CRISPR: The Promise and the Peril

DIETRAM SCHEUFELE, a CALS professor of life sciences communication, serves on a national panel examining the implications of human genome editing.

The committee, appointed late last year by the National Academies of Sciences, Engineering and Medicine, is examining the clinical, ethical, legal and social implications of the emerging technology. Genome editing holds great medical promise but also poses risks of off-target genetic alterations and raises fears it could irrevocably alter the human germline.

Led by UW–Madison law professor Alta Charo and MIT biologist Richard Hynes, the committee will specifically advise on questions about how risks should be quantified and whether some aspects of the technology should or should not go forward.

The ability to “edit” genes to target genetic defects became a much more plausible process with the advent of a technology called CRISPR (an acronym for Clustered Regularly Interspaced Short Palindromic Repeats), which can be used to precisely target and cut portions of a DNA sequence.

Controversy arose last year when a Chinese scientific team used CRISPR genome editing on non-viable human embryos. The experiment produced a number of “off-target events” that altered unintended parts of the genome.

Scheufele has published extensively in the areas of public opinion, political communication and public attitudes toward emerging technologies, including nanotechnology, synthetic biology, stem cell research, nuclear energy, and genetically modified organisms. Web of Science lists Scheufele’s publications among the 1 percent most-cited articles in the fields of general social science and plant and animal science. Scheufele also serves on two other committees for the National Academies of Sciences, Engineering and Medicine: a committee on “The Science of Science Communication: A Research Agenda,” and the Division on Earth and Life Studies (DELS) Advisory Committee.

What’s the focus of your panel?

The committee that I serve on deals with human gene editing research and its potential applications. That includes potential future uses that could alter the human germline, which means that edited genes would be passed on to subsequent generations as part of the human gene pool.

But of course there are a lot of applications of gene editing techniques in agriculture and the life sciences, with the attempts to use genetically modified male mosquitoes to combat the spread of the Zika virus being just one recent example.

What are the potential dangers?

Identifying potential problems or concerns is part of the committee’s charge, and our report will work very carefully through both the scientific complexities of the technology as well as ethical, regulatory or political challenges that might emerge. Many of these challenges are focused on specific applications, such as germline editing. Once germline alterations are introduced into the human population, some have argued, they might be difficult to reverse and to contain within a single community or even country.

In many ways, the benefits are much more clear-cut, especially when it comes to helping parents whose genome puts their biological children at risk of inheriting certain diseases. Many patient advocacy groups are especially excited about the potential for medical breakthroughs in this arena.

What is the charge of your study committee? Are there specific deliverables, and what is the timeline?

The National Academies gave the committee a fairly detailed Statement of Task that can be found on our committee’s web page [link provided below]. In short, we will examine the state of the science of human gene editing as well as the ethical, legal and social implications of its applications in biomedical research and medicine.

Our work actually follows a pretty tight timeline that includes a number of additional meetings and informationgathering sessions. Most of the committee deliberations are open to the public and webcast by the National Academies. Once complete, the draft report will be vetted in a very stringent review procedure. There also have been and will continue to be numerous opportunities for formal public input, including on the draft report. If everything goes according to plan, the report will be released in fall 2016.

What role will you play on it as a communication scientist? What expertise do you bring to the table?

Human gene editing shares a number of characteristics with other recent scientific breakthroughs. One of them is an extremely fast bench-to-bedside transition. In other words, the time it takes to translate basic research into clinical or even market applications is shorter than it has been in the past. New gene editing technologies such as CRISPR provide us with faster, cheaper and more accurate tools for gene editing. But that also means that we as a society must have many of the ethical, legal and social debates surrounding gene editing at the same time that we are developing potential applications.

That is why more and more scientists are calling for what Alan Leshner, former CEO of the American Association for the Advancement of Science, has described as an “honest, bidirectional dialogue” between the scientific community and the public. Interestingly, the 21st Century Nanotechnology Research and Development Act of 2003 legislatively mandated public engagement through “regular and ongoing public discussions.” So the idea is not new, and researchers in the Department of Life Science Communication (LSC) at CALS were in fact involved in two long-term NSF center grants examining the societal impacts of nanotechnology and ways of building a better public dialogue. As a result, much of the research teaching we are doing here in the department focuses on how to best facilitate communication about emerging science among all relevant stakeholders in society.

What experiences from past science communication efforts inform your thinking about how best to communicate about gene editing?

Much of our work in LSC over the last few years has examined emerging areas of science that are surrounded by public opinion dynamics similar to what we might see for gene editing. This research has included work on public opinion on embryonic stem cell research, and also research on how non-expert audiences make sense of the risks and benefits of genetically modified organisms. Our research program has also led to regular engagements with policy communities in Wisconsin and in Washington, DC. When I co-chaired the National Academies’ Roundtable on Public Interfaces of the Life Sciences, for instance, I worked with bench scientists, social scientists and practitioners to build a better dialogue about emerging technologies between scientists and the public. g What aspects of gene editing seem to confuse or frighten people the most? We just collected two representative national surveys, tapping people’s views on synthetic biology, gene editing and other scientific breakthroughs. And our findings show that concerns about overstepping moral boundaries with potential applications of gene editing in humans and “blurring lines between God and man,” as the question was phrased, are definitely on people’s minds when thinking about this new technology. In LSC, we will continue to track public attitudes, especially surrounding the societal, ethical and regulatory questions that arise from applications of gene editing.

Obviously people are already reporting, writing, thinking and talking about CRISPR. Do you have any immediate recommendations for how to communicate about this subject?

It will be particularly important to keep two things in mind. First, this is an exciting area for science, but many of the questions and debates surrounding human gene editing will focus on ethical, moral or political rather than scientific questions. And we as scientists should be prepared to engage in those discussions, making sure that they are based on the best available science.

Second, having an honest dialogue among different stakeholders will require a conversation that is—at least in part—about values. And scientists will have to resist the intuitive urge to try and convince others by offering more scientific facts. Our own research and that of many colleagues has shown that the same scientific information will be interpreted very differently by audiences with different value systems. The same science, in other words, means different things to different people. And public reactions to many potential applications of gene editing will be no exception. g

PHOTO – Dietram Scheufele, professor of life sciences communication.

Photo by Sevie Kenyon

Catch up with Andrea Garber

Andrea Garber, PhD,

Andrea Garber, PhD,

As a professor of pediatrics at the University of California, San Francisco, Andrea Garber BS’92 PhD’99 conducted a groundbreaking study concerning a very vulnerable group: patients hospitalized with anorexia nervosa, an eating disorder that often proves fatal. It is most prevalent among teenage girls and young women.

Garber and her team discovered that the standard “refeeding” protocol used in hospitals nationwide—that is, the necessarily careful reintroduction of food to patients who have been starving themselves—was too low in calories and was in fact causing patients to continue losing weight even during longer hospital stays. The phenomenon had been long observed, but Garber’s study, published in 2012 and based on the largest cohort of its kind, was the first to actually prove it.

What makes this work so critical?

Anorexia nervosa is the most deadly psychiatric illness. It has a mortality rate of 5 to 6 percent, which is the highest among all psychiatric diagnoses, and the recovery rates are really low. Studies that are using the absolute best forms of treatment and psychotherapy still show that maybe only 30 percent at the lowest— but at the highest, half—of patients are recovered at one year. So we absolutely need to develop better treatments.

What protocols do you think will be more effective?

In a new five-year study, we are testing a higher-calorie refeeding protocol, starting at 2,000 calories per day and advancing quickly by 200 calories per day. We’ll compare this to a group receiving a lower-calorie protocol, starting at 1,400 calories per day and advancing slowly by 200 calories every other day. This lower-calorie diet is in fact a little higher than the traditional recommendation to start at 1,200 calories.

Why have such low-calorie diets been used with anorexia patients?

For safety. “Low and slow” refeeding is believed to minimize risk for the refeeding syndrome, which was first documented around the time of World War II. It’s characterized by life-threatening shifts in fluids and electrolytes that can occur when nutrition is reintroduced in starved patients. At UCSF and our collaborating site at Stanford, we are set up for a high degree of medical management and we can carefully monitor for any signs of refeeding syndrome. The main one is electrolyte shifts, which our physicians check every day and correct as needed with supplements. A key question is, how much medical intervention is needed to keep higher-calorie refeeding safe? That’s important to know before disseminating these protocols to other settings, such as residential treatment facilities.

Do these protocols have implications for patients after hospital discharge?

We’re looking very closely at relapse rates. Forty percent of these young people relapse within one year of their first hospitalization. If higher-calorie refeeding gives us shorter hospital stays, but these kids end up coming right back, then we’ve undone any potential benefit of that shorter stay. While the higher-calorie refeeding seems promising in terms of faster weight gain and shorter hospital stay, there are many unanswered questions about potential long-term benefits, long-term risks and the overall effect on recovery.

PHOTO – Andrea Garber BS’92 PhD’99 Nutritional Science

A Jolt to the System

As a linebacker for the UW–Madison Badgers, Chris Borland made a name for himself as a hard-hitting tackler. His senior year, he was selected as a first-team All-American as well as the top linebacker and defensive player in the Big Ten Conference.

A third-round draft pick, Borland seemed destined for a headline career in the National Football League. But during a full-contact practice at the San Francisco 49ers summer training camp in August 2014, Borland got his “bell rung” by a 290-pound fullback during a routine exercise. Though Borland felt dazed, he played through—as he’d done dozens of times before.

Like many football players, Borland had endured his share of hard hits, including two diagnosed concussions. This particular hit, however, got him thinking seriously about the future, and about the negative effects that repeated collisions could have on his long-term physical and cognitive health. Even so, he went on to play a dynamite rookie year.

Then, after the season was over, Borland quit.

The announcement shocked the sports world. Borland was 24 years old and healthy, yet chose to walk away from a $2.3 million, four-year contract.

“I just honestly want to do what’s best for my health,” Borland explained on ESPN’s Outside the Lines. “From what I’ve researched and what I’ve experienced, I don’t think it’s worth the risk.”

With their repeated hits, football players—along with boxers—are at increased risk of developing chronic traumatic encephalopathy (CTE), a degenerative brain disease marked by memory loss, depression, suicidal thoughts, aggression and dementia. Of 91 brains donated to science by former NFL players, 87 have tested positive for CTE. It’s seen as a likely contributing factor to nine suicides by current and retired football players over the past decade.

Borland didn’t want to share that fate.

“To me, Chris Borland is a hero. He walked away before he made the big bucks and he was very explicit about why he quit—that it was not worth it to him,” says CALS genetics professor Barry Ganetzky, whose findings about the central nervous system in fruit flies are shedding light on what hard hits do to humans.

Ganetzky isn’t a sports guy, but he started paying attention to football-related brain injuries after the 2012 suicide of New England Patriots linebacker Junior Seau, intrigued by the biological processes driving this tragic phenomenon.

“I started wondering, what’s the link between a blow to the head and neurodegeneration 10 or 20 years down the line? When I started digging into the scientific literature, it became clear that we know very little,” says Ganetzky, who held the Steenbock Chair for Biological Sciences for 20 years. “And my usual response is, well, if we don’t understand something about the brain, then we should be studying it in flies.”

Fruit flies, officially known as Drosophila melanogaster, are a widely studied model organism, with a vast arsenal of genetic and molecular tools available to support that work. Flies reproduce rapidly and are easy to work with, enabling swift research progress. They are well suited for brain research because they have nerve cells, neural circuitry and a hard skull-like cuticle remarkably similar to our own, allowing scientists to conduct probing experiments that would be difficult in rodent models—and impossible in human subjects.

Fly models already exist to study Alzheimer’s, Parkinson’s and a number of other neurological diseases. Why not concussion? But there wasn’t a model available.

Then Ganetzky remembered work he’d done decades earlier.

“It occurred to me that I knew how to make flies have a concussion, and I had done it 40 years ago as a post-doc,” says Ganetzky. “I thought, ‘That’s it!’”

It was a simple thing: As a post-doctoral researcher at the California Institute of Technology, Ganetzky decided to see if any of his flies happened to be bang-sensitive mutants, flies that display seizures and paralysis after given a high-powered swirl on a vortex machine. But he didn’t have a vortex nearby, so he decided to just bang the vials against his hand.

“After a couple of sharp whacks, some of the flies were hanging out at the bottom of the vial, stunned. Others were on their backs, obviously knocked out. And after a few minutes, they all got up and started walking around again,” recalls Ganetzky.

He immediately knew the flies weren’t bang-sensitive—it’s an extremely rare mutation—but Seau’s death helped Ganetzky realize they had displayed symptoms “very similar in many respects to the empirical definition of a concussion.”

After developing and validating the new fly model, Ganetzky and UW genetics professor David Wassarman have been able to charge forward with brain injury research. The model has already been used to reveal key genes involved in the body’s response to brain injury. It’s also poised to help unlock medical applications, including a genetic test for high-risk individuals and an assortment of promising drugs and treatments.

In addition to helping athletes in contact sports, these advances will benefit the millions of Americans each year who experience traumatic brain injury due to falls, car accidents and violent assaults.

“At the most fundamental level, we just want to understand how traumatic brain injury works,” explains Ganetzky. “However, this is a major medical problem for which there are not many good—or any good—treatments or therapies or preventives, and so that is part of our motivation. If we can learn the genes and the molecules and the pathways, can we come up with interventions?”

Ganetzky was raised in a working-class neighborhood in Chicago by a candy salesman father and a homemaker mother. Growing up, he had an abundance of natural curiosity and asked a lot of tough questions—and often questioned the answers he received. While this trait caused him some problems as a youth, it came to serve him well in science.

At the University of Illinois in Chicago, he figured he’d become a chemist for the good career prospects. He ended up switching to the biological sciences, however, after a 10-week honors biology research experience in a Drosophila lab that expanded into a two-year project. From that point forward he stuck with flies, earning his doctoral degree at the University of Washington and then doing his post-doc work at Caltech.

In 1979, Ganetzky joined the University of Wisconsin–Madison, where he chose to focus his research program on exploring temperature-sensitive paralytic mutants, flies that behave normally at room temperature, but then start to tremble and twitch—or pass out—when things heat up. For each mutant he identified, he sought to uncover the faulty gene involved, and thus better understand how brain cells work.

Over the decades, this approach enabled Ganetzky’s team to discover a number of critical genes and molecular pathways involved in brain cell signaling, including those required for the release of neurotransmitters. That body of work established Ganetzky as one of the foremost leaders in neurogenetics. Some of his findings shed light on human genetic diseases and led to a test that’s now routinely used to assess the safety of new pharmaceutical drugs. For his contributions, Ganetzky was elected in 2006 to the National Academy of Sciences, the nation’s preeminent scientific society.

After Ganetzky’s “eureka moment” about fly concussions in spring 2012, he immediately reached out to colleague David Wassarman, a genetics professor in the UW–Madison School of Medicine and Public Health. Wassarman, who studies human neuronal disorders using fruit flies, had already been attending Ganetzky’s lab meetings for a few years after some of their research findings linking the innate immune response and neurodegeneration dovetailed.

“I did a demonstration of fruit fly concussion for David, and I remember his response very well,” says Ganetzky. “His jaw kind of dropped, and he said, ‘If you’re not going to study that, then I want to.’”

It was exactly the response that Ganetzky had been hoping for. With retirement looming on the horizon, Ganetzky needed a trusted and enthusiastic collaborator to help pursue the work—someone who would be willing to take on more and more as time went on. Wassarman was game.

“I wanted to put both feet in,” says Wassarman. “I said, ‘If we’re going to do it, let’s do it.’”

As a first order of business, Wassarman developed a tool capable of delivering a consistent “dose” of brain injury to flies. The result, known as the High-Impact Trauma (HIT) device, utilizes a metal spring to slam a vial of flies against a firm foam surface. In this setup, it’s important to note, the brain injury the flies experience is caused by the rapid acceleration and deacceleration of their bodies; it’s not necessarily about a direct hit to the head.

“Quite often, as with football players, it can happen because they are running fast and then meet an immovable object. The concussion is caused by a kind of whiplash, where the brain is ricocheting off the inside the skull, and that’s what’s causing the damage,” says Ganetzky. “That’s what we’re doing here with the flies.”

Ganetzky and Wassarman found that flies injured using the HIT device exhibit many of the classic symptoms of traumatic brain injury (TBI) seen in humans. As they reported in the Proceedings of the National Academies of Science in 2013, flies show temporary incapacitation and loss of coordination immediately after injury. Those that survive severe injury go on to develop long-term symptoms: activation of the innate immune response, neurodegeneration and early death.

These TBI flies have the potential to reveal much-needed insights—and medical interventions—for the millions of Americans who experience traumatic brain injury each year. According to the U.S. Centers for Disease Control and Prevention, TBIs cause around 2.5 million emergency room visits, 283,600 hospitalizations and 52,800 deaths each year. Top causes are falls, motor vehicle accidents, and blows or jolts to the head or body, including sports-related concussions. Bomb blasts can cause brain trauma in soldiers in combat zones. Across the country, as many as 6.5 million people are believed to be struggling with the consequences of TBI, and the total economic cost of this health issue is estimated to be $76 billion per year.

In a demonstration of the power of the TBI model, Rebeccah Katzenberger, a senior research specialist in Wassarman’s lab, subjected 179 genetically unique strains of flies to four strikes of the HIT device—meant to simulate a series of severe brain injuries—and then monitored them for death at 24 hours post-injury, a data point that serves as an easy-to-measure proxy for the various negative events unfolding inside the body.

The results revealed a huge diversity of responses, underscoring the fact that genotypes matter when it comes to TBI response. Some strains were particularly susceptible to death, losing as many as 57 percent of the flies in those first 24 hours, while others were much more resilient, losing just 7 percent. The team then identified the genes that possibly made a difference, publishing their findings in eLife in March 2015.

“Now we have these 100 genes, and scientists can start looking at them in more detail,” says Wassarman. “A lot of them are genes that had never really been implicated in traumatic brain injury before. I think this is going to be one of our big contributions.”

These findings, the researchers note, may help explain why people respond so differently to similar brain injury events, and may help lead to a genetic test to identify high-risk individuals.
“Once we understand those genetic links, we’ll be able to test people and tell them, ‘Look, you probably shouldn’t play football. You should play non-contact sports,’” explains Ganetzky.

After identifying the TBI genes, Ganetzky and Wassarman immediately noticed a handful of genes involved in tissue barrier regulation. Tissue barriers—such as the intestinal barrier and the blood-brain barrier—function as biological blockades keeping “bad” things out while allowing “good” things to pass through.

To explore the connection between brain injury and tissue barriers, the duo had Katzenberger conduct a simple, colorful experiment that involves adding bright blue dye to the flies’ food. Under normal conditions, when flies eat the blue-colored food, it stays in the gut, something that is readily observable through the fly exoskeleton. However, after exposure to brain injury—via the HIT device or by having their heads pinched with a forceps—they found that the dye leaks out of the gut and turns the entire body blue, a phenomenon called “smurfing” (after the blue Smurf cartoon characters).

Leaky tissue barriers have previously been observed in rodent models of brain injury as well as in human medical cases. “Somehow this injury to the brain is triggering a series of events that leads to the breakdown of the intestinal barrier,” notes Ganetzky. “So there’s some sort of cross-talk going on between the brain and the intestine, but we don’t fully understand it yet.”

Upon further exploration, Ganetzky and Wassarman were able to confirm that—along with the blue dye—glucose and bacteria were also crossing the intestinal barrier into the fly’s circulatory system, or hemolymph, after brain injury. Homing in on glucose, they found that it plays a causative role in fly death after TBI. “By simply withholding sugar, we were able to keep some of these flies alive, and by a substantial margin,” says Wassarman.

If the findings hold up in rodent models and in human trials, he notes, athletes may one day find themselves advised to avoid certain foods after experiencing concussion.

The bacteria that cross the intestinal barrier appear to be playing more of a long game. Ganetzky and Wassarman believe they are the culprits triggering the innate immune response observed in TBI flies. The innate immune response, also known as the inflammatory response, is the body’s natural reaction to microbial invasion and other stressors. If properly controlled—turned on and off at the right time—it protects the body. If left on, however, it can cause collateral damage throughout the body, including damaging brain cells.

“Here’s what we think is happening: Traumatic brain injury is causing increased intestinal permeability. That causes the bacteria to leak out, which turns on the innate immune response, and that is possibly leading to neurodegeneration down the line,” explains Wassarman.

Ganetzky and Wassarman are intrigued by a concept that is emerging from their work and related studies: that TBI accelerates aging. Some of the key physical outcomes of brain injury—problems with tissue barriers and increased inflammation—are also hallmarks of the natural aging process. More support for this idea came in summer 2015, with the release of a report describing signs of early aging in the brains of war veterans exposed to bomb blasts in Iraq and Afghanistan.

“Somehow a blow to the head is activating all of these pathways related to aging and speeding them all up. Biologically, I think that this is maybe one of the most fascinating things about the whole project,” says Ganetzky, noting that TBI flies are a great model for further exploration.

Even at this early stage, without fully understanding the basic scientific mechanisms involved, the model is already revealing some promising medical applications. As soon as Ganetzky and Wassarman realized that the inflammatory response might lead to neurodegeneration, a treatment suggested itself: Could a simple anti-inflammatory help? They tried giving TBI-injured flies some aspirin mixed in their food. It helped.

“Our studies show that there appears to be a window of time after brain injury when the flies are particularly susceptible to dying. And if we can prevent certain events from happening during this time, then we can prevent death,” says Wassarman. “That’s what we think aspirin is doing—by lowering the innate immune response.”

The next step is to look for drug candidates that work even better than aspirin. Ganetzky and Wassarman are in the process of screening a set of 2,400 compounds, and they’ve already found a handful of very promising ones that can now be tested in rodent models and, ultimately, in human clinical trials.

“It would be wonderful if someday it were possible to offer a simple intervention beyond surgery to help individuals who have suffered a severe traumatic brain injury,” says Wassarman.

There’s a lot left to learn, and Ganetzky and Wassarman are eager to pursue all that the model can tell them. With Ganetzky’s retirement set for early 2016, the work of securing the project’s first federal grant and conducting experiments will largely fall to Wassarman.

But Ganetzky won’t be out of the picture. He continues to keep up on brain injury medical cases and scientific discoveries, and is encouraged by the national conversation about sports and brain injuries that’s starting to gain traction—and by the NFL’s commitment to scientific research in this area.

Some of these advances can be attributed, in part, to Chris Borland, whose post-NFL journey has led him deeper into the world of sports-related brain injury. Borland has submitted to numerous brain scans to support research, and has also become a sought-after speaker, touring the country to raise awareness about the risks of concussion.

It’s that kind of dedication to public service on the part of Borland and many other athletes, along with the excitement of discovery, that’s keeping Ganetzky in the game. Despite his retirement, Ganetzky plans to keep a scaled-back version of his lab running for at least a few more years.

Going for the Gut

How do we keep food animals healthy when bacteria and other pathogens are so good at outsmarting drugs intended to work against them?

In an innovation that holds great promise, CALS animal sciences professor Mark Cook and scientist Jordan Sand have developed an antibiotic-free method to protect animals raised for food against common infections.

The innovation comes as growing public concern about antibiotic resistance has induced McDonald’s, Tyson Foods and other industry giants to announce major cuts in antibiotic use in meat production. About 80 percent of antibiotics in the United States are used by farmers because they both protect against disease and accelerate weight gain in many farm animals.

The overuse of antibiotics in agriculture and human medicine has created a public health crisis of drug-resistant infections, such as multidrug-resistant Staphylococcus aureus (MRSA) and “flesh-eating bacteria.”

“You really can’t control the bugs forever; they will always evolve a way to defeat your drugs,” says Cook.

Cook and Sand’s current work focuses on a fundamental immune “off-switch” called Interleukin 10 or IL-10, manipulated by bacteria and many other pathogens to defeat the immune system during infection. He and Sand have learned to disable this off-switch inside the intestine, the site of major farm animal infections such as the diarrheal disease coccidiosis.

“People have manipulated the immune system for decades, but we are doing it in the lumen of the gastrointestinal system. Nobody has done that before,” Cook says.
Cook vaccinates laying hens to create antibodies to IL-10. The hens transfer the antibody to their eggs, which are then blended, pasteurized and sprayed on the feed of the animals he wants to protect. The antibody neutralizes the IL-10 off-switch in those animals, allowing their immune systems to better fight disease.

In experiments with more than 300,000 chickens, those that ate the antibody-bearing material were fully protected against coccidiosis and other gastrointestinal diseases that commonly affect poultry.

Smaller tests with larger animals also show promise. In one example, animal sciences professor Dan Schaefer and his graduate research assistant, Mitch Schaefer, halved the rate of bovine respiratory disease in beef steers by feeding them the IL-10 antibody for 14 days.

Cook and Sand, who have been working on the IL-10 system since 2011, are forming Ab E Discovery LLC to commercialize their research. One of the four patents they have filed through the Wisconsin Alumni Research Foundation has just been granted, and WARF has awarded a $100,000 Accelerator Program grant to the inventors to pursue the antibiotic-replacement technology. The Discovery to Product partnership between UW and WARF played a key role in helping Cook and Sand prepare it for commercialization.

Cook has already turned his research and some 40 patented technologies into start-up companies including Aova Technologies, which improves animal growth and feed efficiency, and Isomark LLC, which is developing a technology for early detection of infection in human breath.

PHOTO: Eggs from these hens contained antibodies that were used to test the antibiotic replacement. (Photo courtesy of Mark Staudt, WARF)

Bitten

There’s no ignoring it. Some of the students enrolled in this medical entomology class are far more attractive than others. They know it, their classmates know it, and so does Susan Paskewitz, professor and chair of the Department of Entomology.

Paskewitz describes herself as “relatively unattractive,” and she proceeds to prove it using the same test her students have just performed. She fills a small vial with warm water, rubs it between her palms to coat it with volatile compounds from her skin, then places the vial on top of a thin membrane stretched over the top of a plastic container akin to an economy-sized ice cream tub. She invites a visitor to do the same.

Waiting on the other side of that membrane are 20 blood-starved specimens of Aedes aegypti, commonly known as the yellow fever mosquito. Hungry as they are, the insects don’t show a lot of interest in Paskewitz’s vial. They hover near where it touches the membrane, but only two or three land. The visitor’s vial, on the other hand, is a busy spot. At least a dozen have landed and are testing the surface with their needle-like proboscises.

“Wow,” says Paskewitz. “You’re really attractive!”

In another context, those three words could make your day. But not here. Nobody wants this kind of animal magnetism. Nobody wants to be the person who’s cursing and slapping and reaching for the DEET while others are calmly eating their brats and potato salad.

If you’re that person, take heart. Paskewitz can tell you a little bit about why you might have more than your share of interspecies charisma and offer some suggestions on how to scale it back. But first, let’s talk about why this matters.

An average American adult outweighs an average-size mosquito by about 30 million to one. Ounce for ounce, that’s like the USS Nimitz vis-a-vis a good-size duck. But while it’s a safe bet that a 100,000-ton aircraft carrier won’t change course to avoid a six-pound mallard, it’s almost certain that, on a regular basis, you change your behavior to avoid being bitten by a 2.5-milligram mosquito.

Mosquitoes cause us to do things we’d rather not, like dosing ourselves with a repellent that’s sticky and smelly and comes with a sobering warning label (you can apply it to your kids’ skin, but keep the bottle out of their reach), or pulling on long pants, long sleeves, a hat and maybe a head net on a sweltering midsummer day.

Mosquitoes keep us inside when we’d much prefer to go out. In the summer of 2009, Paskewitz and environmental economist Katherine Dickinson, of the Colorado-based National Center for Atmospheric Research, asked a sample of Madison residents how they coped when mosquitoes got fierce.

The second-most-common answer (right after applying repellent) was to stay indoors. About two-thirds of the respondents said they had curtailed outdoor household activities—gardening, yard work, sitting on the deck—in the past month because of mosquitoes, especially in the evening hours, which, for working people, may be the only time available to get a little fresh air. About a third said they had avoided outings, and a similar share said they had avoided outdoor exercise.

Nobody wants to be outside more than John Bates, of Manitowish. An author of seven books about Wisconsin’s north woods and a naturalist by trade, Bates leads interpretive hikes year-round—except in June: “We just kind of throw the month out. The mosquitoes cause too much discomfort for people to listen to interpretation. All we can do is keep walking. People hire me because they want to learn more about the place than they knew before they came. If they can’t stop to listen, what’s the point?”

If we do venture out when mosquitoes are massing, we may not get the experience we were hoping for. Andrew Teichmiller, an outfitter of bikes and paddling gear in Minoqua, recalls mountain biking in 2014, arguably the area’s worst mosquito year ever. “You had to ride the complete trail without stopping, all the way back to the parking lot, and jump in the car, quick, because if you stopped there were 15 or 20 mosquitoes on you immediately.” As for camping: “It’s a different type of experience when you can’t sit by the fire at night and tell stories. You’re forced to run for your tent. It definitely affects the feel of the trip.”

But let’s be clear: A ruined camping trip is far from the worst possible consequence of a mosquito bite.

Mosquitoes transmit diseases that kill nearly a million people every year and sicken hundreds of millions. Tropical and subtropical areas bear the brunt of this, but no place is immune, including Wisconsin. Malaria plagued the immigrants who settled in Wisconsin in the 1800s, and various types of encephalitis are diagnosed on a regular basis.

But today the biggest concern is West Nile virus (WNV). Wisconsin has been relatively lucky since the first case arrived here in 2002, with a total of 230 cases reported through 2014. But all four adjacent states have had bigger outbreaks—notably Illinois, with 2,093 cases total and 884 in its worst year, most of them just across the border in the Chicago area. Wisconsin’s worst year brought 57 cases.

Most cases of WNV bring no symptoms, according to the Centers for Disease Control, but about one in five can involve a fever, headache, body aches, vomiting and a fatigue that can last for weeks or months. Fewer than 1 percent of WNV victims display severe neurologic symptoms, including disorientation, coma, tremors, seizures or paralysis, and of those, about 1 percent die.

Nevertheless, Wisconsin residents are bothered much more by the nuisance of biting mosquitoes than they are worried about West Nile virus. The Madison residents responding to Katherine Dickinson’s 2009 survey said they’d be willing to pay an average of $149 for a hypothetical program to control nuisance mosquitoes, but wouldn’t pay anything for one targeted at mosquitoes carrying WNV when risks were as low as they were at the time (about one case per year in Madison with a population of 250,000).

It’s not surprising to find that attitude in Wisconsin, where mosquito-borne disease is relatively rare, but Dickinson says that people tend to think the same way in places where mosquito bites are often fatal. She observes that in Tanzania, biting mosquitoes were a major factor motivating people to use bed nets. “It was a similar situation to ours,” she says. “Some mosquitoes are more noticeable and more of a nuisance, but those that transmit malaria are kind of sneaky; people don’t feel them biting as much. In areas where mosquitoes were more of a nuisance, people used the bed nets more.”

Biting-wise, there’s an important distinction between nuisance mosquitoes and the ones that transmit WNV. The former come at us aggressively, in such staggering numbers that they’re impossible to ignore. They remind us to protect ourselves. Culex pipiens, the WNV vectors, are more subtle and harder to notice.

Nuisance mosquitoes and the WNV carriers also show up at different times. The most annoying biters—Aedes vexans in particular—are floodwater species that breed after a stretch of wet weather. Culex breed in water that stagnates during a dry spell.

“When it’s been really dry, the water just sits in the stormwater catch basins that are the biggest sources of the WNV vectors,” says Paskewitz. “There’s not enough rain to flush them. Things get more fetid, stinkier. That’s the year when we see a ton of Culex.”

The take-home message: If you only grab the DEET when the biting is so bad that you can’t stand to be without it, you’re not protecting yourself against West Nile virus.
“You need to protect yourself against bites even if you’re not getting a lot of them,” says John Hausbeck, director of environmental health services for Dane County and the City of Madison. “We’ll see summers where it’s really dry and the floodwater mosquitoes are very limited, but we still have plenty of small pools that the Culex can breed in.”

That “biting pressure” is something that Hausbeck needs to stay on top of, and Paskewitz helps with that. She and former grad student Patrick Irwin PhD’10 were able to characterize the types of sites where Culex are most likely to breed and identified alternatives for treating them—for example, introducing fathead minnows to feed on Culex larvae. She and her students analyze the mosquitoes trapped in the area to see how many are Culex and whether they’re carrying WNV. Their data tell Hausbeck whether he needs to issue a public alert.

It’s important to remain vigilant. “When West Nile first came into the country, people doubted it would make it through the first winter,” Paskewitz says. “Well, it did persist, and in a very short period of time it whipped across the whole country. We’ve had a lot of cases in new places. First it was really bad in North and South Dakota. Then Colorado and Arizona. Then Texas, Illinois. It’s really hard to predict. And given the vagaries of climate, we just don’t know whether the next year it might be Wisconsin.”

Maybe WNV hasn’t changed Wisconsin residents’ ideas about why to guard against mosquito bites, but it certainly has spurred a lot of questions about how. There is a seemingly endless list of products and strategies, that, according to somebody, will eliminate mosquitoes or repel them—and since WNV arrived, Paskewitz has been getting questions about pretty much all of them.

“They call me to ask, ‘Would this work or wouldn’t it?’ There is a lot of misinformation out there and not many good sources of information, so I realized I needed to get a better idea of what the science was behind these things,” Paskewitz says.

As she comes up with answers, she posts summaries online. Her website, http://go.wisc.edu/mosquitoes, gets plenty of visits (55,000 last year) and triggers a lot of calls from media from across the nation.

A few of her findings:

• Repellents can be very effective, but comparing them is tricky. There are lots of products with varying active ingredients offered in different concentrations and combinations. Generally speaking, DEET, Picaridin, IR3535, and oil of lemon eucalyptus have good track records. There are also a number of other plant-based compounds—garlic, catnip oil, vanilla and oil of cloves, for example—for which there’s less research and conflicting results. The website sums all this up and gives links to more information.
Yard traps get a thumbs-down. “We tested those and didn’t get any positive outcome,” Paskewitz says. Yard traps lure mosquitoes by releasing C02, light or octenol, a compound contained in our breath and sweat. Sure, they can catch mosquitoes by the hundreds, Paskewitz says. But does this significantly reduce the numbers that bite you? Properly controlled studies say “no.”

• “Sonic” devices—wristbands, smartphone apps, etc.—do better at extracting your money than keeping mosquitoes off your deck. “You can test them yourself,” Paskewitz says. “Sit at the picnic table and count how many mosquitoes land on you, then turn on the device and count again. Or you can trust the research and save your money.”

• Bats are busted. The idea that a colony of bats can consume millions of mosquitoes per night came from a study in which someone put a bat in a room full of mosquitoes and estimated how many it ate. The question is, given the choice, is that what bats eat in the wild? Researchers who examined the stomach contents and fecal pellets of bats have found bigger insects, like butterflies, moths and beetles, but very few mosquitoes. “Bat houses are great for conserving bats,” Paskewitz says, “but not for mosquito control.”

• Avoiding bananas—When she first heard the idea that eating bananas makes you more attractive to mosquitoes, Paskewitz raised her eyebrows. “I thought, okay, we’ll debunk that,” she says. She was teaching medical entomology at the time with 24 students—enough for a robust sample—so she made it a class project. For several weeks, each student ate a banana and then performed an attractiveness assay at prescribed intervals. “We were really intrigued. It did look like we were getting an increase a couple hours after eating the bananas.”

Paskewitz repeated the trial the next two times the course was offered, with a few tweaks to the methodology: Half the students ate bananas, the other half grapes. “The third trial was the best of all—the strongest statistical evidence and the most repeatable,” Paskewitz says. “We did it three times and saw a strong difference between the groups. Grapes didn’t matter, bananas did. At that point I was convinced. I think it’s real,” she says. Does that mean you if you leave bananas out of your picnic fruit salad, you can skip the bug spray? Probably not, Paskewitz says.

Because “less attractive” is not the same as mosquito-proof, Paskewitz gets plenty of mosquito bites, probably more than her share, because she spends a lot of time around mosquitoes—in the woods doing field research, in her garden, and in her lab. When you’re a mosquito researcher, getting bitten comes with the job.

What Makes You Attractive?

It sounds like the topic of an article in Seventeen magazine—and, interestingly, some of the same general categories apply whether you’re talking about your appeal to a mosquito or to a certain someone of your own species.

Your breath. If you breathe, you’re mosquito bait. Every breath adds to a plume of carbon dioxide (CO2 levels in your breath are 100 times that of the atmosphere) emanating from where you stand. “That’s the big signal,” says entomology professor Susan Paskewitz. “Insects are very sensitive to chemical cues. They’ll zigzag to pick up the chemical as it gets stronger and stronger, circling to narrow in on you.”

Your aroma. Once they find you, mosquitoes use chemical cues to decide whether to land and dig in. They have a lot to sort through: You emit roughly 400 different compounds from your skin and 200 in your breath. Many mosquito species won’t land on humans, even if they’re starved for blood. Others will bite us in a pinch but prefer other hosts, Paskewitz says.

Your genes. Perhaps you were born to be bitten. A pilot study at the London School of Hygiene & Tropical Medicine found that identical twin sisters were significantly more alike in their attractiveness to mosquitoes than were non-identical twins. Since identical twins are closely matched genetically, this suggests that some of your Culicidae charisma is inherited. Some volatile compounds on our skin are produced by skin cells (others are produced by bacteria), which would be gene-regulated, the study’s authors note.

Your jeans. What color you wear matters. This is based on a series of studies in which researchers draped different colors of cloth on human volunteers or on robots heated to simulate human body temperatures, then counted mosquito landings. For the most part, darker colors were more attractive. White was least attractive, followed by yellow, blue, red and black.

Your smelly feet. “The malaria mosquito is really attracted to the smell of funky feet,” Paskewitz says. “It’s a classic story in medical entomology. The compound that makes feet smell funky and attractive to mosquitoes is the same one that causes Limburger cheese to smell the way it does.” That compound is produced by bacteria that can accumulate in the moist spots between your toes, and are kin to those used to culture Limburger.

Your drinking habits. A number of researchers speculate that drinking alcohol makes you more attractive to mosquitoes. A team in Japan put this to the test. They asked some volunteers to drink 350 ml of beer while a control subject did not. The percentage of mosquito landings after alcohol consumption increased substantially. Why this happens is unresolved, although some have speculated that people who have been drinking are easier targets because they move more slowly.

Getting Under Your Skin

Maybe you don’t get more mosquito bites than other people. Maybe your body just makes a bigger deal of it. The swelling, redness and itching are signs of your immune system kicking into gear, explains Apple Bodemer, an assistant professor of dermatology at the UW–Madison School of Medicine and Public Health. And some people’s immune systems kick harder than others.

A mosquito bite involves give and take. Before drawing out up to .001 milliliters of your blood, the mosquito injects a bit of its saliva, which contains anticoagulants to prevent clotting. You can spare the blood, but the saliva is a problem. That’s how disease gets transmitted. And the saliva contains foreign proteins, or antigens, that spur your immune system to create antibodies, Bodemer explains. “When antibodies bind to the antigens, it initiates an inflammatory response, which includes the release of histamine, which causes the blood vessels to dilate, which brings the swelling and redness and the inflammatory mediators that are responsible for the itching.”

This doesn’t happen the first time you’re bitten. It’s the second time, when your body has built up the antibodies, that your immune system engages. If you get bitten enough times by the same strain of mosquito, you may become desensitized and have either a very mild reaction or no reaction at all to the bites. “People often have more vigorous immune responses early in the season and then, as the summer goes on, they don’t have as much swelling and redness and itching,” Bodemer says. “But when you go a winter without any exposure, you often become resensitized.”

For the same reason, younger kids tend to have more aggressive reactions. Once they’ve had several years of mosquito exposure, their response tends to die down, Bodemer says.
As for scratching? Doctor’s orders: Don’t! “Scratching really promotes the full inflammatory reaction. It causes more irritation, causing the blood vessels to be more dilated and further dispersing the inflammatory mediators. It initiates a cycle of swelling, redness and itching. If you can avoid scratching, a lot of times the bumps will disappear.”

Antihistamines can ease the itching, she says, or you can try a home remedy: “I paint a little clear nail polish on the mosquito bite. That will stop the itching to some degree and allow the inflammation to clear up more quickly,” Bodemer says. “Some people cover the bite with Scotch tape for two to four hours. The tape stops you from scratching and when you peel it off, it removes some of the mosquito saliva.”

Wisconsin’s Pestilent Past

Wisconsin’s 19th-century settlers knew that mosquitoes were biting them, and they knew that something was making them sick—but they didn’t put the two together.

Their doctors blamed the ailment on “malarial vapors” emitted by decaying vegetation in the swamps, according to Peter T. Harstad, a UW–Madison educated historian who authored several articles on the health of Midwestern settlers. Harstad used reports by military and civilian doctors as well as immigrants’ diaries and letters to chronicle the devastation caused by what was sometimes called “intermittent fever” because the symptoms—chills, aches and a general fatigue—often recurred over a period of months or years.

“I became sick as soon as I came here and have been sick for eighteen months with malarial fever, which is very severe and painful and sometimes fatal,” reads one letter excerpted by Harstad, written in 1941 by a resident of Muskego. “My wife and I are now somewhat better, but far from being well. This year seventy or eighty Norwegians died here … Many became widows and fatherless this year.” About 13 percent of Muskego’s population died that year, Harstad estimates. The town was hard hit because of an abundance of marshes, a relatively warm climate, and the fact that Norwegian immigrants had no resistance to the disease.

Soldiers also suffered. Harstad cites army reports of malaria outbreaks as far north as Ft. Snelling, near present-day St. Paul. Hardest hit was Ft. Crawford, located amid miles of Mississippi River wetlands at Prairie du Chien. In the fall of 1930, there were about 150 cases reported among the 190 soldiers stationed there. To treat the disease, army surgeons were directed to “extract from twelve to twenty ounces of blood, an operation which it is sometimes required to repeat once or twice.” Wisconsin was mostly malaria-free by the end of the 19th century, as farmers drained wetlands and better housing shut out mosquitoes.

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.

Uganda: The Benefits of Biogas

Generating enthusiasm for a new kind of technology is key to its long-term success. Rebecca Larson, a CALS professor of biological systems engineering, has already accomplished that goal in Uganda, where students at an elementary school in Lweeza excitedly yell “Biogas! Biogas!” after learning about anaerobic digester systems.

Larson, a UW–Extension biowaste specialist and an expert in agricultural manure management, designs, installs and upgrades small-scale anaerobic digester (AD) systems in developing countries. Her projects are funded by the Wisconsin Energy Institute at UW–Madison and several other sources. Community education and outreach at schools and other installation sites are an important part of these efforts.

Children get excited by the “magic” in her work, she says. “It’s converting something with such a negative connotation as manure into something positive,” Larson notes. In an AD system, this magic is performed by bacteria that break down manure and other organic waste in the absence of oxygen.

The resulting biogas, a form of energy composed of methane and carbon dioxide, can be used directly for cooking, lighting, or heating a building, or it can fuel an engine generator to produce electricity.

Larson’s collaborators in Uganda include Sarah Stefanos and Aleia McCord, graduate students at the Nelson Institute for Environmental Studies who joined forces with fellow students at Makarere University in Kampala to start a company called Waste 2 Energy Ltd.
Along with another company, Green Heat Uganda, which has built a total of 42 digesters, Waste 2 Energy has helped install four AD systems since 2011.

“Most of these digesters are locally built underground dome systems at schools and orphanages,” Larson explains. Lweeza’s elementary school is a perfect example.

The AD systems use food waste, human waste from pit latrines and everything in between. The biogas generated by the digester is run through a pipeline to a kitchen stove where the children’s meals are prepared. Compared to traditional charcoal cooking, the AD systems greatly reduce the school’s greenhouse gas emissions.

Larson and her team are now focusing on enhancing the efficiency and environmental benefits of these systems. Their goals are to improve the digester’s management of human waste, reduce its water needs, increase the amount of energy it produces and generate cheap fertilizer to boost food crop yields.

“Our overall goal is to create a closed-loop and low-cost sustainability package that addresses multiple local user needs,” Larson says.

The beauty of the project is that all these needs can be met by simply adding two new components to the existing systems: heating elements and a solid-liquid separator.

To help visualize the impact of the fertilizer, Larson set up demonstration plots that compare crop yields with and without it. Down the road, a generator could be added to the system to provide electricity in a country where only 9 percent of the population currently has access.

As a next step, Larson hopes to replicate the project’s success in Bolivia. She is finalizing local design plans with Horacio Aguirre-Villegas, her postdoctoral fellow in biological systems engineering, and their collaborators at the Universidad Amazonica de Pando in Cobija.

Class Act: Keven Stonewall

Some researchers first find success late in their careers. And then there’s Keven Stonewall.

Now a rising junior majoring in biology, Stonewall made news with research he did while still in high school. A headline in the New York Daily News declared, “Meet the Chicago Teen Who May Cure Colon Cancer.”

Stonewall’s research, which he conducted as an intern at Rush University while he was a senior at the Chicago High School for Agricultural Sciences, revealed that an experimental colon cancer vaccine effective in younger mice did not work in older mice. Stonewall won numerous awards for his work and was selected as a finalist for the Intel International Science and Engineer Fair in 2013.

Stonewall, the child of two public school teachers, had always loved science, but while in high school, a close friend’s painful experience losing an uncle to colon cancer made Stonewall determined to fight the disease. “It motivated me to say, ‘Enough is enough, I want to step up and do something about it,’” he says.

More recently Stonewall’s interest has moved toward curing cancer in children. He spent his sophomore year as a student researcher in the lab of Christian Capitini, a pediatric oncologist with the UW–Madison School of Medicine and Public Health. There he worked with mice to study the use of natural killer cells to treat neuroblastoma, a cancer frequently seen in children.

“He has a very advanced understanding of immunology and the immune system,” Capitini says of Stonewall. “He understood the concepts of the project from the beginning, so he could get his hands dirty a lot faster than the typical student.”

And this summer he’s interning with AbbVie, a research-based biopharmaceutical company, at its North Chicago headquarters.

Stonewall is in cancer research for the long haul, and he wants to pursue it as a physician. “My goal is to go to medical school, and I am thinking of going into pediatric oncology afterward,” he says.

Catch Up With … Luxme Hariharan BS’04 Biology

As a pediatric ophthalmologist, clinical researcher and child advocate, Luxme Hariharan has set herself a challenging goal: To prevent childhood blindness globally and help those with imperiled vision to see better. Born in Hyderabad, India, Hariharan graduated with bachelor’s degrees in biology from CALS and in Latin American, Caribbean and Iberian Studies from the College of Letters and Science. She went on to earn an M.D. at the UW School of Medicine and Public Health and a master’s in public health from Johns Hopkins.

While still in medical school she helped establish an eye-care program in Mysore, India, with the organization Combat Blindness International. It was there that she recognized the global impact she could have as an ophthalmologist. “I will never forget witnessing the wonder of a man who received free cataract surgery and exclaimed, ‘Now I can finally see what my granddaughter looks like!’” she says.

Hariharan also has worked on blindness prevention programs in Argentina, El Salvador and Niger and has collaborated on vision-saving initiatives in Armenia and the Philippines.

A recipient of a “Forward under 40” award from the Wisconsin Alumni Association, Hariharan is currently the Pediatric Cornea, Cataract and Glaucoma Fellow at the Children’s Hospital of Los Angeles.

• What led to your interest in pediatric ophthalmology?

I truly love the opportunity to help change the trajectory of a child’s life by helping to maximize
their vision. I remember one child in particular who was held back a grade because teachers thought he was not interested in school. It turned out that he just could not see well. Once he got the correct glasses prescription he was the most lively and participatory child in the class, and his grades drastically improved. I saw the direct impact vision can have on a child’s overall growth and development. I was also excited to learn the intricate surgeries involved to treat pediatric ophthalmic conditions in combination with clinical care.

• What aspects of your work do you find the most challenging?

When we are not able to offer a permanent treatment or cure for certain disorders, and despite our best efforts, a child may eventually go blind. This is very challenging to witness in a young child. According to the World Health Organization, every five seconds a child somewhere in the world goes blind. Over a third of these children never graduate from high school, and half will grow up to become part of the permanently unemployed. The burden that childhood blindness places on society extends far beyond vision impairment alone and has significant social and economic impacts on families, communities and countries worldwide.

• What can we do to help address this problem, beyond making sure every child has regular vision screening?

It’s important for everyone to have an idea of the types of avoidable and treatable causes of childhood blindness. Eighty percent of childhood blindness is preventable. A child’s visual system fully develops by the time he or she is 9 or 10 years old, and up until that time it is possible to improve vision via treatments such as glasses, patching and possible surgery to maximize visual potential. After age 10, however, whatever visual acuity a child has is not likely to change. Therefore, early detection of ophthalmic conditions in children is vital in preventing them from developing further visual impairment and blindness.

To learn more or to donate to childhood blindness prevention programs, Hariharan welcomes your questions at lhariharan@chla.usc.edu.

To Eat It—Or Not

Food engineer Sundaram Gunasekaran, a professor of biological systems engineering, works with gold. But you won’t find the shiny yellow stuff in his lab; instead, the vials on his bench are mostly purple and red. Gunasekaran works with tiny pieces of gold—nanoparticles—that come in almost every color except gold. And those colors can tell a story.

Gunasekaran’s research focuses on food safety concerns, such as whether a food product was transported and stored properly or whether it has become contaminated. But how can a producer or consumer easily know a product’s history and whether it is
safe to eat? That’s where gold nanoparticles come in handy.

“The color of gold nanoparticles will change depending on the size and shape of the particle,” explains Gunasekaran. “At different temperatures, depending on how long you let the particles grow, they acquire different sizes and shapes. And that changes their colors.”

Gunasekaran’s lab is using those color changes for a difficult task—tracking the time and temperature history of a food product as it is packaged, transported and stored. Up to now similar sensors have given consumers some of this information, but they can miss such critical events as, for example, a short temperature spike that could be enough to kick-start the growth of a dangerous microorganism.

The sensors that Gunasekaran and his team are developing provide a more complete and accurate story. The new sensor can differentiate between food stored at high temperatures first and low temperatures second versus a product stored first at low temperatures and then at high temperatures. And that’s thanks to the properties of the gold nano-particles. The color of the first sample would be different than that of the second because of how and when the particles changed size and shape.

“We’re able to do this because the nanoparticle synthesis is affected by how the particles grow initially versus later,” explains Gunasekaran. “We call this the thermal history indicator, or THI.”

These gold nanoparticle sensors are being patented through the Wisconsin Alumni Research Foundation (WARF), and students in Gunasekaran’s lab won a UW–Madison Discovery to Product award. The student team also won a People’s Choice
award in the 2014 Agricultural Innovation Prize competition.

They are now working to further develop and optimize the system. Since different food products travel through different channels, some sensors will be best used to track long-distance travel over the course of a month, while others will track history for only a matter of hours. Some sensors will work best in frozen storage and others will be optimized for various room temperatures.

The goal of optimization is a simple-to-use biosensor customized for any given food product. Gunasekaran envisions the sensors—now being developed as self-adhesive dots or stickers—being used anywhere along the food production channel. Producers, packagers, transporters and even consumers could easily use the biosensors to understand the thermal history of their product, saving time and money and avoiding recalls and health issues.

“There are a number of ways to use this technology, and making a food product’s history easy to see is the key,” says Gunasekaran. “Food is being wasted because people are throwing it out according to an expiration date, or people are getting sick because they eat food that’s gone bad. Those things can be avoided by having a better product safety indicator.”

Unpuzzling Diabetes

The body makes it seem so simple.

You take a bite of supper, and the black-box machinery of metabolism hums into life, transforming food into fuel and building materials. It’s the most primal biology: Every living thing must find energy, and must regulate its consumption.

But for an alarming and ever-increasing number of people, the machinery breaks down. The diagnosis? Diabetes.

Alan Attie, a CALS professor of biochemistry, has been peering into the black box for two decades now, trying to identify the pathways in our bodies by which the disease is formed. “You can’t find a better excuse to study metabolic processes than diabetes,” he says. “It’s very, very rich.”

Type 2 diabetes, caused by an inability to produce enough insulin to keep the body’s blood glucose at normal levels, is a global health crisis that has accelerated at a frightening speed over the last 20 years—roughly the same time Attie has been studying it.

It’s an enormously complex disease driven by both genetics and the environment. A DNA glitch here, an external variable there, and the body slides irretrievably out of balance. But only sometimes. Most people who develop type 2 diabetes are obese, yet most people who are obese don’t actually wind up diabetic.

Tracking this riddle has led Attie and his lab to several major discoveries, chief among them identifying two genes associated with diabetes: Sorcs1 and Tomosyn-2. Through years of elaborate experimentation, Attie and his team teased them from the genetic haystack and then relentlessly deciphered their role in metabolic malfunction.

Science has uncovered more than 140 genes that play a role in diabetes, yet genetic screening still has little value for patients. As with any part of a large and complicated puzzle, it’s hard to see precisely how Sorcs1 and Tomosyn-2 fit in until we have more pieces. The biology of diabetes is so complex that we can’t be certain what the discoveries may ultimately mean. But both genes have shed light on critical stages in metabolism and offer intriguing targets for potential drugs.

Attie need not look far to replenish his motivation. His own mother suffers from diabetes, and she used to quiz him weekly about when he would cure her. “The painful answer is that translation of basic research into cures takes a long time,” Attie once told the American Diabetes Association. “The most important clues that can lead to cures do not necessarily come from targeted research or research initially thought to be relevant to the disease.”

Alan Attie grew up an expatriate in Venezuela, where his father, Solomon, originally from Brooklyn, New York, ran a textile factory (Attie’s mother had family in South America). Poverty and then World War II had kept Solomon from traditional schooling, but he managed to put himself through high school at night, and he nurtured a deep passion for literature, poetry, history and politics. At home he ran the family dinner table like a college seminar. “Our evening meal was like a 20-year course,” recalls Attie. “It was the most stimulating part of our day growing up. I was reading Shakespeare with my father and my siblings when I was 10 years old.”

Still, Attie wasn’t quite prepared for the academic rigor of UW–Madison when he arrived in 1972. He’d never had to work particularly hard in high school and was shocked by how much time and effort college required. His grades were poor and his introduction to chemistry lackluster.

But the BioCore curriculum—an intercollege program focusing on doing science, not memorizing facts—turned Attie’s natural inquisitiveness and enthusiasm toward science. During a cell biology course where his lab reports had to be written like journal articles, Attie decided he really wanted to be a biologist. Following graduate school at the University of California, San Diego, he found himself back at UW– Madison as a young assistant professor. Ten years had passed since his freshman matriculation.

Attie’s first research focus was cholesterol metabolism, but his curiosity led him elsewhere. Until 2001 he held a joint appointment with the School of Veterinary Medicine, where he taught an introductory class in biochemistry. While preparing for the class he read broadly in metabolism and found himself continually drawn toward the quandary of diabetes.

Increasingly he found himself suffering from “discovery envy,” he says. “And then I finally decided one day I do want it to be me.” Midcareer course changes are never easy, but Attie plotted a careful transition that gained momentum with hard work and good fortune.

In 1992 Dennis McGarry, a prominent diabetes scholar, published a provocative thought experiment in Science. It had been observed for centuries that diabetics had sweet urine, and one of the earliest researchers in the disease, Oskar Minkowski, had surmised that diabetes was therefore a dysfunction of sugar metabolism. McGarry speculated that if Minkowski had had no sense of taste and had relied instead on the smell of a diabetic’s urine, he would have smelled ketone bodies, a hallmark of lipid metabolism. Might he have concluded instead that diabetes was a defect in lipid metabolism?

Soon afterward, McGarry and Attie wound up at the same research symposium in Edmonton and shared breakfast every morning. “I’m really interested in diabetes,” said Attie. “Is there room for someone like me who has been working on lipid metabolism for 20 years?” McGarry encouraged Attie, a pep talk that gave him confidence that maybe he wasn’t committing career suicide.

Gradually Attie’s new focus gathered steam. When another UW diabetes researcher left for Washington state, Attie was able to bring on researcher Mary Rabaglia from that lab. She was highly skilled in the lab manipulation of pancreatic islets, the home of the beta cells that produce insulin. Her arrival jump-started Attie’s efforts. “It was an unbelievable stroke of luck because she brought all that expertise,” says Attie.

Attie also felt he needed a new analytical toolbox, and he saw real potential in using mouse genetics to study diabetes. With one small problem: He didn’t know any genetics. So he went to the Jackson Laboratory in Bar Harbor, Maine—a global center of mouse research—and took a mouse genetics course (which he now teaches there).

The learning soon paid off. Gene chip technology was just becoming available, and industry pioneer Affymetrix was looking to commercialize the expensive technology. The company was interested in funding labs to demonstrate that the power was worth the price. Attie proposed looking at how genes were turned on and off in the fat-storing cells of diabetic mice, and Affymetrix approved the project.

Exploring gene expression—which genes get turned on and off—was an important first clue in figuring out which genes might contribute to diabetes. With thousands of proteins and a still unknown quantity of genes in play, diabetes is vexingly elaborate. Gene chip technology brought previously unimaginable power to the equation. “The reason for doing genetics is we can’t imagine the complexity of these processes,” Attie explains. “We really do need the serendipity of genetics to find our way.”

Attie sent Sam Nadler, a new M.D./ Ph.D. candidate, off to Maryland and California for training. It was an ambitious project, and the old analytical tools broke under the mountain of new data. Enlisting the help of Brian Yandell, a CALS professor of horticulture with a joint appointment in statistics, they were able to interpret their data.

In late 2000 the team published the first paper on genome expression changes in diabetes using gene chip technology. It was premature to get too excited—they were, in effect, translating a book of unknown length, and had only finished the first of many chapters.

But it was an important demonstration of the power of their new tools. And Attie and his lab were now a known quantity in the world of diabetes research, and part of the conversation.

Attie’s team could now assess any DNA they got their hands on, but there was still too much static hiding the working genes. Only by basing his experiments on other, more tangible clues could Attie find anything useful.

He decided to tackle the obesity link. “Most people who have diabetes are obese, but most people who are obese don’t have diabetes,” he notes. To get at the problem, Attie’s team took two strains of lab mice: a standard control strain known as “black 6” (B6) and a diabetic strain (BTBR) that, when the mice became obese, were diabetic. The team intercrossed the two strains for two generations, testing the second generation of mice for diabetes. Offspring were strategically bred to enable the lab to pinpoint the genes responsible for diabetes susceptibility.

The collaboration that had begun with Brian Yandell now expanded to include Christina Kendziorski, a professor of biostatistics with the School of Medicine and Public Health. Teasing conclusions from large data sets was an exciting new field, and the team saw real potential for developing new techniques— and they had the statistics grad students to do it. Some even took up residence in Attie’s lab to be closer to the puzzles cascading from each successive experiment. It was like game after game of Clue, only with a half million possible rooms, a half million possible murder weapons, and a half million possible suspects. And as many homicides as you wanted to look for. Some computations took days.

Ultimately they were looking for genes, but what they found at first were just general target zones, located on chromosomes 16 and 19. That was a big first step, but chromosomes are constructed of many millions of base pairs—the building blocks of DNA . Considered relatively small, chromosome 19 still runs to about 61 million base pairs. The first round of sifting reduced the search zone to a neighborhood with only 7 million base pairs, an almost 90 percent narrowing of the field.

Pinpointing the gene required a constant shuffling of the genetic deck, counting on the random nature of sexual reproduction to winnow away the chaff, revealing the kernel of the gene. It’s a process that can take years, measured in mouse generations. Finally, in 2006, they were able to pinpoint the precise location of Sorcs1. It was a triumph, but it also set the stage for heartbreak.

Meanwhile, other projects kept rolling. Sushant Bhatnagar, a postdoctoral scholar in biochemistry, was working on the other target zone—chromosome 16. In 2011 he zeroed in on Tomosyn. “It was crazy,” he says of the work needed to sift through so many mouse generations.

But in the end they discovered that Tomosyn-2 played a critical role in diabetes. Tomosyn was also more willing to give up its secrets. Most of the myriad proteins in a beta cell are positive regulators, which means they facilitate flipping the insulin switch to “on.” Tomosyn is an off switch—one of very few known to exist.

Though mouse and human diabetes are different, the lab confirmed that the human version of Tomosyn plays a similar role. Now the challenge is using the clue to develop a targeted therapy. “Loss of insulin secretion leads directly to diabetes,” Bhatnagar explains. “If you can fix insulin secretion you can fix the majority of diabetes.”

Finding Sorcs1 had been difficult enough, but unlocking how it worked would prove devilishly complex. Two students tried and failed, and eventually left research altogether, demoralized by the dead ends. Attie felt terrible. “I always feel responsible for everything that goes on in the lab,” he says.

Then, in 2012, Attie welcomed a new postdoctoral scholar. The only problem was that Sorcs1 was a beta cell problem, and Melkam Kebede did not come to Madison to work on beta cells.

A child prodigy from Ethiopia by way of Australia, Kebede was through college by age 18 and had her Ph.D. at 23. After spending most of her career on beta cells, she was looking for something  different in her second postdoctoral position. Able to go almost anywhere, she chose Madison, and Attie.

“Of all the places I interviewed, Alan was the most passionate about teaching,” Kebede says. And she liked the way he encouraged people. She’d always been told that she was exceeding expectations, and nobody challenged her during interviews. Except for Attie. “I wanted someone to push me more, so I can do more than what I’ve been doing,” she says.

Pushing people, of course, is a delicate process, and easily fumbled. Attie instead seems to pull with a magnanimous curiosity. And with Kebede he was patient but persistent. Attie would keep asking: Why were the Sorcs mice diabetic? “You still have the parents of these mice waiting in the hallway at the hospital,” he would say. “They are buying so many coffees. You’ve got to come up with a reason why they are diabetic.”

Finally, Kebede couldn’t resist the puzzle—the opportunity to find the link between obesity and diabetes. While the lab hadn’t cracked Sorcs, they had narrowed the focus. And Angie Oler, an invaluable technician with 20 years of experience, would help her get the end game rolling.

In an obese person, cells do not respond completely to normal insulin levels—this is called insulin resistance. To compensate, the body typically produces more insulin. Type 2 diabetes develops when the insulin resistance outpaces the body’s attempt to make more. Sorcs seemed to play a role, but how?

“There are so many things in the body that contribute to controlling glucose levels in the blood,” Kebede explains. A beta cell has to sense an increase in glucose and secrete insulin, which then triggers other reactions that lead, ultimately, to glucose being removed from the blood and absorbed by the cells that need it. Sorcs1 could work anywhere in this great game of cellular call and response.

Despite all of the genetic and biochemical tools at Kebede’s disposal, it was ultimately a simple observation in a microscope that yielded the key. Insulin is manufactured in advance and stored by beta cells in the pancreas, then released as needed. Typically only 1 to 4 percent of the insulin is released at any one time, and a healthy beta cell would simply reload and release more insulin as needed. Examining hundreds upon thousands of cells, Kebede realized that the diabetic beta cells were partly emptied of insulin—but not enough to reveal an insulin secretory dysfunction.

The problem was that a standard lab testing for insulin production was a one-shot deal. The Sorcs1-deficient cells could handle that first test, but not a second test. Finally she understood: The diabetes was caused not by a lack of insulin, but by a failure to reload in a timely way.

The team had the answer—but after their first submission to the prestigious Journal of Clinical Investigations, they were asked to do 22 more experiments.

Kebede had been thinking along the same lines and had already begun the additional work. “We wanted to make sure we got the story right,” she says.

It took an extra eight months, but in August 2014 the paper was finally released. It was an exciting and novel find. In type 2 diabetes, it often seems as if the insulin-producing pancreatic beta cells are wearing down. The Sorcs1 discovery suggests a possible explanation for that, and also provides an important change in how to work with beta cells.

Around the same time, a related discovery came from, of all things, a single-celled organism called Tetrahymena thermophila being studied at the University of Chicago. Attie and Kebede went down to brainstorm with Aaron Turkewitz, a professor of molecular genetics and cell biology. It was an inside-baseball connection, the kind that might take pages to explain and doesn’t show up in grants or co-authored papers. But it personifies the role of a researcher like Attie in an endeavor as complex as decoding diabetes.

“His interests at the most basic scientific level have immense medical implications, and in that way, he connects to a large swath of investigators,” explains Peter Arvan, M.D., Ph.D., director of the University of Michigan Comprehensive Diabetes Center. “There are few like him, but he is a model investigator for the 21st century. As the science gets more complex, the field needs investigators like Alan to connect us.”

Once upon a time, Alan Attie had a bumper sticker that said, “Don’t believe everything you think.”

And Attie thinks about so many things. He makes very good wine and is an accomplished amateur photographer. As much as he loves research, he’s passionate about teaching. Conversations glide from the unification of Germany and money in politics, to Ebola and science funding, to income inequality and student debt.

Attie’s not the happiest of scientists right now. As the United States has reduced its lead in science funding, he’s become acutely aware that the kind of midcareer leap he made into diabetes would be impossible in today’s funding environment. He’s got fewer mice in inventory than at any time in recent memory—and to him that means discovery is languishing.

“We can’t pursue all of our good ideas. We can’t pursue all of our bad ideas, either. But we don’t know which ideas are good or bad until we try. The thing is, we’re not trying as much,” he concludes, frustrated. He worries that we’re losing our edge.

For example, he has a lead on a protein that appears to be involved in both Alzheimer’s and diabetes—perhaps the two greatest challenges to health care financing. “I won’t write the grant because it has zero chance of receiving funding,” Attie says. I

In an age where science seems so often a political pawn, it’s refreshing to hear it talked about as a human ideal.

In Attie’s vision, scientific thinking isn’t just running the numbers and picking the ones you like. It’s about “being self-critical, being introspective about how you think and what algorithm you’re using to arrive at a conclusion about anything in the world,” says Attie. “If that were a widespread value, I think our society would be different, better. We would have less hatred, less racism. We would be more nuanced in the way we judge other people.”

Meanwhile, there are mice to study and students to train. Attie’s been involved in the Collaborative Cross, a massive multi-institutional effort to refine mouse genetics to better allow the study of human disease. Using new mice strains, his team is beginning a major fishing expedition, a multiyear project focusing on insulin secretion and beta cell biology in general—utilizing brand new genetic techniques that already are being hailed as game-changing.

Attie knows there will likely be moments of eureka as well as dead-end heartbreak. The team that he loves so much will grow and change as members adapt to the shifting landscape of discovery. He’ll miss the old students and technicians as they move on, but he’ll gain new students and collaborators as he keeps asking the questions that come so naturally to him.

“Being in science is very humbling because I’ve been wrong about a lot of things over time,” says Attie. “That’s part of learning to be a scientist—and yet I think it’s also part of learning to become a better human being.”