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.”

Of Pests and Pathogens

Imagine you’re standing in a sun-drenched field full of lettuce plants. There’s a gentle breeze and a smattering of tiny insects flitting about. It’s a pleasant scene, right?

Now let’s say one corner of the plot is contami- nated with the deadly human food-borne pathogen Salmonella enterica, due to dirty irrigation water.

Could the insects, which hop from leaf to leaf feeding on plant sugars, play a role in spreading the contamination further afield?

“As insects feed and wander around on contami- nated plant surfaces, it’s possible that they pick up Salmonella, so we decided to ask if they are playing a role in food safety,” says Jeri Barak, a CALS professor of plant pathology.

It’s an important question. Salmonellosis is one of the leading causes of acute bacterial gastroenteritis in the United States, responsible for an estimated 1.4 million illnesses, 15,000 hospitalizations and 400 deaths annually. In recent years, there have been more salmonellosis illnesses linked to fresh produce than to animal products. Yet very little is understood about produce-associated outbreaks, including basic information about how human pathogens survive and spread on plants.

To test whether plant-eating insects play a role, Barak teamed up with CALS entomology professor Russell Groves to design experiments assessing the ability of two common crop insect pests—the aster leafhopper and the green peach aphid—to pick up and spread Salmonella.

“Because we work with human pathogens in my lab, we never get to do research outside, so it was important to have someone like Russ Groves on board,” notes Barak. “He’s a UW–Extension ento- mologist, so he has a lot of experience in the field, and he pushed us to be as practical as possible.”

In one set of experiments, insects were given a piece of Salmonella-contaminated leaf material to munch on for 24 hours. In another, they spent the day drinking Salmonella-laced sugar water through a protective barrier that prevented physical contact between Salmonella and

insect. In both setups, the insects were then transferred into a series of clean environ- ments over the next 48 hours to see if—and how long—the contamination lasted.

In both cases, the insects readily picked up Salmonella, and once contaminated, most stayed contaminated.

Those that ate tainted leaves became contaminated inside and out, harboring Salmonella in their guts as well as on their feet and antennae, and when they were trans- ferred to clean tubes, they spread the bacteria to fresh leaf material.

For insects that drank the Salmonella- laced sugar water, the bacteria got into their guts—and also found a way out.

“They excreted the Salmonella through honeydew—that’s a nice word for insect poop. Even after 48 hours, they were still pooping it out,” says Barak.

Honeydew, they also found, serves as a nutrient-rich fertilizer that helps Salmonella grow on plant surfaces that would otherwise be inhospitable.

On the practical side, notes Barak, farmers can now add these insects to the list of risk factors they consider when making crop management decisions.

“Now when a raw-produce grower looks out and it’s been a bad year for insect infestation, it might sig- nal to them that they may have a higher food safety risk,” she says.

Five things everyone should know about . . . Milkweed

1. It is the stuff of life for monarch butterflies. Monarchs lay their eggs on milkweed, and milkweed leaves serve as nearly the sole food of monarch caterpillars. But many species benefit from the bounty of milkweed. Milkweed flowers produce nectar that other kinds of butterflies, honey bees, native bees and other pollinators enjoy. Hummingbirds line their nests with floss from milkweed seed pods.

2. It’s both medicine and poison. Milkweeds—there are more than 100 species—belong to the genus Asclepias, named after the Greek god of medicine and healing. Milkweeds have been used in medicine for thousands of years because their tissue contains cardiac glycosides, which increase the heart rate and in a purified form are useful in treating such conditions as cardiac arrhythmia and congestive heart failure. As a crude extract, cardiac glycosides are toxic and have been used as poison. Monarch larvae retain the toxins they consume in milkweed leaves and as butterflies remain toxic to predators.

3. Its presence is dwindling, along with the monarchs. The first decade of this century saw a 58 percent decline in milkweeds in the Midwest, according to a 2012 study—a time when we’ve also seen a whopping 81 percent decrease in monarch production. Factors often cited for milkweed’s decline include loss of habitat as grasslands and conservation reserves have been converted to farmland for corn and soybeans as well as increased use of herbicides on those crops.

4. There’s a growing movement to bring it back. Researchers at CALS and elsewhere have noted an increase in biodiversity, pollination and other ecosystem services that come from establishing or maintaining a mix of perennial native plants near cropland—and milkweed, they say, should be part of it. Vigorous efforts are taking place throughout the Midwest to plant large areas of milkweed along the monarchs’ migration path to Mexico, where they spend the winter.

5. Milkweed will enliven and beautify your garden—but keep your gloves on when handling. The toxins that protect the monarch can harm humans. Make sure the sap doesn’t get into your eyes, and if it does, seek medical attention as it can cause significant damage. While not all milkweeds are equally toxic and some kinds can be eaten, great care must be taken when selecting and preparing it.

 

Five things everyone should know about gluten

1. What is it? Gluten is a substance composed of two proteins—gliadin and glutenin—that are found in the endosperm (inner part of a grain) of wheat, rye, barley and foods made with those grains, meaning that gluten is widespread in a typical American diet.

2. Is it harmful? People who suffer from celiac disease, an autoimmune digestive disorder, are unable to tolerate gluten. Even a small amount of it (50 milligrams) can trigger an immune response that damages the small intestine, preventing absorption of vital nutrients and potentially leading to other problems such as osteoporosis, infertility, nerve damage and seizures.

3. How widespread is celiac disease? An estimated 1.8 million Americans have celiac disease; as many as 83 percent of those suffering from it remain undiagnosed or are misdiagnosed with other conditions. Another 18 million (about 6 percent of the population) do not have celiac disease but suffer from gluten sensitivity. They report such symptoms as diarrhea, constipation, bloating and abdominal pain—which also are symptoms of celiac disease—but do not experience the same intestinal damage. For those with celiac disease or gluten intolerance, a gluten-free diet is beneficial.

4. Should you cut gluten from your diet even if you don’t have these conditions? Probably not. Restriction of wheat in the diet often results in a decrease in the intake of fiber at a time when most Americans consume significantly less than the recommended amount. Low-fiber diets are associated with increased risk of several acute gastrointestinal diseases (examples: constipation, diverticulosis) and chronic diseases such as heart disease and colon cancer. If not done carefully, gluten-free diets also tend to be low in a number of vitamins and minerals.

5. Don’t diagnose yourself. The broad range of symptoms associated with celiac disease and gluten sensitivity may be due to other causes; self-diagnosis and treatment of perceived gluten intolerance may delay someone from seeking more appropriate medical care. The only way to know for certain if you have celiac disease is from a blood test for the presence of specific antibodies followed by a biopsy of the small intestine. If you are experiencing the symptoms described above, please seek medical care.

Beth Olson is a professor of nutritional sciences. Her principal research areas concern breastfeeding support and improving infant feeding practices in low-income families.

Upping the Orange

Sherry Tanumihardjo is a CALS professor of nutritional sciences and director of the Undergraduate Certificate in Global Health, a popular new program that draws participants from majors all across campus. She has almost three decades of experience working with vitamin A, and her research team has conducted studies in the United States, Indonesia, South Africa, Ghana, Burkina Faso and Zambia. Tanumihardjo has acted as a consultant to many studies throughout the world to assist with study design and appropriate standardization. She is a strong advocate for the promotion of nutritionally enhanced staple foods, vegetables and fruits to enhance overall health and well-being.

Describe your work with orange vegetables.
I have worked for a number of years on carrots of many colors as well as on orange-flesh sweet potato and, more recently, orange maize. Basically we are trying to improve the vitamin A status of individuals by having them consume more orange fruits and vegetables in general.

Can you give us an idea of how you go about doing that?
For many years I have worked with carrot breeder Phil Simon in the Department of Horticulture. He was breeding carrots for more orange color. We did a series of studies in both an animal model and in humans, trying to look at the uptake and distribution of the carotenoids that give the vegetables their orange color—and the vitamin A that is made from the carotenoids. Then we moved on to orange vegetables in humans in Africa. I have worked with orange-flesh sweet potato in South Africa and with orange maize in Zambia.

Can you describe the connection between the color and the nutritional value?
There are three well-known precursors of vitamin A that are called pro-vitamin A carotenoids. Those are beta-cryptoxanthin, alpha-carotene and beta-carotene. Many of you may have heard of beta-carotene because it is one of the compounds found in many over-the-counter supplements. But those are also the compounds that give carrots and orange maize their bright orange color.

What happens if there is not enough vitamin A in the diet?
The most drastic thing that can happen is death. So we go around trying to get people to improve their vitamin A intake not only to prevent death—there are many steps before that happens, and one of them is blindness. Vitamin A is extremely important in vision and it also helps us ward off disease, so it’s a very important vitamin.

How did you get started in Africa?
It actually started very slowly. I used to be a consultant and I would fly back and forth to different countries to help them look at study design. The sweet potato study was funded by the International Potato Center. I helped them design the study, they did the school implementation—a feeding study—and then I helped them get the work published. My work with orange maize started in 2004 in collaboration with HarvestPlus, a project managed by the International Food Policy Research Institute. We started working with animal models and then progressed to full-fledged feeding trials, the latest of which we finished in 2012.

What were some of the challenges in your work in Africa?
The challenge is that feeding trials, if they’re going to show what we call efficacy, have to be highly controlled. So that means you have to keep the children for long periods of time and feed them all of the foods—and the foods need to be the same across the group except your test food. So in South Africa we fed orange-flesh sweet potato to half the children and white-flesh sweet potato to the other half. And then when we moved on to orange maize we did two studies. One study was similar to the sweet potato study where we fed white maize and orange maize. And then we did a second study where we had three groups, which got a little more complicated. We had white maize, orange maize and then white maize with a vitamin A supplement.

Another challenge is that all of the human work that I do involves blood—so we have to take blood from these children. Vitamin A in the human body is stored in the liver, and we use indirect markers of liver reserves of vitamin A that you can pick up from the blood.

Looking down the road what kind of goals do you have for your research?
We would like for people to have optimal health by having a diet that has not only all the nutrients you need but also some of the potential compounds that gear us toward optimal health. So it’s not just about fighting blindness anymore, but to see if we can get people into a new nutritional state where they are actually able to ward off diseases such as cancer.

What kind of progress have you made?
We have had significant progress with sweet potato. Most people in Africa used to eat white sweet potato, not the orange sweet potato we eat here in the United States. Many countries in Africa have now adapted the vines to be orange-flesh sweet potatoes. We think that’s a success story. Regarding orange maize, there are three lines of orange maize that have been released by the Zambian government. Currently orange maize is available to consumers. Right now it’s at a premium price, but hopefully with time the price will come down to the level of white maize.

How did you get interested in this line of work?
It chose me. It wasn’t something that I was looking for, but I was working with vitamin A and if you’re working with vitamin A and status assessment, it’s going to draw you to the countries that may have a history of vitamin A deficiency.

Can you talk a little more about the international nutritional programming you’ve been involved in?
Most of the work that I’ve done is to support biochemical labs. We have not done a lot of nutrition education on the ground, although that is a goal of mine, especially in Zambia. We have discovered that Zambians actually have really good sources of vitamin A in their daily diets, so we want to help them continue to eat the fruits and vegetables that are good sources of those phytonutrients and vitamins and minerals.

The other thing that I work on is isotope methods, which sounds a little scary!

What are isotope methods and what do they do?
We work with a compound called 13C. Typical carbon in the human body is 12C and radioactive carbon is 14C. We are working with the form of carbon that constitutes 1 percent of the human body. It’s perfectly safe to use, but it also has allowed me to work with the International Atomic Energy Agency. That’s the same agency that oversees radioactive bombs in different countries, so it’s kind of interesting that they have something called Atoms for Peace. And they actually received the Nobel Peace Prize one year based on the safe use of isotopes in nutrition.

I have worked in several countries trying to help them understand isotope methods and to apply isotope methods at the population level to inform public health policy. It’s a very technical method, but it can answer questions of public health significance.

So it’s a research tool. And what kinds of questions does it answer?
It is the most sensitive marker of liver reserves of vitamin A. Basically what we do is we give a dose of vitamin A that has a slightly higher amount of 13C than what’s found naturally in the environment, and then we can follow the uptake and the clearance of that 13C in the human body. And from that we can calculate total body stores of vitamin A—how much is in the whole body.

To conclude here, there’s an interesting story about your office and a more recent career development of yours—serving as director of the Undergraduate Certificate in Global Health, a program you helped develop and launch in 2011.
Yes. The Nutritional Sciences Building was originally a children’s hospital, and this particular office that I sit in sat idle for many, many years, used only for small committee meetings and things like that. When we received funding for the Undergraduate Certificate in Global Health, I looked in this office again and realized that it now fits my purpose. Originally it was the viewing room for children who had died from a variety of diseases, and the parents would sit in this room and mourn their lost child. I decided that this room fit my new mantra at the university, which is to empower undergrads, to mobilize them, to try to change the world. And while I’m sure we won’t have 100 percent participation, we’ve already had about 1,000 students go through the program.

The Mysteries of RNA

For people who know about RNA mostly from its place in the central dogma of biology—DNA➙RNA➙Protein—this story may hold a number of surprises.

That handy equation, taught in Biology 101 courses around the globe, sums up the flow of genetic information in living organisms: how our DNA gets copied into RNA, which then gets converted into proteins, the building blocks of our cells, our bodies.
Originally, the RNA referred to in this equation—messenger RNA, or mRNA, the type that codes for proteins—was the only kind known to science. However, over the years, it has become clear that there are many, many other kinds.

“The world of RNA has proven to be a big and fascinating place,” says Marv Wickens, a CALS professor of biochemistry and leading pioneer in RNA research. “I’ve come to think of it as a Fellini movie, full of strange and unexpected characters.”

These Felliniesque characters are all the non-coding RNAs that exist in nature—the kinds that don’t code for proteins. They go by names like small interfering RNA, piwi-interacting RNA, microRNA, long non-coding RNA, small nuclear RNA—the list goes on and on. Together they far outnumber messenger RNAs in the cell; while only 3 percent of the human genome gets made into proteins (via messenger RNA), a full 80 percent gets copied into RNA.

What are all of these other RNAs doing? Lots of important and surprising things, scientists are discovering.

Over the past few decades, RNA, a close chemical cousin of DNA, has proven itself to be a much more versatile molecule than originally thought—far more than just a passive messenger.

The first big surprise came in the 1980s when it was shown that RNA can have catalytic activity, meaning that it can perform chemical reactions inside the cell. Originally assumed to be inert, like DNA, scientists found RNA molecules that could edit their own sequence—expunging a segment of their own genetic code.

Later, RNAs were discovered at the heart of important cellular machines, or enzymes, performing critical catalytic reactions, including those at the heart of the cell’s information transfer system. Previously only proteins were thought capable of such enzymatic feats.