Research

Why self-defense turns self-attack

By Jinghong Chen | Bond Life Sciences Center

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Mahmoud Khalafalla, a Ph.D. student at Weisman’s lab, is isolating RNA from salivary glands of Sjögren’s syndrome mouse model to look for the expression of pro-inflammatory genes. | photo by Jinghong Chen, Bond LSC

Our immune system is often the key to our health. Everyday, it works to protect us from foreign invaders such as bacteria and virus, but what happens when it attacks our own tissues?

Gary Weisman, a Curator’s Distinguished Professor of Biochemistry at the Bond Life Sciences Center, is working to advance our understanding of the mechanisms behind immune system function and autoimmune diseases such as Sjögren’s syndrome.

In our immune system, B cells are responsible for producing antibodies to recognize foreign invaders. However, in many autoimmune diseases, B cells produce autoantibodies that recognize our own proteins, causing inflammation and tissue damage. In Sjögren’s syndrome (SS), they attack the glands that produce saliva and tears.

Patients with SS often suffer chronic dry eyes and dry mouth, which might lead to bacterial infection, difficulties in swallowing and speech.

“The symptoms decrease the quality of life rather than the length of life,” said Lucas Woods, research lab manager in Weisman’s lab.

Although SS patients are at higher risk of developing lymphoma cancers and other concurrent autoimmune diseases that may increase mortality, Woods further explained.

According to the Sjögren’s Syndrome Foundation, there are an estimated four million people living with the disease in the U.S. For unknown reasons, 90 percent of them are female.

Yet, current clinical treatments only reduce symptoms by using artificial saliva and tears or cholinergic agents to promote fluid secretion, but there is no approved treatment to reduce the inflammation of the glands themselves. This is the focus of Weisman’s lab.

Sensor of danger

There are 15 different types of nucleotide receptors in humans that regulate numerous cell processes from inflammatory responses to tissue regeneration. Those receptors are stimulated by nucleotides such as ATP. In the past three and a half years, Mahmoud Khalafalla, a Ph.D. student in Weisman’s lab, has focused on one of them in particular – the P2X7 receptor.

Previous studies show increased P2X7 expression in salivary glands from SS patients, as compared to healthy individuals. To understand the reasons behind this, Weisman’s lab used genetically modified mice that develop disease traits similar to SS patients.

In this mouse model, Sjögren’s-like disease occurs when the immune cells invade salivary glands and damage the tissue, leading to decreased saliva production. The invasion of immune cells is triggered by proinflammatory cytokines, a type of signaling molecule that promotes the recruitment of immune cells to the inflamed areas.

But what induces those cytokines?

Weisman’s lab tries to piece together the answer. For the first time, they found that the P2X7 receptor is responsible for the release of these proinflammatory molecules from salivary gland epithelial cells.

To function, most cell-surface receptors require ligands that bind to the receptor to induce cellular responses. The ligand for the P2X7 receptor is ATP – the “energy currency inside of cells.” P2X7 receptors are activated when high concentrations of ATP are released to the outside of the cells, which typically occurs when the cells are injured during inflammation.

“P2X7 receptors [act like] the sensor of danger,” Khalafalla said.

After identifying the role of the P2X7 receptor, the lab then asked: if we stop its activation, what would happen?

Using a drug that inhibits P2X7 receptor activation, they blocked the receptor in their SS mouse model to determine its effect on the development of autoimmune disease. Interestingly, saliva secretion was restored when the P2X7 receptor is blocked while the levels of invading immune cells in salivary glands were dramatically reduced.

“This gives us the thought that [blockade of the] P2X7 receptor is really a promising strategy to reduce salivary inflammation. This may not only relate to Sjögren’s syndrome, but to other autoimmune diseases as well,” Khalafalla said.

Our receptor

Another similar receptor that plays a role in autoimmune diseases is the P2Y2 receptor, which has been referred to as “our receptor” by Weisman’s lab.

As one of the researchers who proved the existence of this receptor, Weisman has spent most of his career studying it.

One of his research projects investigating P2Y2 receptors in human disease recently gained a grant extension for another five years from the National Institutes of Health. The lab found that in a mouse model of SS, similar to the P2X7 receptor, the expression of P2Y2 receptors was increased in both the salivary gland epithelial cells and immune cells.

Furthermore, after they knocked out the P2Y2 receptor in the SS mouse model by breeding them with genetically-modified P2Y2 receptor knockout mice, the inflammation of salivary glands was dramatically reduced.

“The very next step is that we are going to isolate these immune cells out of the diseased mouse salivary glands, and characterize what kinds of cells they are. We want to know exactly which ones are controlling the development of autoimmune diseases, and how P2Y2 receptors and nucleotides like ATP in general are contributing to the diseases,” Woods said.

 

Gary Weisman is a Curator’s Distinguished Professor of Biochemistry at the Bond Life Sciences Center. His research focuses on the relationship between inflammatory diseases and nucleotide receptors. He currently works on a collaborative research project with Dr. Carisa Petris, an eye surgeon at the MU Hospital, to understand the mechanism of how Sjögren’s syndrome damages the tear-secreting lacrimal glands in mice.

 

Planting a seed for sciences

Jinghong Chen | Bond Life Sciences Center

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Plants on the left grow with rhizobia bacteria, one type of fixing nitrogen bacteria, in the greenhouse, while the plants on the right grow without the bacteria. | photo by Jinghong Chen, Bond LSC

Since eight years old, Beverly Agtuca knew she wanted to be a scientist.

A trip to Philippines changed Agtuca, an American-born Filipino, and inspired her passion on plants.

“My grandma always told me to work in the field all day so that they can have enough food for us to eat,” Agtuca said. “The life [in Philippines] is so different from here…I want to not just provide food but be that scientist trying to figuring something out, and hopefully saving the world.”

Agtuca is on her way to her dream. She is now a third year doctoral student in Gary Stacey’s lab at Bond Life Sciences Center with a focus on nitrogen-fixing bacteria.

Although she has been involved in research since high school, Agtuca recently faced a new challenge of telling people about her work. The Preparing Tomorrow’s Leaders of Science class tasked her with making a 90-second video to explain her two-year study to the general public.

Her team, “The A Team,” chose to go with the benefits of having nitrogen-fixing bacteria.

For decades, people have been adding nitrogen fertilizers to plants to improve yields, but this can lead to pollution in water systems and ecosystems. Scientists need to enhance plant productivity to meet a huge food demand by the year of 2050.

One little bacteria might make this possible and save the world. Rhizobia, a type of natural bacteria in soil, are able to fix nitrogen via biological nitrogen fixation. These bacteria can convert nitrogen gas into ammonia as a plant nutrient source, while the plants give all the carbon sources back to the bacteria.

“It is like a walky-talky,” Agtuca said. “They are communicating with each other.”

Yet before speaking to the public, Agtuca needs to explain the plant-bacteria interaction to her teammates. Students less well versed in science like Jessica Kaiser, a strategic communication student, thinks of science differently.

“The biggest issue we ran into is jargon, like basic science words that [my teammates] are so comfortable with,” Kaiser said. “We need to focus on what people care about instead of the technical sides, to focus on why it matters to anybody rather than just to a science person.”

Within two weeks, they produced the video “Good Microbes: reducing pollution one farm at a time.” Along with two other teams, their videos will be commented and judged by representatives from Monsanto.

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“The A Team” stands together at Bond Life Sciences Center. From left to right: Jessica Kaiser, Sven Nelson, Anna Glowinski, Eleni Galata and Beverly Agtuca. | photo by Jinghong Chen, Bond LSC

The 90-second video is just a glimpse of Agtuca’s study. In the last two years she has been focusing on the use of a new technique — laser ablation electrospray ionization mass spectrometry (LAESI-MS) — that does in situ metabolic profiling of tissues. The lab is using LAESI-MS to investigate the metabolites in a well-characterized model plant-rhizobium system, specifically nitrogen-fixing soybean nodules resulting from root infection by the symbiotic bacterium Bradyrhizobium japonicum.

This work includes a huge collaboration that was developed through a Department of Energy (DOE) grant involving the George Washington University, Washington D.C. and the Environmental Molecular Science Laboratory (EMSL), Pacific Northwest National Laboratory, Richland, WA.

LAESI-MS works like a superhero’s laser-like beams. You first aim the laser on the sample, which then heats it and causes neutral particles to be released into the air. This plume of neutrals is then captured and ionized by the electrospray, and finally analyzed by the spectrometer to figure out the exactly what metabolites in nodules are involved in biological nitrogen fixation.

“It takes about three seconds to analyze one sample using this LAESI-MS technique,” Agtuca said. Other metabolic techniques require extensive pre-treatment of the sample before analysis.

By analyzing the data collected via LAESI-MS, the lab is able to confirm that future plant studies could apply this new approach to understand the interactions between plant and bacteria.

Agtuca’s research is a long way from her first experiences with plants. She still remembers the moment she found her plants in her own garden died. She was less than 10 years old, yet devoted to taking care of her plants with water and fertilizers.

“I was really sad. I could not get my tomatoes, peppers and eggplants to live.…That makes me think that I want to answer why they didn’t grow,” Agtuca said.

More than ever, her future is helping her answer those question for herself.

 

Gary Stacey is a Bond LSC investigator and MU curators’ professor of plant science and MSMC endowed professor of soybean biotechnology. Read more here about Stacey lab.

Sven Nelson is a USDA/ARS postdoctoral research scientist at the University of Missouri. Anna Glowinski is a Ph.D. student in the USDA/ARS lab. Jessica Kaiser is a graduate student in strategic communication. Eleni Galata works as the team mentor and she is a Ph.D. student in agricultural and applied economics at MU.

Art of balance

Jinghong Chen | Bond Life Sciences Center

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Vinit Shanbhag mixes the CRISPR plasmid DNA with cells. The lab will test whether the gene of interest has been knocked out of the cells later. | photo by Jinghong Chen, Bond LSC

It might be strange to say, but in a way the Australian soil led scientist Michael Petris to where he is now.

In certain areas of Australia, soils suffer from extremely low level of copper bioavailability, resulting in poor growth and neurological problems on sheep.

Petris, a Bond LSC investigator and professor of biochemistry who was born in Australia, now spends his time studying how copper, an essential mineral in human body, works in cells to build and maintain essential functions.

Recently published work from his lab focuses on how the ATP7A protein, one of the major proteins, cycles within the cell.

“Copper is solely acquired from diet. The absorption of copper from the intestine in the blood needs ATP7A,” Vinit Shanbhag, a Ph.D. biochemistry student at Petris’ lab and an author of the study, said. “It transports copper to different copper dependent enzymes and exports free copper from the cell to the outside.”

After exporting copper at the cell membrane, ATP7A needs to come back to its steady-state location within the Golgi apparatus of cells – via a process called retrograde trafficking. But one question baffled scientists: what are the key elements that lead ATP7A coming back?

Back in the late 90s, Petris discovered the importance of one single di-leucine in retrograde trafficking of ATP7A. For those of you wondering, leucine is an amino acid that forms the building blocks of proteins like ATP7A, while di-leucine consists of two of them connected via a peptide bond.

His team wished to identify other signals for retrograde trafficking, but one technical hurdle stood in the way— the ATP7A gene is unstable when grown in bacterial plasmids, the traditional way of amplifying genes in the lab.

Commercial DNA synthesis was the answer. This method could create artificial genes in the laboratory.

“We reasoned that if we introduced enough silent mutations into a DNA sequence, we could avoid or change the region of instability in the native sequence without affecting the encoded protein,” Petris said.

To stabilize the gene, they changed more than 1,000 nucleotides within a 3,000 nucleotides segment, and thus solving the problem of instability of the ATP7A gene. In doing so, they subsequently found that in fact multiple di-leucines that are required for retrograde trafficking of ATP7A. This approach could be used by other laboratories whose gene of interest is also unstable.

An overlooked mineral

“If you ask [people], is it important to understand iron nutrition? Is it important to understand calcium nutrition? Most people would say of course! … But, perhaps you would not get the same answer for copper, despite the fact there is a little dispute that copper is important,” Petris said.

As an essential micronutrient, copper performs central functions to develop and maintain human skin, bones, brains and other organs.

“If you don’t have enough copper in your body, you cannot use oxygen to make energy,” Petris said. “If you don’t have copper, you would not survive.”

Pregnant women who carry a mutated ATP7A gene on their X chromosome can pass it on to their children in the form of Menkes disease.

Menkes disease is a genetic disorder that results in poor uptake and distribution of copper to cells. The incidence of this disease is estimated to be one in 100,000 newborns, according to U.S. National Library of Medicine.

Infants with Menkes disease typically begin to develop symptoms during infancy and rarely live past the first few years of life. Abnormally high accumulation of copper in kidneys and low-level accumulation in the liver and brain, cause visible symptoms like sparse hair, loose skin and failure to grow.

Despite copper’s importance, it also can be a potentially toxic nutrient.

“Copper deficiency can be a problem but too much copper is also a problem. There should be a balance,” Shanbhag explained.

The liver normally stores excessive copper and excretes it into bile to release it out of the body. Yet people with genetic disorders that preventing copper excretion might suffer Wilson’s disease, leading to life-threatening organ damage.

Shanbhag said people with Wilson’s disease accumulate toxic amounts of copper in liver and other organs, causing Kayser–Fleischer rings that encircle the pigmented regions of the eye, a hue caused by copper deposits in the cornea.

Its clinical consequences differ from chornic liver failure to neurological sysmptoms like tremors, dystonia, ataxia and cognitive deteriortation.

About one in 30,000 people have Wilson disease, according to National Institute of Diabetes and Digestive and Kidney Diseases.

Starving tumors

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Vinit Shanbhag mixes the CRISPR DNA with mammalian cells to specifically delete a gene in these cells in lab hood. | photo by Jinghong Chen, Bond LSC

In 2013, Petris’ lab published the first direct evidence suggesting ATP7A is essential for the dietary absorption of copper. Since then they have dug deeper into this copper transporter, and his lab now sets their sights on a greater enemy of human health — cancer.

Tumor growth requires access to large amounts of nutrients. Without an adequate supply of oxygen and nutrients, tumors fail to grow and survive. Scientists have identified that by preventing access to nutrients—for example by blocking the growth of new blood vessels—they could starve the tumor of nutrients.

Copper is a key nutrient for tumor growth. With the new-introduced system CRISPR-Cas9 — a genome editing tool to knock out specfic genes — his lab has explored how to exploit understanding of copper metabolic pathways to withhold copper from cancer cells.

“Copper starvation might be a good approach as an anti-cancer strategy,” Petris said.

Weapon of the immune system

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Michael Petris, a professor of biochemistry at MU, stands with his lab. From left to right: Vinit Shanbhag, Nikita Gudekar, Michael Petris, Kimberly Jasmer-McDonald, Aslam Khan. | photo by Jinghong Chen, Bond LSC

Currently, four members study in Petris’ lab to tackle the relationship between copper and various diseases. Petris plans to expand his research to another area: the role of copper in innate immunity against bacterial pathogens.

This is the topic of Petris’ next grant. Nutritional immunity, which describes how the mammalian host withholds nutrients from the invading bacteria during infection, is very well-described for iron and zinc.

Yet copper performs differently.

During infection, the level of copper in blood actually goes up instead of going down. The immune system concentrates copper at sites of infection and within regions where the bacteria are engulfed.

“We speculate that copper is being used as weapon by the host to kill the bacteria,” Petris said. “That is the area we are trying to develop further.”

 

Michael Petris is a Bond LSC investigator and a professor of biochemistry. The study “Multiple di-leucines in the ATP7A copper transporter are required for retrograde trafficking to the trans-Golgi network” was published by The Royal Society of Chemistry in September 2016.

 

 

Chemical persuasion

Scientists prove parasite mimics key plant peptide to feed off roots
By Roger Meissen | Bond LSC

A nematode (the oblong object on the upper left) activates the vascular stem cell pathway in the developing nematode feeding site (syncytium) on a plant root. | contributed by Melissa Mitchum

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.

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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.”

Mitchum is a Bond LSC investigator and an associate professor of Plant Sciences in the College of Agriculture, Food and Natural Resources. The study Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation” recently was published by the journal PLOS Pathogens in February 2017.

Cornelison receives highest honor from White House

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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.

If you’d rather hear about it from her mouth, listen to Cornelison’s interview on KFRU Wednesday morning.

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.

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.

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.