A Return on Investment Understanding responses to environmental changes and improving the health and lifespan of ecosystems and people are among the potential benefits of systems biology.

As ORNL researchers seek answers to life sciences' persistent questions, some are struck by how systems biology applies to life on a variety of scales. Rich Norby, a physiological ecologist in ORNL's Environmental Sciences Division (ESD), is one of those scientists. At the annual American Association for the Advancement of Science meeting in 2004 in Seattle, Norby heard LeRoy Hood, pioneer of the DNA sequencer, "talk about systems biology in a way that made me think, 'That's what I do.'"

The effects of elevated levels of carbon dioxide on a forest are being determined at this ORNL sweetgum stand.

Hood studies living cells and immunology while Norby focuses on forests and global change.

"Dr. Hood was talking about DNA, proteins, and underlying networks in a live cell and I talked as a panelist at the AAAS conference about trees, roots, microbes, air temperature, and carbon dioxide in a small forest ecosystem. There's a huge gulf in scale there, but I think the basic principles are still the same. In both cases the objective is to identify components of the system and analyze their interactions to reveal emergent properties of the system as a whole."

Systems biology research at ORNL has applications at different scales, as shown in a few examples in this article. The research described relates to ecosystems and global change, energy production, bioremediation, and human health.

Ecosystems and Global Change

Norby is one of many ESD researchers who studied individual seedlings in chambers to determine how their physiology was affected by exposure to one pollutant such as ozone. The scientists studied a simple, controlled system that was a long way from today's endpoint of a global forest.

FACE experiment. After a number of years experimenting with small tree seedlings in growth chambers and saplings in field chambers, Norby became involved in the much larger, more complex but controlled Free-Air Carbon Dioxide (CO₂) Enrichment (FACE) experiment, a Department of Energy user facility at ORNL. The purpose of this experiment, now in its seventh year, has been to help DOE understand more completely the consequences of elevated atmospheric CO₂. Whereas in the past, the focus of experiments was on individual components of the system, the current focus is on integration of component organisms and processes into a system-level response.

In the FACE experiment, tons of CO₂ are pumped into plots of sweetgum trees, so that the concentration of CO₂ in the tree stand is almost 50% higher than the ambient level. Norby and his colleagues then compare the responses of the forest ecosystems exposed to elevated levels of CO₂ with the responses of the forest in ambient air.

Each year, ORNL staff measure net primary productivity (NPP), or the total amount of carbon fixed into organic matter in the ecosystem, above and below ground. They have found that the NPP of the plots exposed to elevated CO₂ was enhanced by about 23% annually over six years. But how that carbon was allocated changed over time.

"In the first year," Norby says, "the trees exposed to elevated CO₂ had a 35% increase in wood production—above ground trunks and branches. In the second year it was 15% and then in the past four years we measured only a 5 to 7% annual increase in wood production.

A new experiment at ORNL is measuring the growth of roots of seven species of grasses, herbs, and weeds.

Instead, the NPP shows up in fine root production. Our technician Joanne Ledford has measured and documented increases in production of fine roots over six years, which is a significant and unprecedented response."

Fine roots are an important component of a forest system because they regulate the cycling of carbon, water, and essential nutrients. Unfortunately, for DOE's interest in removing CO₂ from the air, fine roots do not store carbon for much more than a year, unlike the wood in tree trunks, which can store carbon for decades.

"Fine roots have a short life, and when they die, microbes digest them to get energy" Norby says. "Much of the carbon in fine roots is returned to the atmosphere as CO₂. That's not a good story if your interest is net removal of CO₂ from the atmosphere."

However, because considerable carbon is moving through the soil system, an opportunity exists for some of it to be trapped in longer-lived soil organic matter pools. One important research challenge at the FACE facility is to quantify the amount of carbon that remains in or gains access to these pools. Researchers are seeing indications that, compared with the ambient plots, the FACE plots show an increase in 'protected' carbon—soil carbon that will not decompose right away.

"Through understanding the interrelationships between components within the system and how they work together to get an integrated response, we will have a stronger basis from which to project the responses of forests to global change," Norby says. "Systems biology applied at a large scale can help DOE better understand biological impacts of atmospheric and climatic change."

Underground activities. The FACE experiment demonstrated the importance of whole-system analysis, including responses below the ground. The experimental system, however, is very simple—one dominant species (sweetgum) and one environmental change (CO₂).

A new study at ORNL is investigating the responses of a more complex community to multiple environmental change factors. Near the FACE facility is the Old-Field Community, Climate, and Atmospheric Manipulation (OCCAM) experiment, a new joint project between ORNL and the University of Tennessee. ESD's Steve DiFazio is the principal investigator of an internally funded Laboratory Directed Research and Development Program project on ecosystem genomics that makes use of the OCCAM experiment.

DiFazio is testing genomics in a systems-level approach on abandoned land to determine the amount of growth of roots of seven different species of grasses, herbs, and weeds subjected to three different treatments— ambient and elevated atmospheric CO₂, ambient and increased temperature (higher by 3°C), and ambient and decreased soil moisture. The ORNL researchers are interested in how the different combinations of treatments change the composition of the plant community, and how this transformation alters ecosystem responses.

The different species are not difficult to distinguish above ground, but observing community composition changes below ground presents a challenge. "In the OCCAM plots, you cannot tell which species the intertwined roots belong to because there is no easy way to distinguish, based on a root's appearance, which of the aboveground species it came from," DiFazio says.

To solve the problem, he and his colleagues are taking a novel approach based on the new field of ecosystem genomics. The approach views ecosystems not as a web of habitats for a variety of species but rather as a stage on which genes, proteins, and living cells interact.

"We are investigating a method in which characteristic DNA from the roots of the individual plants is used to identify the species," DiFazio says. "We will take a plug of soil, grind it up and, using our DNA-based technique, determine the relative abundance of each species present. By comparing the different treatments, we will know more about how different species respond to changes in carbon dioxide levels, temperature, and soil moisture."

Another aspect of the project is an assessment of the indirect effects of the plant responses to ecosystem perturbations. For example, researchers are uncertain how microbial populations will respond to increased productivity and competitiveness of individual plant species. Also, if the plants have a higher rate of photosynthesis under changing conditions, will microbial populations that fix nitrogen for plants adapt fast enough to meet plants' nutritional needs?

To address these questions, ESD's Jizhong Zhou is using microarrays to assess the responses of microbial populations in this experiment. His team will determine how the microbial populations change in response to the treatments, and whether changes in plant populations are reflected in the composition and functioning of the microbial communities. Detailed, integrated studies such as these are required to achieve a systems level understanding of the effects of climatic change.

Improving the efficiency of biological hydrogen production is an ORNL research goal.

Energy Production

A systems biology approach to understanding a protein complex could unlock a source of energy, according to Brian Davison, director of ORNL's Life Sciences Division (LSD). One complex is hydrogenase, an enzyme that can take electrons and protons from other enzymes and compounds and use them to release hydrogen for use in power-producing fuel cells. "This is a way to produce energy in the form of hydrogen using a biological system," Davison adds.

Systems biology may enable researchers to find smarter ways to harness the ability of microbes to produce hydrogen under certain limited conditions. "Everything in the life of some microorganisms has tended to limit their ability to produce hydrogen," Davison says. "Some microorganisms, we think, use hydrogenases to deal with excess energy and prevent the buildup of protons and other free radicals floating around inside their cells. We want the process to produce hydrogen all the time, so we are studying the enzyme's active site and its partners to see how hydrogen is generated."

Scientists have found evidence that when hydrogenase is used to produce hydrogen from water, the oxygen that is formed can hurt the hydrogenase. Research at ORNL and elsewhere seeks to solve this problem to improve the efficiency of biological hydrogen production.

"When we fully understand natural systems," Davison says, "I believe we can coax them to do our bidding in a smarter, better way than in past biological approaches where success came from doing experiments and getting desired results by accident."

Bioremediation

Bioremediation is the use of microorganisms to eliminate, contain, or reduce the concentration of contaminants in soil and water. One of DOE's missions is to use bioremediation to clean up waste sites or immobilize wastes so they do not migrate.

Computer visualization of a Shewanella bacterial protein.

Using a systems biology approach, ORNL researchers have been helping DOE identify bacteria that can immobilize and make less bioavailable any compounds containing radionuclides, such as uranium and strontium, and toxic metals, such as chromium, technetium, and mercury.

ORNL researchers are helping DOE search for bacteria that show great promise for changing uranium compounds from a soluble to an insoluble chemical state. Such transformed uranium compounds are more likely to stay put in soil or sediments rather than dissolve in groundwater and flow off-site. If scientists can find a bacterium that is especially effective at reducing uranium compounds, the discovery could well meet DOE's environmental goals and save millions of dollars in potential cleanup costs.

Researchers could characterize the capabilities of this new bacterium and try to identify the genes that enable the reduction of each uranium atom by donating two electrons. Such an interaction with metal enables the bacterium to extract energy from carbon.

Jizhong Zhou, Dorothea Thompson, and others on Zhou's team have been studying the bacterium Shewanella oneidensis strain MR-1, whose genome was completely sequenced by The Institute for Genomic Research (TIGR) and annotated by TIGR as well as LSD's Frank Larimer and others. Shewanella is able to make uranium less soluble in the laboratory, but further research is needed to determine how well the bacterium responds at a toxic waste site.

"Because of DOE's mission, we are trying to understand how Shewanella responds to environmental stresses such as high and low pH, high temperature, high salt, and metal toxicity," Thompson says "DOE wishes to know how Shewanella transforms, detoxifies, and reduces metals in the environment. DOE also seeks to understand the relationship to environmental stresses and which stresses make the process less effective in bioremediation."

DNA microarray technology allows Zhou's team to place an array of at least 20,000 DNA probes, each corresponding to a single microbial gene, on a glass microscope slide. They can look at the global expression—all the responses of genes at the messenger RNA level—in a bacterial cell exposed to a toxic metal such as strontium or chromium. Then, by interacting with the mass spectrometry group at ORNL, they can determine if the switched-on, or up-regulated, genes produce corresponding increases in the encoded protein products.

Working with Steve Brown, an ESD postdoctoral researcher, Thompson found in some cases that a limited set of genes in Shewanella revealed greater than a hundredfold increases in expression in response to exposure to strontium. Some of these differentially expressed genes encode enzymes that synthesize siderophores, low-molecular-weight compounds that show a high affinity for binding iron.

"By disrupting a gene in Shewanella, we have produced a mutant that is unable to produce the siderophore," Thompson says. "We found this mutant displays an increased sensitivity or lower tolerance to strontium than the normal bacterium, suggesting that siderophores may be involved in the resistance mechanism."

In Zhou's laboratory, researchers have built microarrays for a mixture of microbes in contaminated samples to determine which genes in bacteria have been turned up or down by exposure to the contaminated site. They have found that many of the different bacteria have adapted to the contaminated site by changing the amounts of specific proteins produced.

The researchers have determined how the microbe population changes in soil when nitrogen is added or when a contaminated site is remediated. No other group has been able to characterize in detail how a community of different species of microbes changes in a contaminated soil or groundwater sample and how that community differs from a community of the same species in a clean reference sample.

Zhou and his colleagues have been analyzing microbes present in groundwater at an Oak Ridge site that has legacy wastes with a high concentration of toxic metals and radionuclides. The site is a Field Research Center (FRC) of DOE's Natural and Accelerated Bioremediation Research (NABIR) Program, located at the Y-12 National Security Complex on DOE's Oak Ridge Reservation. Zhou believes that microarray analysis of many samples taken from the NABIR site will produce the microbe that is the most effective at reducing uranium.

"It is very expensive to pump and treat groundwater, so DOE would like to speed the growth of bacteria that can convert uranium and technetium compounds to materials that are less mobile and less toxic," says ESD's David Watson, manager of the FRC in Oak Ridge. "We hope that this bioremediation strategy for wastes containing metals and radionuclides will economically reduce risks to human health and the environment."

Human Health Benefits

Live-cell nanobiosensor. The first observation of programmed cell death in a single live cell, or apoptosis, was made recently at ORNL by Corporate Fellow Tuan Vo-Dinh and two colleagues.

ORNL's nanobiosensor penetrates a cell without destroying it and targets a specific protein.

Vo-Dinh has led the development of "nanobiosensor" technology for investigating vital biomolecular processes, including interactions between proteins in living cells.

Vo-Dinh, leader of LSD's Advanced Biomedical Science and Technology Group; Paul Kasili, a Ph.D. degree candidate at the UT-ORNL Graduate School of Genome Science and Technology, and postdoctoral researcher Joon Myong Song recently published papers on the optical nanobiosensor for measuring apoptosis in a single living cell in the Journal of the American Chemical Society and in Nature.

"This minimally invasive nanotechnology allows scientists to go inside a live cell and follow its molecular processes in real time," Vo-Dinh says.

The nanobiosensor is a tiny fiber-optic probe drawn to a tip of only 40 nanometers (nm) across—a thousand times smaller than a human hair. Experiments have demonstrated that such a probe is small enough to be inserted into a cell and withdrawn without destroying it. Light from a laser can be directed through the fiber-optic probe.

Because the 40-nm width of the probe tip is much more narrow than the 400-nm wavelength of the light, only molecules near the tip are excited by the laser signal. In this way, scientists can target specific molecules inside the cell, such as proteins, enzymes, or DNA strands.

Vo-Dinh and his colleagues have demonstrated that a fiber-optic probe with a bioreceptor molecule at its tip can be manipulated inside a cell to find a target protein. When the protein binds to the bioreceptor, a laser signal excites the target molecule, causing it to fluoresce. The resulting glow is detected.

The team recently detected the signaling process involved in apoptosis—a key process in an organism's ability to prevent disease. The programmed cell-death mechanism causes the cell to self-destruct before it can introduce disease to the organism. "When a cell in our body receives insults such as toxins or inflammation and is damaged, it kills itself so that it does not propagate," says Vo-Dinh. "The loss of cells' ability to undergo apoptosis is one cause of uncontrollable cell growth leading to cancer. For the first time we have seen apoptosis occur within a single live cell."

Environmental and engineered nanoparticles. Some particles in the air we breathe are smaller than one 100 billionths of a meter in diameter. To improve our understanding of these "nanoparticles" and their impacts on human health and the environment, ORNL researchers led by ESD's Mengdawn Cheng have developed special technologies.

Nanoparticles generated by ultrasonic atomization are studied at ORNL to determine their potential health effects.

Their inventions produce well-defined nanoparticles of a known size and composition and measure the responses of lung cells to particles of different sizes. The research is of particular importance to DOE because emissions from internal combustion engines in automobiles, trucks, off-road vehicles, and aircraft are known to contain nanoparticles.

"Our research suggests that the environmental and health effects of nanoparticles are different from the impacts of particles in the micrometer size range, even when particles of different sizes have an identical chemical composition," Cheng says.

"Using a direct air-cell exposure approach, we found that human lung cells exposed to 10-micron titanium dioxide particles showed little damage. But other lung cells died when exposed briefly to 20-nanometer titanium dioxide particles. It appears that the size of the particles, their surface properties, and area of exposure affect cellular response and increase the nanoparticles' toxicological potency toward biological tissues."

Cheng believes that nanoparticles emitted from engines as by-products are potentially more dangerous than titanium particles. The reason: engine nanoparticles are complex mixtures of organic chemicals and toxic metals.

The work of Cheng's group has a systems biology flavor when the group applies precision aerosol science and technology to detect and characterize the biomarkers generated by human lung cells exposed to nanoparticles. The group collaborates with university researchers as well as scientists at DOE and Department of Defense laboratories. Cheng expects results from the research will have significant impacts on future emissions controls, environmental and occupational health regulation, and defense work.

Combating blindness. Plant proteins might someday provide higher-resolution vision for the legally blind than current and near-term artificial retina implants such as electrode arrays. Recent research at ORNL showing that plant molecules can be fused with mammalian cells suggests this exciting possibility could be realized soon.

DOE's Artificial Retina Program, previously managed by ORNL, focuses on construction of microelectrode arrays that would directly stimulate surviving retinal tissue in people who become blind as a result of retinal degenerative diseases. Courtesy of Lawrence Livermore National Laboratory.

The research was led by Eli Greenbaum, a corporate fellow in ORNL's Chemical Sciences Division (CSD) and former leader of DOE's Artificial Retina Program, which involves research by several national laboratories.

"Mammalian and plant systems have been separated by two billion years on the evolutionary time scale, but we showed it is possible to combine them," Greenbaum says. "This work fits well into systems biology because large multicomponent systems are involved."

Greenbaum and CSD's Tanya Kuritz showed that a spinach protein—a light-absorbing pigment, or "photosynthetic reaction center," called Photosystem I (PSI)—could be incorporated in a liposome, an artificial membrane made of lipids. In collaboration with Professor Ida Lee of the University of Tennessee, the team demonstrated that a voltage high enough to make a nerve cell fire is generated by PSI inside a liposome when exposed to light. They then inserted the PSI-containing liposomes into membranes of retinablastoma cells, which are cancerous versions of cells in the eye's retina. The process demonstrated that the presence of PSI molecules is essential to making eye cancer cells respond to light.

"What we do not know is whether these spinach proteins are stable enough to last a long time and whether they would undergo immune rejection by the eye," Greenbaum says.

Longevity and genes. "Aging is a perfect example of systems biology," says Dabney Johnson, leader of LSD's Mammalian Genetics Group. "Like mice, people are predetermined to live a long life or a short life, depending on whether they have a network of longevity or 'shortevity' genes."

Two years ago, members of the Tennessee Mouse Genome Consortium (TMGC) were surprised to learn that the National Institute on Aging was less interested in knowing which diseases shorten a lifespan or impair health and wellness and more interested in finding out which genes increase a healthy lifespan.

Dabney Johnson and her colleagues are studying old mice to determine which genes increase a healthy lifespan.

The NIA was responding to findings that 200 genes in a recently sequenced worm are related to lifespan and that making a mutation in any one of these genes will alter the worm's longevity. The NIA funded a TMGC study to identify mouse genes that affect lifespan. TMGC researchers targeted different longevity genes for mutation in individual mice by using ENU, the chemical mutagen discovered 25 years ago at ORNL.

The offspring affected by the ENU treatment are being aged to their full lifespan at a UT mouse facility. Researchers compared the blood chemistry and other characteristics of the mice to try to predict which mice might live longer. Johnson says that two factors related to a network of genes are known to affect the lifespans of mice, as well as worms, fruit flies, and—probably— people.

"Individuals who are smaller and thinner live longer," she notes. "Individuals that are big, heavy, bulky, and tall for their species tend to die young."

"The other factor associated with longevity is resistance to stress. Individuals who are usually on an even keel live longer. The theory is that individuals subjected to prolonged stress make oxygen radicals inside their cells. The radicals damage proteins, DNA, and lipids inside cells. Damage accumulated over a lifetime diminishes the functioning of cells."

In about two years, the researchers will know which of the mice with "mutated longevity genes" lived a significantly long life. They then can determine which gene or genes are involved in longevity.

Cell Communication. Understanding information processing within living cells and communication between them is a goal of researchers in the Molecular-Scale Engineering and Nanoscale Technologies Research Group of ORNL's Condensed Matter Sciences and Engineering Science and Technology divisions. Mike Simpson, who leads this group and holds a joint faculty appointment at the University of Tennessee and ORNL, is spearheading an effort to use computational, analytical, and experimental tools to simulate genetic circuits and genetic networks in cells and predict how they will respond to signals generated by the environment or other cells.

"Computation and simulation will help us select the most important experiments to perform and decide the most intelligent ways to do them to learn more quickly about information processing within cells," Simpson says.

Instead of wires, components within a genetic circuit are interconnected by molecular interactions such as regulatory protein-DNA interactions to control gene expression; RNA polymerase-DNA interactions that produce messenger RNA (mRNA) during gene expression; and ribosome-mRNA interactions that produce proteins that carry out cellular functions. A small subset of interconnected reactions that carry out a single function is considered a genetic circuit; a genetic network, which hooks together multiple genetic circuits, is responsible for complex cellular functions.

Simpson's group has written mathematical expressions to represent the components of genetic circuits and genetic networks to understand better the biological functions of bacterial cells. The approach is one of many used in systems biology, an emerging discipline that endeavors to apply analytical tools and approaches more familiar to the physical sciences to biological problems.

For a U.S. Defense Advanced Research Projects Agency project funded jointly with the National Science Foundation, UT graduate students in Simpson's group have developed software tools that simulate and analyze "stochastic fluctuations"— random noise in cellular biomolecular populations that may be important to genetic circuit fluctuations. For example, stochastic fluctuations are vital to the decision-making process in the phage virus's infection of E. coli bacteria. To examine such processes and gain new insight into biological function, the UT-ORNL collaboration developed and published papers on the Exact Stochastic Simulator that simulates noise and its effect on biological systems.

The group is refining an experiment in which genetic components of a cell-cell communication system found in the marine bacterium Vibrio fischeri are inserted into E. coli. V. fischeri cells do not give off light unless a large enough population of cells is present, such as a squid's "light" organ that offers them a nutrient-rich environment. When populated with V. fischeri, this otherwise dark organ suddenly becomes luminescent. Thus, the squid's predators lurking below cannot distinguish the "camouflaged" squid from starlight above.

Simpson's group is interested in the genetic circuits that process cell-cell communication like that found in V. fischeri. "Cell-cell communication is the mechanism that allows groups of cells to coordinate their activities and produce the complex group behaviors that lead to infection, biofilm formation, and functioning tissues, organs, and organisms," Simpson says. "By looking at the more primitive communication systems in bacteria, we hope to develop an understanding of information processing in cellular communication systems of more complex organisms, especially those that impact human health."

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