynh explained that the more enzyme you add in ratio to the substrate, in this instance blood, the faster the conversion process will go — “maybe just 30 minutes.”
Hsieh wrote that “Blood type conversion would be the ultimate challenge for our bioreactor, because it has so many clinical aspects to be concerned [about] and conquered. It is a challenge but our [multi-disciplinary] team is willing to take it on and make it work.”
Lin said that the team has already submitted a letter of intent to the U.S. Department of Defense, “hopefully to secure some support for the blood-conversion application.”
Hien Huynh packs a bio-reactor column with carbon fiber bio-reactor sites that look like feathers. The sites are coated in enzyme before being packed into the column. | Phillip Sitter, Bond LSC
Enzymes in action
There are other potentially massive implications for the battlefields of the future.
“You can immobilize anti-microbial, anti-fungal and anti-inflammatory enzymes on a surface to use as a wound-healing patch,” Lin said, noting that such a patch could be used on the battlefield, as well as for cosmetic surgery recovery.
But the applications don’t stop there. Other uses could use enzymes to clean up TNT residues leeching out of unexploded ordinance like cluster bomblets, mortars, rocket-propelled grenades and landmines buried in the ground before the toxic residues contaminate groundwater.
Even within the confines of biofuels, there’s a strong military market. By 2020, the Navy wants 50 percent of its total energy consumption to come from alternative sources as opposed to petroleum-based fuels — part of a broader strategy to go green. The U.S. military in the near future wants to reduce the cost of its energy consumption and secure a stable domestic supply of energy.
According to the U.S. Government Accountability Office, from fiscal years 2007 to 2014, the Department of Defense bought 32 billion gallons of petroleum-based fuels at a cost of $107.2 billion.
Away from the military sphere, Lin detailed other uses for cheaper, higher quality enzymes. It could purify and recycle urine into clean water on space flights on for astronauts or convert waste into energy with an ammonia fuel cell that’s already available.
Mass-produced enzymes can be used for water treatment on earth, too. Pollutants like dioxin and herbicides like atrazine that contaminate soil can be bio-remediated in the same way that TNT residues can be cleaned up.
The food industry already uses enzymes as flavor removers to remove strong tastes from products like beer.
Minh Ma simulates the end result of a successful operation of the bio-reactor. She extracts and separates samples of real glucose product produced by the reactions in the column. A stronger yellow color in the solution indicates a higher concentration of glucose. | Phillip Sitter, Bond LSC
A bright bioreactor future
To call the team’s work revolutionary might be a bit premature.
There is a whole process ahead of them, including patent filing and university reviews, before the team can approach investors with the assurance their discoveries are legally protected. And, future investors will ultimately help determine how the technology is used.
But, Lin and the others might just have found themselves in the right place at the right time to make major breakthroughs, and that’s not all due to just advancements in technology.
“We have identified new directions and found a new niche to be competitive. I think the most important resource we have is people, and their brains,” Lin said.
Hsieh wrote that “To assemble a successful team is to put the right talent in the proper position and to inspire them to challenge themselves. I was lucky to come across so many young, talented students who are eager to learn and work hard for their bright future on MU’s campus.”
Hsinyeh Hsieh, a veterinary pathobiology research scientist in George Stewart’s Bond LSC lab, coordinates this project. Hsieh is an expert in gene fusion, enzyme production and characterization and enzymatic blood type conversion. Stewart is a medical bacteriologist, McKee Professor of Microbial Pathogenesis and chair of Veterinary Pathobiology at MU.
Lin works with Stewart and Hsieh to develop concepts, design prototypes and assemble the rest of the team — students and recent graduates — that optimizes the enzymatic reactions and the physical and chemical aspects of their bioreactor system. Minh Ma is a junior studying bio-chemistry. Mason Schellenberg studies bio-engineering, will be a senior and worked to find the most efficient platform design that the team’s 3-D printer could produce. Hien Huynh is a recent graduate who works on immobilizing enzymes. In addition to his MBA, Gupta also has a background that includes nano-technology, molecular engineering and financing. He concentrates on the feasibility and market potential of the team’s work.
Tiger Energy Solutions, LLC is the team’s industry partner — a spinoff startup from the team’s research project . Their focus in the development of a cheaper and higher quality method of converting cellulose into glucose for biofuels is to produce aviation biofuel. Tiger Energy serves as the interface between the team and industry while the team’s work is scaled-up for commercialization.
From left to right: Minh Ma, Hien Huynh and Sagar Gupta are three of the teams members, standing here in George Stewart’s lab. Ma is junior studying bio-chemistry. Huynh is a recent graduate who works on enzyme immobilization. Gupta is also a recent graduate — he obtained his MBA in May — and he focuses on the financial feasibility and market potential of the team’s work. | Phillip Sitter, Bond LSC
NASA, NIH-funded work seeks to understand bio-chemical mechanisms of life on Earth, and among the stars
Donald Burke-Agüero stands in his office in Bond LSC, holding a model of an RNA protein structure. Burke-Agüero studies the bio-chemical functions of RNA, and how those functions might be able to be artificially designed or replicated. | Phillip Sitter, Bond LSC
By Phillip Sitter | Bond LSC
Any child obsessed with Legos knows the fun of creation bound only by imagination and the size or variety of the blocks within their pile.
For some scientists, that spirit extends into adulthood, but instead of plastic parts they think about arranging blocks of nucleic acids.
Scientists may not be able to create dinosaurs, dragons or mythical sea creatures the way kids with Legos can. Through the manipulation of nucleic acid building blocks though, they may be better able to understand how the processes of life on Earth work, as well as out among the stars.
“I have a lot of fun asking what is possible,” said Donald Burke, a Bond Life Sciences Center investigator who spends his time researching the building blocks of life.
Burke said he has been interested in the origins of life for 40 years, and he has been associated with NASA for about 20 years.
NASA’s interest in understanding the origins of life is pretty straightforward. It wants to know what clues to look for on other worlds to figure out if those planets also support life.
Many of Burke’s previous discoveries at Bond LSC are funded by NASA’s exobiology and evolutionary biology program.
“No, I have not thought of an excuse to fly anything up there. I’ve tried to think ‘which of my experiments would make sense to do in a micro-gravity or zero gravity environment?’” he explained of the prospect of sending some of his work into orbit, with a wry smile.
But, there’s even more to understanding the building blocks of life than looking for bio-chemical signatures out among the stars. Knowing how these parts are put together allows scientists like Burke to understand the origins and processes of Earth’s biology, and, conceivably create chemical and biological processes or even organisms not found in nature in the near future.
A quadrillion arrangements of blocks, one arrangement at a time
“Many of the molecules of life are built from strings of amino acids, or nucleotides or other building blocks,” Burke explained. He also noted that these buildings blocks are not just strings, but fold up into three dimensional shapes.
RNA, or ribonucleic acid, stands out as an essential building blocks in the bio-chemical processes of life.
Put simply, RNA is a kind of molecular structure of nucleic acids similar to DNA (deoxyribonucleic acid) that comes in many combinations. These combinations are at the core of every cell, and play a role in coding, decoding, regulating and expressing the basic operating instructions for each cell — its genes.
The molecules we’re talking about are almost unimaginably small. In one test tube, Burke said there can be one quadrillion of them — that’s a one with 15 zeroes after it. Put another way, that’s roughly equivalent to the estimated number of ants that live on Earth.
Burke’s work focuses on the end goal of being able to artificially create original RNA combinations. In what’s known as experimental evolution, “the population of molecules in the tube is evolving as a result of us imposing experimental constraints upon it.”
This artificial synthesis of RNA molecules looks to create random sequences or variations on natural RNA to create new ones non-existent in nature. A second route aims to selectively choose molecules with certain properties, and use them to build altogether new combinations.
“Their string-like properties allow us to copy them, and make more copies, and make more copies, and make more copies. Their shape-like properties allow us to observe the bio-chemical behaviors they may have,” Burke explained how he and other scientists interact with RNA’s structure in the lab.
“I don’t think we know what those limitations are yet,” he said of the capabilities of RNA.
The motivation for wanting to be able to intentionally design RNA molecules is so that it “can do the things we want it to do under the conditions where we want it to do those things,” he explained of the process of the process of selecting RNA sequences for specific properties.
“I want the ones that will bind a tumor cell. I want the ones that will bind a viral protein. I want the ones that will catalyze useful chemical reactions.”
RNA’s path to the future following in biology’s footsteps
The National Institutes of Health and other organizations recognize that engineered forms of RNA have the potential to fight diseases, and they have funded Burke’s work.
He has studied RNA that instructs human cells on how to defend themselves from HIV and is now looking at other RNA that interferes with the proteins of the Ebola virus.
The expectation is that such therapeutics would work in conjunction with other treatments. In the future, they could be expanded to help fight other viruses, cancers and other diseases.
RNA could also be used to start, or catalyze, chemical reactions. As Burke explained, catalysts remove barriers to chemical reactions — “they don’t make things happen that wouldn’t otherwise happen, but they speed up the process.”
Synthetic RNA could be used to accelerate removal of toxins from soil or to get the bacteria in our guts to recognize cancerous tumor cells and kick-start an immune response.
But, the future of RNA research may soon reveal a few different Holy Grail moments on its horizon.
One such Holy Grail that Burke said will definitely happen will be observations consistent with the presence of life on other worlds, based on evidence like an atmosphere having certain chemical compositions.
Another likelihood could involve construction of a self-replicating, fully-artificial organism, either created from scratch or reverse-engineered from other organisms.
For those of you already anticipating the plot of a low-budget sci-fi thriller, Burke offered to assuage your fears.
“The notion of it escaping out in the world and taking over Los Angeles is [only] good 1950’s B-movie” material, because the conditions under which this artificial organism would survive would probably be difficult to maintain even in the controlled environment of lab, he said.
Instead of B-movie science, Burke explained that “really, I’m thinking about what kinds of chemistries we want to see take place, and then building the enzymes that would make it possible.”
“Biology has had a few billion years to work on this, but we’re just starting to figure it out.”
Donald Burke-Agüero is a professor of molecular microbiology and immunology and joint professor of biochemistry and biological engineering.
