The Inner World of Athletes

So many things typically distinguish accomplished athletes from the rest of us—greater strength and endurance, better balance, faster reactions—but one of the more surprising differences is that, according to dental studies, they also tend to get more cavities.

This intriguing phenomenon was the subject of a capstone course in microbiology this past spring, offering undergrads a chance to be part of a burgeoning worldwide scientific effort while using cutting-edge technology.

There are trillions of microbes in the human body; the community of microbes that lives in each of us is our microbiome. As more and more research focuses on microbiomes, it’s becoming clear they play a significant role in human health and wellness. Microbiology 551 students worked to add to that body of research using a next-generation DNA sequencer manufactured by the California-based company Illumina.

“It’s only our department and maybe one or two in California that are doing hands-on work with undergraduates in teaching this technique,” says co-instructor Melissa Christopherson. Christopherson teaches the course with Tim Paustian, both faculty associates in the Department of Bacteriology. “Having students conduct meaningful research with these modern techniques makes them more competitive in the job market and better able to navigate the field of microbiology.”

Students were tasked with comparing the oral microbiomes of athletes and nonathletes, using saliva samples. They sampled a range of students, from UW athletes to occasional exercisers to students who hadn’t exercised for at least five weeks. Once students collected and prepared the samples—including their own oral microbiomes—they sequenced the DNA and determined which microbes were present in each sample.

With so many samples, the students were able to look beyond the question of exercise to test other hypotheses they developed themselves.

“We wound up taking the same data set and asking other questions,” explains Samantha Gieger, who graduated in May with a BS in microbiology and genetics. “In groups of four or five, we looked at the effects of dairy, caffeine or using an electric toothbrush.”

Students presented their projects at a poster session last semester, and their work is currently being analyzed for publication. Their findings will become part of the growing research into microbiomes. Student Sophie Carr BS’16 and Christopherson were invited to the White House last spring for a summit announcing the launch of the National Microbiome Initiative.

As a capstone class, the course offered a research experience requiring students to integrate diverse bodies of knowledge to solve a problem. And it quickly proved invaluable as students considered next steps in their careers.

“I’ve learned so much—how to go about research, what to do when encountering a problem. Troubleshooting is such an important technique,” says Isaiah Rozich BS’16, then a senior majoring in microbiology and Spanish. “Figuring out which solution is best takes a lot of time, and it opened my eyes to what life as a researcher will be like. While it’s overwhelming, I think the end result is gratifying.”

PHOTO: On the case: Students compared the oral microbiomes of athletes to figure out why athletes get more cavities.
Photo by Sevie Kenyon

To Market, to Market

If you’re familiar with the College of Agricultural and Life Sciences (CALS), you no doubt know all about Stephen Babcock and his test that more than 100 years ago revolutionized the dairy industry by providing an inexpensive, easy way to determine the fat content of milk (thus preventing dishonest farmers from watering it down). What you might not know is that his great discovery went unpatented. The only money Babcock received for his invention was $5,000 as part of a Capper Award—given for distinguished service to agriculture—in 1930.

Just years before Babcock received that award, another entrepreneur was hard at work in his lab—and his discovery would break ground not only in science, but also in direct remuneration for the university.

In 1923, Harry Steenbock discovered that irradiating food increased its vitamin D content, thus treating rickets, a disease caused by vitamin D deficiency. After using $300 of his own money to patent his irradiation technique, Steenbock recognized the value of such patents to the university. He became influential in the formation in 1925 of the Wisconsin Alumni Research Foundation (WARF), a technology transfer office that patents UW–Madison innovations and returns the proceeds back to the university.

Discoveries have continued flowing from CALS, and WARF plays a vital role for researchers wanting to patent and license their ideas. But today’s innovators and entrepreneurs have some added help: a new program called Discovery to Product, or D2P for short.

Established in 2013, and co-funded by UW–Madison and WARF, D2P has two main goals: to bring ideas to market through the formation of startup companies, and to serve as an on-campus portal for entrepreneurs looking for help. Together, WARF and D2P form a solid support for researchers looking to move their ideas to market. That was the intent of then-UW provost Paul DeLuca and WARF managing director Carl Gulbrandsen in conceiving of the program.

“The idea of D2P is to make available a set of skills and expertise that was previously unavailable to coach people with entrepreneurial interests,” explains Leigh Cagan, WARF’s chief technology commercialization officer and a D2P board member. “There needed to be a function like that inside the university, and it would be hard for WARF to do that from the outside as a separate entity, which it is.”

D2P gained steam after its initial conception under former UW–Madison chancellor David Ward, and the arrival of Rebecca Blank as chancellor sealed the deal.

“Chancellor Blank, former secretary of the U.S. Department of Commerce, was interested in business and entrepreneurship. D2P really started to move forward when she was hired,” says Mark Cook, a CALS professor of animal sciences. Cook, who holds more than 40 patented technologies, launched the D2P plan and served as interim D2P director and board chair.

With the light green and operational funds from WARF and the University secured, D2P was on its way. But for the program to delve into one of its goals— helping entrepreneurs bring their ideas to market—additional funding was needed.

For that money, Cook and DeLuca put together a proposal for an economic development grant from the University of Wisconsin System. They were awarded $2.4 million, and the Igniter Fund was born. Because the grant was good only for two years, the search for projects to support with the new funds started right away.

By mid-2014, veteran entrepreneur John Biondi was on board as director, project proposals were coming in and D2P was in business. To date, 25 projects have gone through the Igniter program, which provides funding and guidance for projects at what Biondi calls the technical proof of concept stage. Much of the guidance comes from mentors-in-residence, experienced entrepreneurs that walk new innovators down the path to commercialization.

“For Igniter projects, they need to demonstrate that their innovation works, that they’re not just at an early idea stage,” explains Biondi. “Our commitment to those projects is to stay with them from initial engagement until one of three things happen: they become a startup company; they get licensed or we hand them over to WARF for licensing; or we determine this project might not be commercial after all.”

For projects that may not be destined for startup or that need some additional development before going to market, the collaboration between WARF and D2P becomes invaluable. WARF can patent and license discoveries that may not be a good fit for a startup company. They also provide money, called Accelerator funding, for projects that need some more proof of concept. Innovations that may not be ready for Igniter funds, but that are of potential interest to WARF, can apply for these funds to help them move through the earlier stages toward market.

“Some projects receive both Accelerator and Igniter funding,” says Cagan. “Some get funding from one and not the other. But we work together closely and the programs are being administered with a similar set of goals. We’re delighted by anything that helps grow entrepreneurial skills, companies and employment in this area.”

With support and funding from both WARF and D2P, entrepreneurship on campus is flourishing. While the first batch of Igniter funding has been allocated, Biondi is currently working to secure more funds for the future. In the meantime, he and others involved in the program make it clear that the other aspect of D2P—its mission to become a portal and resource for entrepreneurs on campus—is going strong.

“We want to be the go-to place where entrepreneurs come to ask questions on campus, the starting point for their quest down the entrepreneurial path,” says Biondi.

It’s a tall order, but it’s a goal that all those associated with D2P feel strongly about. Brian Fox, professor and chair of biochemistry at CALS and a D2P advisory board member, echoes Biondi’s thoughts.

“D2P was created to fill an important role on campus,” Fox says. “That is to serve as a hub, a knowledge base for all the types of entrepreneurship that might occur on campus and to provide expertise to help people think about moving from the lab to the market. That’s a key value of D2P.”

Over the past two years, D2P, in collaboration with WARF, has served as precisely that for the 25 Igniter projects and numerous other entrepreneurs looking for help, expertise and inspiration on their paths from innovation to market. The stories of these four CALS researchers serve to illustrate the program’s value.

Reducing Antibiotics in Food Animals

Animal sciences professor Mark Cook, in addition to helping establish D2P, has a long record of innovation and entrepreneurship. His latest endeavor, a product that has the potential to do away with antibiotics in animals used for food, could have huge implications for the animal industry. And as he explains it, the entire innovation was unintentional.

“It was kind of a mistake,” he says with a laugh. “We were trying to make an antibody”—a protein used by the immune system to neutralize pathogens—“that would cause gut inflammation in chickens and be a model for Crohn’s disease or inflammatory bowel disease.”

To do this, Cook’s team vaccinated hens so they would produce a particular antibody that could then be sprayed on feed of other chickens. That antibody is supposed to cause inflammation in the chickens that eat the food. The researchers’ model didn’t appear to work. Maybe they had to spark inflammation, give it a little push, they thought. So they infected the birds with a common protozoan disease called coccidia.

“Jordan Sand, who was doing this work, came to me with the results of that experiment and again said, ‘It didn’t work,’” explains Cook. “When I looked at the data, I saw it was just the opposite of what we expected. The antibody had protected the animals against coccidia, the main reason we feed antibiotics to poultry. We knew right away this was big.”

The possibilities of such an innovation—an antibiotic-free method for controlling disease—are huge as consumers demand antibiotic-free food and companies look for ways to accommodate those demands. With that potential in hand, things moved quickly for Cook and Sand. They filed patents through WARF, collaborated with faculty colleagues and conducted experiments to test other animals and determine the best treatment methods. More research was funded through the WARF Accelerator program, and it became clear that this technology could provide the basis for a startup company.

While Cook didn’t receive funds from D2P to bring the product to market, he and Sand used D2P’s consulting services throughout their work—and continue to do so. Between WARF funding and help from D2P, Cook says starting the current company, Ab E Discovery, has been dramatically different from his previous startup experiences.

“D2P is a game changer,” says Cook. “In other cases, there was no structure on campus to help. When you had a technology that wasn’t going to be licensed, you had to figure out where to get the money to start a company. There were no resources available, so you did what you could, through trial and error, and hoped. Now with WARF and D2P working together, there’s both technical de-risking and market de-risking.”

The combination of WARF and D2P has certainly paid off for Cook and Sand. They have a team and a CEO, and are now producing product. Interest in the product is immense, Cook says. He’d like to see the company grow and expand—and stay in Wisconsin.

“It’s been a dream of mine to make Wisconsin a centerpiece in this technology,” Cook says. “I’d like to see the structure strong here in Wisconsin, so that even when it’s taken over, it’ll be a Wisconsin company. That’s my hope.”

Better Corn for Biofuel

Corn is a common sight in Wisconsin and the upper Midwest, but it’s actually more of a tropical species. As the growing regions for corn move farther north, a corn hybrid has to flower and mature more quickly to produce crop within a shorter growing season. That flowering time is determined by the genetics of the corn hybrid.

Conversely, delayed flowering is beneficial for other uses of corn. For example, when flowering is delayed, corn can produce more biomass instead of food, and that biomass can then be used as raw material to make biofuel.

The genetics of different hybrids controls their flowering time and, therefore, how useful they are for given purposes or growing regions. Shawn Kaeppler, a professor of agronomy, is working to better understand those genes and how various hybrids can best fit a desired function. Much of his work is done in collaboration with fellow agronomy professor Natalia de Leon.

“We look across different populations and cross plants to produce progeny with different flowering times,” Kaeppler explains. “Then we use genetic mapping strategies to understand which genes are important for those traits.”

Throughout his work with plant genetics, Kaeppler has taken full advantage of resources for entreprenuers on campus. He has patents filed or pending, and he has also received Accelerator funds through WARF. For his project looking at the genetics behind flowering time, Kaeppler and graduate student Brett Burdo received Igniter funds from D2P as well. The Igniter program has proven invaluable for Kaeppler and Burdo as they try to place their innovation in the best position for success.

“I found the Igniter program very useful, to go through the process of understanding what it takes to get a product to market,” says Kaeppler. “It also includes funding for some of the steps in the research and for some of the time that’s spent. I can’t fund my graduate student off a federal grant to participate in something like this, so the Igniter funding allowed for correct portioning of funding.”

The end goal of Kaeppler’s project is to develop a transgenic plant as a research model and license the technology, not develop a startup company. His team is currently testing transgenic plants to work up a full package of information that interested companies would use to decide if they should license the technology. For Kaeppler, licensing is the best option since they can avoid trying to compete with big agricultural companies, and the technology will still get out to the market where it’s needed to create change.

“In this area of technology transfer, it is important not only to bring resources back to UW but also to participate in meeting the challenges the world is facing with increasing populations,” says Kaeppler. “Programs like D2P and WARF are critical at this point in time to see the potential of these discoveries realized.”

A Diet to Treat Disease

Around the world, about 60,000 people are estimated to have phenylketonuria, or PKU. Those with the inherited disorder are unable to process phenylalanine, a compound found in most foods. Treatment used to consist of a limited diet difficult to stomach. Then, about 13 years ago, nutritional sciences professor Denise Ney was approached to help improve that course of treatment.

Dietitians at UW–Madison’s Waisman Center wanted someone to research use of a protein isolated from cheese whey—called glycomacropeptide, or GMP—as a dietary option for people living with PKU. Ney took on the challenge, and with the help of a multidisciplinary team, a new diet composition for PKU patients was patented and licensed.

“Mine is not a typical story,” says Ney, who also serves as a D2P advisory board member. “Things happened quickly and I can’t tell you why, other than hard work, a good idea and the right group of people. We’ve had help from many people—including our statistician Murray Clayton, a professor of plant pathology and statistics, and the Center for Dairy Research—which helped with development of the foods and with sensory analysis.”

Being at the right place at the right time had a lot to do with her success thus far, Ney notes. “I’m not sure this could have happened many places in the world other than on this campus because we have all the needed components—the Waisman Center for care of patients with PKU, the Wisconsin Center for Dairy Research, the clinical research unit at University of Wisconsin Hospitals and Clinics, and faculty with expertise in nutritional sciences and food science,” she says.

Ney is currently wrapping up a major clinical trial of the food formulations, referred to as GMP medical foods, that she and her team developed. In addition to those efforts, the new diet has also shown surprising promise in two other, seemingly unrelated, areas: weight loss and osteoporosis prevention.

“My hypothesis, which has been borne out with the research, is that GMP will improve bone strength and help prevent fractures, which are complications of PKU,” explains Ney. “I have a comprehensive study where I do analysis of bone structure and biomechanical performance, and I also get information about body fat. I observed that all of the mice that were fed GMP, whether they had PKU or not, had less body fat and the bones were bigger and stronger.” Interestingly, the response was greater in female compared with male mice.

To support further research on this new aspect of the project, Ney received Accelerator funds from WARF for a second patent issued in 2015 titled “Use of GMP to Improve Women’s Health.” Ney and her team, including nutritional sciences professor Eric Yen, are excited about the possibilities of food products made with GMP that may help combat obesity and also promote bone health in women.

“There is a huge market for such products,” says Ney. “We go from a considerably small group of PKU patients who can benefit from this to a huge market of women if this pans out. It’s interesting, because I think I’m kind of an unexpected success, an illustration of the untapped potential we have here on campus.”

Fewer Antibiotics in Ethanol Plants

Bacteria and the antibiotics used to kill them can cause significant problems in everything from food sources to biofuel. In biofuel production plants, bacteria that produce lactic acid compete with the wanted microbes producing ethanol. At low levels, these bacteria decrease ethanol production. At high levels, they can produce so much lactic acid that it stops fermentation and ethanol production altogether.

The most obvious solution for stopping these lactic acid bacteria would be antibiotics. But as in other industries, antibiotics can cause problems. First, they can be expensive for ethanol producers to purchase and add to their workflow. The second issue is even more problematic.

“A by-product of the ethanol industry is feed,” explains James Steele, a professor of food science. “Most of the corn kernel goes toward ethanol and what remains goes to feed. And it’s excellent animal feed.”

But if antibiotics are introduced into the ethanol plant, that animal feed byproduct can’t truly be called antibioticfree. That’s a problem as more and more consumers demand antibiotic-free food sources. But Steele and his colleagues have a solution—a way to block the negative effects of lactic acid bacteria without adding antibiotics.

“We’ve taken the bacteria that produce lactic acid and re-engineered it to produce ethanol,” says Steele. “These new bacteria, then, compete with the lactic acid bacteria and increase ethanol production. Ethanol plants can avoid the use of antibiotics, eliminating that cost and increasing the value of their animal feed by-product.”

The bacteria that Steele and his team have genetically engineered can play an enormous role in reducing antibiotic use. But that benefit of their innovation didn’t immediately become their selling point. Rather, their marketing message was developed through help from D2P and the Igniter program.

“Learning through D2P completely changed how we position our product and how we interact with the industry,” says Steele. And through that work with D2P, Steele plans to later this year incorporate a company called Lactic Solutions. “D2P has helped us with the finance, the organization, the science, everything. Every aspect of starting a business has been dealt with.”

Steele and his collaborators are now working to refine their innovation and ideas for commercialization using Accelerator funds from WARF. Steele’s work, supported by both WARF and D2P, is a perfect example of how the entities are working together to successfully bring lab work to the market.

“There is no doubt in my mind that we would not be where we are today without D2P,” says Steele. “On top of that you add WARF, and the two together is what really makes it so special. There’s nothing else like it at other campuses.”

With such a strong partnership campaigning for and supporting entrepreneurship at UW–Madison, CALS’ strong history of innovation is poised to endure far into the future, continuing to bring innovations from campus to the world. And that is the embodiment of the Wisconsin Idea.

 

The New Old Forest

Jodi Forrester got the call while she was in the forest. The loggers were ready to go. So on a cold winter day in northern Wisconsin, she found herself riding shotgun in a harvester. Forrester, a research scientist in forest and wildlife ecology, watched as the loggers cut down the trees she and her team had carefully selected in the Flambeau River State Forest. Another huge vehicle, a forwarder, clambered behind, pinching the cut trees in its claw and moving them to where they were needed. All the while, the loggers played a little game, dodging between laundry baskets placed around the forest floor to catch leaves and falling debris. In the end, they managed to avoid all but a few.

It was not a typical job for the loggers. Instead of harvesting trees for timber, they were taking part in an experiment—the second phase of a research project on a large scale. Under the supervision of CALS forest and wildlife ecology professor David Mladenoff, Forrester and her colleagues had already been working for years to plan a forest experiment that would stretch over almost 700 acres. The loggers were there to implement that plan. Because all the wood they were cutting was going to be left in the forest as part of the experimental setup, the loggers were not able to remove any of it. It went against their nature.

“Every once in a while, the loggers had to cover their eyes,” says Forrester with a smile. “There are a lot of beautiful, valuable trees in that forest, and I think they weren’t too sure about what they were being asked to do.”

But the loggers had agreed to the job because they knew it was part of an experiment that would push the science of forest management in Wisconsin forward. All the work, including the tough job of watching the wood get left behind, was being done in the name of science—specifically, in the name of bringing the characteristics of old-growth forests back to the state.

Old-growth forests have been a scarce sight in Wisconsin since the early 20th century. Clear-cutting in the late 1800s and early 1900s left few old-growth stands. In the Upper Midwest, most big trees had been cut down by the 1930s. In the place of those stands, younger second-growth forests emerged.

Starting in the 1980s, a push to promote and protect old-growth forests picked up steam. It started in the Pacific Northwest, where obligate species, such as the spotted owl, live only in old-growth forests. As the interest in these forests moved east, people in the Midwest began recognizing the valuable ecosystem services provided by old-growth forests, such as storing carbon, maintaining soils and fostering biodiversity in plants, animals and microbes by offering needed habitats.

In Wisconsin it wasn’t a matter of protecting old-growth forests, it was a question of creating them again, or at least some of the functions they provide. And that was no small task. Creating old-growth forests requires defining them, and even that can be difficult. It’s not just a matter of age—and age doesn’t always mean the same thing. A 40-year-old aspen forest would be old, notes Mladenoff; a 40-year-old sugar maple forest, on the other hand, would be quite young.

“It’s not always the age that matters,” says Mladenoff. “Sometimes what really matters are the characteristics and features of the forest.”

With the features of Upper Midwestern old-growth forests unclear, Mladenoff and scientists at UW–Madison, other UW campuses and the Wisconsin Department of Natural Resources (DNR) in 1992 started Phase 1 of what was dubbed the Old Growth Project.

Phase 1 was a comparative study. The researchers looked at forests of various ages and histories—a total of 46 different areas—to determine what was unique to the older, unmanaged forests. They considered features like plant and tree species and sizes, woody debris on the ground, snags or standing dead trees, soil characteristics and forest wildlife. Different scientists looked at different aspects, the collaboration creating a complete picture of the forests.

After a decade of collecting and comparing enormous amounts of data, Mladenoff and his colleagues found that many of the features of old-growth forests had to do with two structural elements: the size and distribution of gaps in the forest canopy and coarse woody debris—sizable logs—on the forest floor.

Gaps are openings in the forest canopy caused when large trees fall. With sunlight able to reach the forest floor, these areas become places of regeneration and growth, and the diversity of understory plants is often higher in gap areas than in the surrounding forest.

Coarse woody debris, meanwhile, provides shelter for salamanders, insects and other small animals as well as food for fungi, insects and even other trees like hemlock and yellow birch. Logs also sequester carbon on the forest floor and reduce the amount of carbon dioxide returning to the atmosphere.

“We wanted to explore the importance of those two elements in more detail,” explains Mladenoff. “We wanted to know if creating those structural elements in second-growth northern hardwood forests could restore functional old-growth characteristics.”

Phase 2—The Experiment

Mladenoff, Forrester and their colleagues—including Craig Lorimer and Tom Gower, emeritus and former CALS professors of forest and wildlife ecology, respectively—wanted to address that question using an experimental setup. Phase 2 of the Old Growth Project, the Flambeau Experiment, was born. The first step of that phase, however, was not a trivial one. They had to find a piece of land on which to conduct the experiment. They needed a site that was big enough for all the treatments they envisioned and that would otherwise be undisturbed for a long period of time—50 years, in fact.

With help from the DNR, Mladenoff and his colleagues used geographic information systems—GIS—to look at forests at different sites to find one that would fit the bill. After two years of looking, the researchers, including a postdoctoral student dedicated to the project, finally chose the site in the Flambeau River State Forest—a hardwood stand around 100 years old, dominated by sugar maples.

Before the experimental treatments were applied to the newly found forest, pretreatment data were collected. Scientists could then compare the data collected after treatment to this baseline information. Forrester and her colleagues, including several graduate students, used grids that they laid on the forest floor to count and catalog understory plant species such as trout lilies, wild leeks, nodding trillium and jack-in-the-pulpits. They also observed and measured tree species and diversity, leaf litter that fell in the forest, nutrient cycling, activity of soil microbes and more.

Finally, after spending two years looking for a site and two more years collecting pre-treatment data, the Flambeau site was ready for treatment in January 2007. In came the loggers and machinery to create the canopy gaps and coarse woody debris. The researchers also put up fences surrounding some of the plots to exclude deer and remove their influence from those treatment areas.

For five years after Forrester first rode shotgun in the harvester, she, graduate students and other scientists worked year-round to collect data. In the winter, researchers made the four-hour trip from Madison to Flambeau to check equipment, take measurements, replace batteries and mend fences. Once the spring thaw came, their work ramped up.
A typical summer day in the forest lasted about 10 hours. The scientists would ride from their rented cabins to the Flambeau Forest, walk about a half-mile to the research site and start collecting data. These days would last until October or November, when the researchers would start to see the orange vests of hunters.

“We’d head out in the morning and take our lunch and everything we needed for the day,” says Forrester. “We’d walk into the site, do our work, then head back to the cabins and crash.”

Their work included collecting a huge number of plant and soil samples. Without any university buildings at the Flambeau site, Forrester and her colleagues had to transport all of those samples back to Madison in their vans. Once back on campus, the samples and data needed to be analyzed and entered into spreadsheets.

“We have gobs of soil and wood samples, and we employed a lot of undergrads to help us,” says Forrester, laughing. “Some folks would help in the field in the summers and then continue working in the lab in the fall while they took classes.”

Ten years into Phase 2, Forrester, Mladenoff and their collaborators are just now beginning to shape a picture of the effects of their treatments. While a decade seems like a long time for research, they have another 40 years ahead of them. Such is the course of a 50-year experiment. And researchers have a vast array of forest components to consider and measure.

At this point they have some preliminary data and even some surprising results. One of the unexpected outcomes has been in the plots with coarse woody debris. While the researchers were expecting that the effects of woody debris would take years to recognize as the wood decayed, they are already beginning to see changes in the carbon dynamics. The woody debris affected rates of decomposition and what kinds of microbes were present in the soil, for example, within just a few years after being left on the forest floor.

“I thought someone else would be seeing what happens to the wood in the future, that I would just be seeing the effects of the canopy gaps,” explains Mladenoff. “But it didn’t turn out that way.”

The researchers are also seeing more expected results. Saplings and understory vegetation are growing more quickly in areas with canopy gaps and more light, for example. Also, the deer exclusion fences make a difference. In areas without the fences, the deer are eating all of the sprouts growing from the stumps of harvested trees, which can change the composition of the forest, leaving more of the less palatable and lower value trees such as ironwood.

After five years of intense sampling after treatment, the researchers are now spacing out their measurements and sampling to allow the forest time to grow, settle, decay and cycle. With such a long-term experiment, some of the time must be spent waiting.

That time will also be spent securing funding for the project as it goes forward. The DNR provided money both for Phase 1 of the project and to get the experimental Phase 2 going. That initial funding for Phase 2 allowed the researchers to do the preliminary work, after which other funding started flowing in.

“The DNR was really helpful in getting this project started,” says Forrester. “They provided all that base funding for us to get established, and only once we started were we able to get other money.”

The USDA has provided a five-year grant, and Mladenoff and his colleagues have also received funding from the Department of Energy and USDA McIntire-Stennis grants for graduate students. Forrester is now working to secure funds for the years ahead.

The USDA grant afforded Forrester and her colleagues an unexpected benefit—the opportunity to teach a new generation of forest ecologists. The grant was awarded based on their proposal to integrate an educational component into their research, and to fulfill that aspect, Forrester created a summer internship program. Undergraduate students from around the country and the world, most with little experience in forest research, joined the scientists in the Flambeau.

“Initially we taught them the basics of forest ecology measurements and had them help us with our measurements,” explains Forrester. “As summer rolled on, we helped them focus on a topic and develop an independent study project.”

Around 40 students participated in the program over the four years it was available. At the end of each summer, they’d hold a symposium to allow the students to present their work and interact with the scientists. The graduate students gained valuable mentorship experience. It was a beneficial experience for all involved, and one that both Forrester and Mladenoff discuss with pride.

“It was an important part of the project, and it turned out to be a really great component of those summers,” says Forrester.

DNR Collaboration

In addition to providing funding, scientists at the DNR are also long-term collaborators with CALS researchers. They are working on a parallel 50-year project called the Managed Old-Growth Silviculture Study, or MOSS. Silviculture is the practice of managing forests to meet various needs or goals.

Having worked with Mladenoff and his team from Phase 1 of the project and into Phase 2, the DNR wanted to look at many of the same elements of old-growth forests, but with a more operational spin. They wanted to find out how to create the characteristics of old-growth forests while also allowing for economically beneficial harvesting of timber.

“There were three objectives for the MOSS project,” says Karl Martin BS’91, a former wildlife and forestry research chief at the DNR who is now with UW–Extension as state director of the Community, Natural Resource and Economic Development (CNRED) program. “We wanted the study to be applicable to the forest industry, we wanted to do something on a large scale so we could look at impacts on wildlife, and we wanted to show this was economically viable from a commercial standpoint.”

Martin worked closely with Mladenoff and other CALS and UW scientists to collaborate on the parallel MOSS project. One of the three MOSS sites is just north of the CALS site in the Flambeau River State Forest, with the two other sites located in the Northern Highland American Legion State Forest and the Argonne Experimental Forest.

Many of the treatments used on those three tracts of land are the same as those the CALS team is using in their experiment—canopy gaps, coarse woody debris and deer exclosures. The MOSS project also considered snags, or standing dead trees, which are another feature of old-growth forests.

Before establishing the treatments, Martin and his team spent several years surveying and measuring the trees. Because they wanted to harvest timber, they had to carefully consider which trees would be cut down and which would be left behind. Yellow birch trees were rare in the sites, so those were immediately off the table for harvesting. They also wanted to avoid cutting down the largest trees in the stands. To establish snags, the researchers chose crooked or highly branched trees that were of low economic value. While such trees make good habitat for wildlife, they are most likely to be used for low-valued pulpwood or firewood if harvested.

“We took three or four years before treating to really get things in place,” says Martin. “The problem with a 50-year study is that if you rush into it, you’re going to look back and wish you’d done something differently. We really wanted to cover all our bases.”

As with the CALS study, MOSS is in the early stages of gathering data and there are many angles to consider. The economic viability of silviculture that encourages old-growth characteristics is one of the main questions MOSS aims to answer, and Tom Steele MS’83 PhD’95, director of the Kemp Natural Resources Station in Woodruff, has been instrumental in finding that answer. Early data suggest that treatment cost of traditional harvests and the MOSS harvests is similar. In addition, the difference in timber revenue that a landowner would receive is quite minimal—just a few percent.

With years ahead to uncover the economics of such a system, MOSS is well positioned to understand and implement silviculture systems that are both economically and ecologically viable. That, in the end, is what the CALS–DNR collaboration is all about. It’s a partnership that brought about an otherwise unlikely project.

“The idea behind the collaboration is to leverage the resources of both organizations to help the citizens of the state,” explains Martin. “The scale of this study would not have been possible without the partnership of the university and the DNR. You need those resources, both intellectual and financial, to come together in a cohesive project.”

The size and scope of the Flambeau Experiment and MOSS are what make the projects so powerful—and so promising. There are decades of study ahead for researchers, and many of the original scientists will have to pass the project on to new researchers before it’s over. But the goal is clear: To determine if diverse ecosystems of old-growth forests can be developed through management while allowing for sustainable timber harvests. The outcome of the projects will have major impacts on forest management and harvests as well as on property owners, residents and visitors.

“With long-term studies, we work in the present, build on those that came before us, and count on colleagues in the future to continue the work,” says Mladenoff. “This research will be essential for long-term sustainable ecosystems and the services they provide.”

Forestry technician Donald Radcliffe BS’15, who graduated with CALS degrees in forestry and life sciences communication, contributed to reporting this piece.

Stealth Entry

Many human diseases—including cancer—are caused by protein malfunctions. Those malfunctions, in turn, are caused by damaged DNA that gets translated into the damaged proteins. While many clinicians and scientists are trying to treat those diseases by fixing the DNA, Ron Raines is taking a different approach—he’s looking to replace the proteins directly.

“Our strategy is to do gene therapy without the genes,” explains Raines, a professor of biochemistry. “We want to skip the genes and go right to the proteins.”

The strategy is intriguing, but there’s a problem. Proteins have a hard time getting into cells where they would do their work. The lipid bilayer of a cell membrane serves as a barrier that keeps the inside of the cell in and the outside out. That membrane stops potential intruders—including uninvited proteins—from entering.

Raines and his team have found a way around this in what amounts to a kind of biochemical calling card. They can attach “decorations,” using what is called an ester bond, to the protein to change its characteristics. The ester bonds link the protein to a “moiety,” a molecule that gives the protein a desired attribute or function.

“Moieties could encourage cell entry, which is one of our major goals,” says Raines. “But moieties could also enhance the movement of the protein in an animal body. Or they could be agents that target the protein, for example, to cancer cells specifically.”

Modifying proteins to give them these attributes has been done using other approaches, but those changes are permanent and can cause problems. The modified protein might not function normally, or the immune system might see the protein as foreign and mount an attack.

Raines’ strategy avoids these problems by using reversible modifications. Because the moieties are added using ester bonds, they are removed once inside a target cell. Naturally occurring enzymes in the cell—called esterases—sever the ester bonds and break off the moieties. What’s left is the normal protein without any decorations. That protein can then do its job.

“We don’t have the problem of damaging the function of the protein or of an immune response because what we ultimately deliver will be the wild-type protein, the protein as it’s naturally found in cells,” explains Raines.

The strategy is promising, and the Wisconsin Alumni Research Foundation (WARF) already has patent applications for it on file. Raines’ lab is now working to make adding the decorations as straightforward and user-friendly as possible. That way, scientists and clinicians could add a moiety of their choosing and get the protein to perform its desired function.

Raines sees innumerable possibilities.

“We’re very excited about this because it has a lot of potential,” he says. “We can now decorate proteins reversibly with pretty much any molecule you can imagine. We are exploring the possibilities to try to bring something closer to the clinic.”

Second Life for Phosphorus

Phosphorus, a nutrient required for growing crops, finds its way from farm fields to our food and eventually to our wastewater treatment plants. At the plants, the nutrient causes major problems, building up in pipes or going on to pollute surface waters.

Brushite bounty: Phil Barak displays brushite produced during trials at the Nine Springs Wastewater Treatment Plant of the Madison Metropolitan Sewerage District. Each jar contains brushite harvested from 30 gallons of anaerobic digest. Photo courtesy of Phil Barak

Brushite bounty: Phil Barak displays brushite produced during trials at the Nine Springs Wastewater Treatment Plant of the Madison Metropolitan Sewerage District. Each jar contains brushite harvested from 30 gallons of anaerobic digest.
Photo by Rick Wayne

But soil science professor Phil Barak has an idea about how to retrieve the nutrient from wastewater in a valuable form—and it started from a basic lab experiment. “I was doing some work on crystallizing phosphorus, just out of pure academic interest,” explains Barak. “That led me to crystallize a mineral called struvite. Then I realized it was forming in wastewater treatment plants as a nuisance.”

If he could form crystals in the lab, he reasoned, why couldn’t it be done in the wastewater treatment plants in a controlled way? It could. And, even better, if he collected the phosphorus early on in the treatment process in the form of a mineral called brushite, he could harvest even more of it.

Beyond removing phosphorus from wastewater, brushite can serve as a nutrient source for growers. While Barak will do further testing to prove its utility, brushite is a phosphate mineral that’s actually been found in agricultural fields for years.

“When conventional phosphorus fertilizers are added to soil, brushite forms. I maintain that we’ve been fertilizing with brushite for decades, but nobody’s been paying attention to it,” says Barak.

Being able to remove phosphorus from wastewater and supply it back to growers is a win-win situation, Barak notes. “We’re collecting phosphorus where it’s localized, at really high concentrations, which is the most economical place to collect it,” says Barak. “This works out in just about every dimension you can consider, from the treatment plants to the cost of recycling phosphorus as opposed to mining it new.”

Graduate students in Barak’s lab suggested that he commercialize the technology and start a company. After the Wisconsin Alumni Research Foundation (WARF) passed on the patent, Barak and his students sought help from the UW Law and Entrepreneurship Clinic. They received two federal Small Business Innovative Research grants, and, with some additional funds from the state, including the Wisconsin Economic Development Corporation, their efforts have turned into a spinoff company: Nutrient Recovery & Upcycling, LLC (NRU).

The company’s next step was a big one. This summer, a phosphorus recovery pilot plant is being implemented in a wastewater treatment plant in Illinois. The pilot project will test the research ideas on a larger scale.

Additionally, the NRU team will participate in the Milwaukee Metropolitan Sewerage District’s granting system to determine if a pilot project would be a good fit in Milwaukee. They hope to start collecting and analyzing data from Illinois by September, using that pilot system to lay the groundwork for others in Milwaukee and beyond.