Live long and prosper: healthy mitochondria, healthy motor neurons?

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Chris Lorson (front) and Mark Hannink (back) collaborate to study the role of mitochondria in motor neuron health, particularly in relation to spinal muscular atrophy, a neuromuscular disorder | photo by Jen Lu, Bond LSC

Chris Lorson, a professor of veterinary pathobiology, and Mark Hannink, a professor of biochemistry, want to find a new way to help motor neurons live a long and healthy life. Their question: what’s the relationship between motor neuron sruvival and a cellular component called mitochondria?

The two researchers at the Bond Life Sciences Center were awarded preliminary funding from the Bond LSC to pursue this question. Their findings could lead to new targets for therapies to treat a type of muscular dystrophy called spinal muscular atrophy, or SMA.

Spinal muscular atrophy, a genetic disease characterized by the death of motor neurons in the spinal cord, is caused by a mutation in the Survival Motor Neuron 1, or SMN1, gene. Patients with SMA develop muscle weakness and deterioration that spread inwards from the hands and feet, which progresses to interfere with mobility and breathing. The severity of symptoms and time of onset depend on how well a related gene is able to compensate for the lack of SMN1. As a result, treatment strategies usually focus on improving the activation of SMN1’s back-up gene.

Hannink and Lorson, however, are interested in a different pathway that is related to mitochondria dsyfunction.

Mitochondria are like the cell’s battery packs. Produced in the cell body, mitochondria migrate to the other end of the motor neuron to provide the energy to send electrochemical signals to recipient muscles and nerves. When mitochondria break down, the cell packs them into vacuoles that return to the cell body for recycling or removal.

“I saw a report that said that in SMA, there’s evidence for dysfunctional mitochondria in spinal motor neuron atrophy,” Hannink said. “My lab knows something about how mitochondria respond to stress.”

“There’s a lot of information out there that hints at it,” Lorson, an expert in SMA, said. “A number of the same responses you see in the stress pathway are also activated in neurodegeneration.”

To test their hypothesis, Hannink and Lorson plan to make motor neurons from pluripotent stem cells taken from people with and without SMA, and compare mitochondrial function and cell survival between the two groups. Then, they will test if a number of different genes that are known to be important for mitochondrial function will affect motor neuron health in both SMA and non-SMA derived cells.

“If you look at the tool chest of SMA therapeutics right now,” Lorson said, “you have a number of very obvious targets.”

Most approaches aim to boost the performance of the SMN or its back-up gene, but there are also options like neuroprotectants and skeletal muscle activators. Molecules that maintain healthy mitochondrial function could be another possibility.

“These are things that don’t worry about the state of the SMN gene and are targeting something in addition to, supplemental to or as an alternative to SMN,” Lorson said. “And that’s where this project would fall.”

This seed funding is one of seven awarded this year at the Bond Life Sciences Center. These awards, which range from $40,000 to $100,000 in funding, foster inter-laboratory collaboration and make possible the development of pilot projects.

Mizzou Epigenetics 2016

Five faculty speakers from five different universities, along with two trainees selected based on the merits of their poster abstracts, presented on current topics in epigenetics. The daylong symposium, titled Mizzou Epigenetics, took place on Wednesday, Nov. 9 at the Bond Life Sciences Center.

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Kenote speaker Dr. Jean-Pierre Issa talks about epigentic drift at the epigenetics symposium on Nov. 9th, 2016 | photo by Jen Lu, Bond LSC

Dr. Jean-Pierre Issa of Temple University, the keynote speaker, said he was a stickler for the definition of classical epigenetics: stable, long-term changes in gene expression. Textbook examples of epigenetics include X-inactivation, an irreversible process that happens at the beginning of gestation, and imprinting, where certain genes are not expressed based on their parental origins.

DNA methylation is one mechanism that cells use to control whether genes are activated. The presence of methyl tags—single carbons bonded to three hydrogen atoms—act like “off” switches when attached to a region of the gene called the promoter.

Enzymes that add or remove tags are normally busiest during the embryonic development. Cancer is the exception to the rule. According to Issa, cancer presents a “chaotic picture” where methyl tags get added to regions where they don’t belong, and removed from regions where they ought to be, resulting in epigenetic shift.

The greater the epigenetic shift, it seems, the greater the age of the cell. Regardless of whether you look at mice, monkeys or humans, Issa said, from a methylation perspective, “cancers look like very very very old cells.”

He also drew connections between epigenetic shift and other conditions related to aging. For example, specimens with chronic inflammation, infection or the introduction of a new microbiome to a germ-free body tended to show a higher than average amount of epigenetic shift as their cells age. Meanwhile, mice and monkeys who were exposed to calorie restriction tended to have lower amounts of epigenetic shift over time.

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Poster session from the epigenetics symposium held Nov. 9th, 2016 | photo by Jen Lu, Bond LSC

Other speakers who presented on epigenetics included:

  • Dr. Rick Pilsner, from the University of Massachusetts, on how paternal exposure to plasticizers affect sperm DNA methylation
  • Dr. Bob Schmitz, from the University of Georgia, on the identification of mechanisms behind spontaneous epigenetics variation
  • Dr. Zohreh Talebizadeh, from Children’s Mercy Hospital, on the genetics of autism
  • Dr. Andrew Yoo, from Washington University, on microRNA-mediated changes in chromatin during neuronal reprogramming of human fibroblasts

The event was sponsored by Mizzou Advantage, the School of Medicine, the College of Agriculture, Food & Natural Resources, the Bond Life Sciences Center and the Chancellor’s Distinguished Visitors Program.

 

 

 

 

 

 

 

 

The eyes have it

Bond LSC scientist works with MU eye surgeon to help people suffering from autoimmune-disease Sjögren’s syndrome

By Phillip Sitter | Bond LSC

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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

 

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.

Sjögren’s attacks the glands in our bodies that produce tears and saliva. Without tears and saliva, people suffer chronic dry mouth and eyes and are more susceptible to infections. Sjögren’s itself can also spread to other parts of the body, including the lungs, kidneys and digestive organs. An estimated four million people in the United States live with the disease, according to the Sjögren’s Syndrome Foundation.

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.

 

Gary Weisman was recently recognized for his career studying auto-immune responses with an award at an international conference in Seoul, Republic of Korea. Read more about Sjögren’s syndrome and Weisman’s work here.

This seed funding is one of seven awarded this year at the Bond Life Sciences Center. These awards, which range from $40,000 to $100,000 in funding, foster inter-laboratory collaboration and make possible the development of pilot projects. Read more about another Bond LSC seed funding grant-supported collaborative project here.

“Bucket” actor kicked Hollywood career, has enjoyed life in veterinary medicine

Dr. Peter Ostrum spoke at Bond LSC in celebration of World One Health Day

By Phillip Sitter |Bond LSC

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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

 

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.

 

Ostrum was invited to speak at Bond LSC in celebration of World One Health Day — an effort to spur collaboration between experts in human, animal and environmental health. Attendees at the lecture could purchase chocolate bars for three dollars, and five bars out of 200 available contained golden tickets that entitled winners to prize baskets. Proceeds went to MU’s Veterinary Health Center’s Barkley House project — a guest house for families of pets receiving treatment.

Close encounters of the plant protein kind

Bond LSC researchers David Mendoza (left) and Scott Peck (right) are collaborating to develop a new method for studying plant signaling pathways as they happen inside the cell. | photo by Jennifer Lu, Bond LSC

Bond LSC researchers David Mendoza (left) and Scott Peck (right) are collaborating to develop a new method for studying protein signaling pathways inside plant cells. | photo by Jennifer Lu, Bond LSC

By Jennifer Lu | Bond LSC

Sometimes, timing is everything.

That was the case in what led to a new collaboration between the Mendoza and Peck laboratories. The two researchers were recently awarded $48,250 in seed money from the Bond Life Sciences Center to adapt a new technology to the study of signaling pathways in plant cells.

David Mendoza, a Bond LSC researcher and assistant professor of plant sciences who is interested in nutrient uptake in plants, got the idea for the project when he attended the Trace Elements in Biology and Medicine conference in June. There, he kept hearing about an enzyme called BioID used to identify protein interactions in mammalian cells.

“In plants, we have a hard time figuring out how proteins interact with each another to transfer information within the cell,” Mendoza said. BioID could be the key.

BioID works like a spy slipping a small tracker into the coat pocket of every person it encounters, but instead of a tracker, BioID transfers a unique molecular tag onto every protein that comes near. It’s a speedy process, no matter how brief the interaction between BioID and the incoming protein. But once the proteins are tagged, they can be rounded up and identified later, even if they’ve moved elsewhere in the cell.

Scientists can study which proteins interact with their protein of interest by linking BioID to their protein. This lets them track the signals being communicated to and through their protein without disrupting what’s happening inside the cell.

Although BioID has exclusively been used in animal systems, Mendoza talked to the scientist behind BioID to see if it could be used in plants.

Incidentally, BioID has been publicly available for several years but the enzyme was impracticable for plant experiments. It needed a lot of raw material on hand before it could start tagging proteins, much more material than what is normally found within plant cells.

However, research on a more suitable candidate called BioID2 was published just months before the conference. Unlike its predecessor, BioID2 required very little starting material to function in plants.

“Like a lot of things,” Mendoza said, “timing was key.”

When he approached Scott Peck, a colleague at the Bond LSC and professor of biochemistry specializing in plant proteomics, with the news, Peck saw immediate applications for BioID2.

With currently available methods, plant scientists have to look at protein interactions in artificial environments, such as in a test tube or in yeast systems. A real-time protein-tagging method would allow plant scientists to observe signaling pathways in their native environment–the cell–under a variety of conditions.

“It allows the contextual information within the plant to still be present,” Peck said.

For example, with BioID2 the Peck lab, which studies plant resistance to bacteria, could watch how incoming stimuli such as plant pathogens or stress from drought affect overall protein-to-protein interactions within plants, compare these protein interactions across different cell types, or even discover previously unknown protein interactions, he said.

“You know you have a good idea when the other person gets excited right away,” Mendoza said.

Peck also had a suitable model handy in which they could test BioID2 at work, but the two researchers first had to make sure plant cells could produce functional BioID2. Mission accomplished, the next step is to make plants produce BioID2 that is linked to their protein of interest.

“The nice part of this seed grant is it lets us get a jump on some new technology to develop here,” Peck said.

Using BioID2 in plants is an interesting and novel idea, Mendoza said. “For me, that’s enough to try.”

 

This seed funding is one of seven awarded this year at the Bond Life Sciences Center. These awards, which range from $40,000 to $100,000 in funding, foster inter-laboratory collaboration and make possible the development of pilot projects.

 

The seeds of progress

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

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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.

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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

Expert Comment: How science made the “three-person” baby possible

Family silhouette

Stock image via iStock

By Jennifer Lu | Bond LSC
A new in-vitro fertilization technique that uses genetic material from three persons made the news last week following the announcement of the successful birth of a now five-month-old baby boy. The process allowed the mother, who had a rare mitochondrial disease known as Leigh Syndrome, to have a child without passing her faulty mitochondrial genes.  The nucleus from the mother’s egg was inserted into a prepared donor egg that had healthy mitochondria to make a cybrid, or cytoplasmic hybrid, egg that was then fertilized.

We asked Mark Hannink, Bond LSC scientist and professor of biochemistry, who studies oxidative stress in mitochondria, what this all means.

This is not the first “three-person” baby. Why is this technique new?

It’s another way of getting a healthy mitochondrial genome into the baby.

You have to bring together three parts: nuclear DNA from the mom, nuclear DNA from the dad, and mitochondrial DNA from the donor. The question is whether you bring together the mitochondrial DNA from the donor and the nuclear DNA from the mom first, and then add the DNA from the father.  The other way is making the diploid nucleus first (combining the mother and father’s DNA,) and then putting that into the donor.

Wait, so we have two types of DNA in our cells?

Way way early in evolution, a bacteria got together with a cell that had a nucleus, and they decided to cooperate. Over time, many of the genes that were originally in that bacteria’s genome moved to the nuclear genome. But some of them haven’t. The mitochondrial genome in humans has some 37 genes. But the mitochondria itself has about 1000 different proteins so those other proteins are encoded by the nuclear genome. Together, those proteins work together to form healthy mitochondria that, among other important jobs, provide energy for the cell.

What makes this procedure controversial?

Any time you manipulate the sperm and the egg, there is a chance that you will generate subtle alterations which result in defects in the child during development or after it’s born. Even in vitro fertilization, which has been shown to be effective and works, has a higher rate of diseases associated with it.

Now you’re doing a whole set of complicated manipulations before you get to IVF….You take out the existing nucleus from the donor. You put in the nuclear genome from the mother. And you hope that it all comes back together and then you do the IVF…. Any time you do a manipulation like that, you may cause subtle mistakes that you’re not aware of.

Then there’s the other concern. The mitochondrial proteins encoded by the nuclear genomes and the mitochondrial proteins encoded by the mitochondrial genomes  have to work together to form functional mitochondria that make energy, regulate signaling, regulate calcium, regulate nerve transmission and cell survival.

Your nuclear genes have been interacting with your mitochondrial genes throughout your entire natural lineage, so they’ve coevolved to work together. If, let’s say, there’s a minor mistake made in one of the nuclear genes that encodes a mitochondrial protein in your grandma, you might still get selected for a compensatory mutation in the mitochondrial genome that would still allow a functional mitochondria to be made….But the nuclear genome of one person may not be compatible with the mitochondrial genome of another person even though that mitochondrial genome is normal and works just fine in the context of that person’s nuclear genome. But there’s no way to know that in advance. So you may end up with a healthy baby, or you may end up with a baby in which the nuclear genome and the mitochondrial genome are not compatible.

An enzymatic future

Inter-departmental MU team aims to improve enzyme use and recovery for spectrum of industrial, medical and military applications

By Phillip Sitter | Bond LSC

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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.

 

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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.

 

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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.”

 

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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.

 

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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.

 

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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

Saturday Morning Science returns to Bond LSC

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This past weekend not only ushered in Mizzou’s first home game of the season, but the return of Saturday Morning Science. The weekly lecture series connects the Columbia community with MU scientists and their research, from bio-engineering to volcanology to anthropology and linguistics.

Elizabeth G. Loboa, dean of the College of Engineering, kicked off the semester with her talk on tissue engineering in the age of drug-resistant bacteria.

Tissue engineering is about turning cells into tissues and organs, for example, fat-derived stem cells into muscle, bone and cartilage. The tissues take shape on tiny scaffolds that are bio-compatible and biodegradable.

The Loboa lab does this, but they’ve added an extra layer to their research: Loboa’s scaffolds also act as pipelines that deliver wound-healing and anti-bacterial compounds to cells as they grow into tissue. The idea is to reduce infection, inflammation and scarring as the wound heals.

“We’re trying to kill these bacteria while helping these stem cells become the cells we want to create,” Loboa said, about her research at the University of North Carolina-Chapel Hill and North Carolina State University.

Using a process called electrospinning, Loboa’s group makes scaffolds shaped like porous fibers, sheaths, or hollow sheaths. Depending on their structure, these scaffolds act like faucet taps that control the rate and timing at which anti-bacterial compounds are released.

“I look at our fibers as delivery platforms,” Loboa said.

Saturday Morning Science takes place 10:30 a.m. Saturday at the Bond LSC’s Monsanto Auditorium. Coffee and bagels are available preceding the talks. This semester’s schedule is as follows:

9/17: Carolyn Orbann, Assistant Teaching Professor, Department of Health Sciences, “Historical Epidemics, Novel Techniques: Using Historical and Ethnographic Materials to Build Computer Simulation Models”

9/24: Michael Marlo: Associate Professor of English, “Documenting linguistic diversity: a view from the East African Great Lakes”

10/1: Steve Keller, Associate Professor of Chemistry, “The 20 Greatest Hits in Science…In an Hour”

10/8: Manuel Leal, Associate Professor of Biological Sciences, “Are Lizards Smarter Than Those Who Study them?”

10/15: Stephan Kanne: Executive Director and Associate Professor, Thompson Center for Autism & Neurodevelopmental Disorders, “What Do We Look For When We Diagnose Autism?”

10/29: Libby Cowgill, Assistant Professor Anthropology, “Fitness for the Ages: How to Lift Like a Neanderthal?”

11/5: Arianna Soldati, Ph.D. Candidate, Department of Geological Sciences, “Living in a Viscous World: A Volcanologist’s Perspective”

11/12: Frank Schmidt and Gavin Conant, Professor of Biochemistry (Schmidt); Associate Professor of Bioinformatics, Department of Animal Science (Conant), “Networks in Biology and Beyond”

12/3: Elizabeth King, Assistant Professor, Division of Biological Sciences, “What’s the Best Way to Divide up the Pie: The Price of Long Life”

Building blocks of life in the lab could revolutionize life for us all

NASA, NIH-funded work seeks to understand bio-chemical mechanisms of life on Earth, and among the stars
By Phillip Sitter | Bond LSC

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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

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.