In cells, self-destructive behavior suggests strategy for fighting cancer

SOMETIMES, TO SURVIVE, our cells destroy their own ribonucleic acid (RNA), the part of our genetic instruction code that helps turn genes into proteins. Cells do this as part of the first line of defense against pathogens and damage. New research suggests how this mechanism could form the basis of a strategy against cancer.

The mechanism is part of the body’s innate immune system, which kicks into gear minutes after infection. The innate immune system holds pathogens at bay for the critical few days needed to make antibodies.

One of the mysterious weapons in the innate immune system is an enzyme called RNase L that chops up strands of RNA, an intermediate step between genes and their final products, proteins. Because viruses lack the machinery to make copies of themselves to spread infection, they hijack a cell’s reproductive technology and force it to make viral RNA and proteins. To fight back, the body sends RNase L. (The “ase” is a common ending for enzymes, and the “L” is for latent, because RNase L lies in wait for infection or damage signals.)

RNase L chops up the viral RNA, but it also shears the body’s own RNA. This self-mutilation is necessary for good health. Mice that lack RNase L are obese, diabetic and have signs of inflammation.

“This cleavage of the cell’s own RNA has a protective function. It does not always kill the cell, but when it does it eliminates only damaged or infected cells, which repairs the tissue. It ultimately means animals survive better,” said Alexei Korennykh, an associate professor of molecular biology. His lab was the first to solve the crystal structure of human RNase L, a result they published in the journal Science March 14, 2014.

Korennykh’s research is funded by the National Institutes of Health, the Sidney Kimmel Foundation, the Burroughs Wellcome Fund and the Vallee Foundation.

With the 3-D structure in hand, Korennykh and his team turned to the question of how RNase L works. Korennykh wondered if RNase L chops up every RNA that comes along, or just certain ones. To find out, the researchers sequenced every piece of RNA made in a typical human cell and evaluated them to see which ones were susceptible to RNase L.

What they found surprised them. RNase L chops up RNA strands that govern the rapid division of cells, as well as how well cells stick to each other. These two activities — when cells proliferate and attach themselves in new locations — are two of the key steps in the spread of cancer. They published the findings Dec. 29, 2015, in the Proceedings of the National Academy of Sciences.

The link between RNase L and cancer made sense because RNase L mutations are common in individuals from families with a hereditary predisposition for prostate cancer, Korennykh said. The team is exploring how to turn this discovery into a strategy for inhibiting cancer’s spread using small drug-like molecules to boost RNase L’s activity against cell proliferation and attachment.

“We really stumbled on this approach very unexpectedly,” Korennykh said. “Often the best experiments are the ones where you didn’t get the results you expected.” –By Catherine Zandonella

Bright future: Princeton researchers unlock the potential of light to perform previously impossible feats

By Bennett McIntosh

One hundred years ago, Italian chemist Giacomo Ciamician predicted a future society that would run on sunlight.

In a paper presented in 1912 to an international meeting of chemists in New York City, he foresaw a future of vibrant desert communities under “a forest of glass tubes and greenhouses of all sizes” where light-driven chemical reactions would produce not just energy but also wondrous medicines and materials.

Ciamician’s vision has not yet arrived, but a handful of Princeton researchers have succeeded with one part of his legacy: they are harnessing light to perform previously impossible feats of chemistry. In Princeton’s Frick Chemistry Laboratory, blue LED lamps cast light on flask after flask of gently stirring chemicals that are reacting in ways they never have before to create tomorrow’s medicines, solvents, dyes and other industrial chemicals.

The leader in this emerging field is David MacMillan, who arrived in Princeton’s chemistry department in 2006. He was intrigued by the potential for using light to coax new chemical reactions. Like most chemists, he’d spent years learning the rules that govern the interactions of elements such as carbon, oxygen and hydrogen, and then using those rules to fashion new molecules. Could light help change these rules and catalyze reactions that have resisted previous attempts at manipulation?

Changing the rules

The idea for using light as a catalyst had been explored since Ciamician’s time with limited success. Light can excite a molecule to kick loose one or more of its electrons, creating free radicals that are extremely reactive and readily form new bonds with one another. However, most chemists did not think this process could be controlled precisely enough to make a wide variety of precision molecules.

But that changed in the summer of 2007.

MacMillan and postdoctoral researcher David Nicewicz were working on a tough problem. The two scientists wanted to create chemical bonds between one group of atoms, called bromocarbonyls, and another group, known as aldehydes. “It was one of those longstanding challenges in the field,” MacMillan said. “It was one of those reactions that was really useful for making new medicines, but nobody knew how to do it.”

Nicewicz had found a recipe that worked, but it involved using ultraviolet (UV) light. This high-energy form of light causes sunburn by damaging the molecules in the skin, and it also damaged the molecules in the reaction mixture, making the recipe Nicewicz had discovered less useful. MacMillan, who is Princeton’s James S. McDonnell Distinguished University Professor of Chemistry, asked Nicewicz to investigate how to do the transformation without UV light.

Nicewicz recalled some experiments that he’d seen as a graduate student at the University of North Carolina-Chapel Hill. Researchers led by chemistry professor Malcom Forbes had split water into oxygen and hydrogen fuel using visible light and a special molecule, a catalyst containing a metal called ruthenium. The approach was known as “photoredox catalysis” because particles of light, or photons, propel the exchange of electrons in a process called oxidation-reduction, or “redox” for short.

David MacMillan

David MacMillan is a leader in developing the use of light to catalyze chemical reactions — a technique called photoredox catalysis. (Photo by Sameer A. Khan/Fotobuddy)

Visible light is lower in energy than ultraviolet light, so Nicewicz and MacMillan reasoned that the approach might work without damaging the molecules. Indeed, when the researchers added a ruthenium catalyst to the reaction mixture and placed the flask under an ordinary household fluorescent lightbulb, the two scientists were astounded to see the reaction work almost perfectly the first time. “More times than not, the reaction you draw on the board never works,” Nicewicz said. Instead, the reaction produced astonishing amounts of linked molecules with high purity. “I knew right away it was a fantastic result,” he said.

With support from the National Institutes of Health, MacMillan and Nicewicz spent the next year showing that the reaction was useful for many different types of bromocarbonyls and aldehydes, results that the team published in Science in October 2008. Research in the lab quickly expanded beyond this single reaction, and each new reaction hinted at a powerful shift in the rules of organic chemistry. “It just took off like gangbusters,” MacMillan said. “As time goes on you start to realize that there are nine or 10 different things that it can do that you didn’t think of.”

Old catalysts, new tricks

At the time that Nicewicz and MacMillan were making their discovery, chemistry professor Tehshik Yoon and his team at the University of Wisconsin-Madison found that combining the ruthenium catalyst with light produced a different chemical reaction. They published their work in 2008 in the Journal of the American Chemical Society the same day MacMillan’s paper appeared in Science. Within a year of MacMillan publishing his paper, Corey Stephenson, a University of Michigan chemistry professor, and his team found yet another photoredox-based reaction.

With these demonstrations of the versatility of photoredox catalysts, other chemists quickly joined the search for new reactions. About 20 photoredox catalysts were already available for purchase from chemical catalogs due to previous research on watersplitting and energy storage, so researchers could skip the months-long process of building catalysts. However, by designing and tailoring new catalysts, the chemists unlocked the potential to use light to drive numerous new reactions, and today there are more than 400 photoredox catalysts available.

The secret to these catalysts’ ability to drive specific reactions lies in their design. The catalysts consist of a central atom, often a metal atom such as ruthenium or iridium, surrounded by a halo of other atoms. Light frees an electron from the central atom, and the atoms surrounding the center act as a sort of channel that ushers the freed electrons toward the specific atoms that the chemists want to join.

One scientist who became intrigued with the power of photoredox catalysts was Abigail Doyle, a Princeton associate professor of chemistry. Doyle, whose work is funded by the National Institutes of Health, uses nickel to help join two molecules. In 2014, she was searching for a way to conduct a reaction that had long eluded other scientists. She wanted to find a catalyst that could make perhaps the most common bond in organic chemistry — between carbon and hydrogen — reactive enough to couple to another molecule. Perhaps a photocatalyst could make a reactive free radical, allowing her to then bring in a nickel catalyst to attach the carbon-carbon bond.

Abigail Doyle

Abigail Doyle is one of a handful of Princeton professors to quickly adapt the use of blue LED light and photoredox catalysis to rewrite the rules of organic chemistry. Drug companies have taken notice. (Photo by Sameer A. Khan/Fotobuddy)

Unbeknownst to Doyle, the MacMillan lab had recently turned their attention to combining photoredox and nickel catalysts on a similar reaction, coupling molecules at the site of a carboxylate group, a common arrangement of atoms found in biological molecules from vinegar to proteins.

Given the similarities in their findings, the MacMillan and Doyle labs decided to combine their respective expertise in nickel and photoredox chemistry. Together, the teams found a photocatalyst based on the metal iridium that worked with nickel to carry out both coupling reactions — at the carbon-hydrogen bond and at the carboxylate group. Their collaborative paper, published in Science July 25, 2014, showed the extent of photoredox catalysis’ power to couple molecules with these common features.

The ability to combine molecules using natural features such as the carbon-hydrogen bond or the carboxylate group makes photoredox chemistry extremely useful. Often, chemists have to significantly modify a natural molecule to make it reactive enough to easily link to another molecule. One popular reaction — which earned a Nobel Prize in 2010 — requires several steps before two molecules can be linked. Skipping all these steps means a far easier and cheaper reaction — and one that is rapidly being applied.

“It’s one of the fastest-adopted chemistries I’ve seen,” Doyle said. “A couple of months after we published, we were visiting pharmaceutical companies and many of them were using this chemistry.”

The search for new drugs often involves testing vast libraries of molecules for ones that interact with a biological target, like trying thousands of keys to see which ones open a door. Pharmaceutical companies leapt at the chance to quickly and cheaply make many more kinds of molecules for their libraries.

Merck & Co., Inc., a pharmaceutical company with research labs in the Princeton area, was one of the first companies to become interested in using the new approach — and in funding MacMillan’s research.The company donated $5 million to start Princeton’s Merck Center for Catalysis in 2006, and recently announced another $5 million in continued research funding.

In addition to aiding drug discovery, photoredoxcatalyzed reactions can produce new or less expensive fine chemicals for flavorings, perfumes and pesticides, as well as plastic-like polymer materials. And the techniques keep getting cheaper. MacMillan published a paper June 23, 2016, in Science showing that with the aid of a photoredox catalyst, a widely used reaction to make carbon-nitrogen bonds can be carried out with nickel instead of palladium. Because nickel is thousands of times cheaper than palladium, companies hoping to use the reaction were contacting MacMillan before the paper was even published.

Spreading the light

Doyle has continued to explore photoredox chemistry, as have other Princeton faculty members, including two new assistant professors, Robert Knowles and Todd Hyster.

Hyster combines photoredox catalysis with reactions inspired by biology. Drugs often function by fitting in a protein like a hand fits in a glove. But just as placing a left hand in a right glove results in a poor fit, inserting a left-handed molecule into a protein designed for a right-handed molecule will give poor results. Many catalysts produce both the intended product and its mirror image, but by combining photoredox catalysts with artificial proteins, Hyster is finding reactions that can make that distinction.

Hyster, who arrived at Princeton in summer 2015, was drawn to Princeton’s chemistry department in part because of the opportunities to share knowledge and experience with other groups researching photoredox catalysis. “The department is quite collegial, so there’s no barrier when talking to colleagues about projects that are broadly similar,” he said.

Students from different labs chat about their work over lunch, teaching and learning informally — and  formally, as the labs encourage collaboration and sharing expertise, said Emily Corcoran, a postdoctoral researcher who works with MacMillan. When Corcoran was trying to determine exactly how one of her reactions  worked, she was able to consult with students in Knowles’ lab who had experience using sensitive magnetic measurements to find free radicals in the reaction mixture.

“If you have a question, you can just walk down the hall and ask,” Corcoran said. “That really pushes all the labs forward at a faster pace.”

A bright future

After the graduate students go home at night, the blue LEDs continue to drive new chemical reactions and new discoveries. “This is really just the beginning,” Doyle said.

Hyster thinks that within a few years, manufacturers may take advantage of photoredox chemistry to produce biological chemicals — such as insulin and the malaria drug artemisinin — to meet human needs. For his part, MacMillan envisions zero-waste chemical plants in the Nevada desert, driven not by fossil fuels but by the sun.

MacMillan’s vision echoes that of the original photochemist, Ciamician. The Italian’s optimistic vision of a sunlit future is brighter than ever.

Tiny delivery capsules for new drugs

‘Jack’ Hoang Lu researches nanoparticles for drug delivery

Graduate student ‘Jack’ Hoang Lu works on engineering nanoparticles for targeted drug delivery and diagnostics in the laboratory of Robert Prud’homme, professor of chemical and biological engineering. PHOTO BY CATHERINE ZANDONELLA

Some drugs cannot be delivered via a normal pill or injection because they cannot readily dissolve in water. About 40 percent of new pharmaceuticals have this hydrophobic (water-fearing) character, and like a globule of oil in water, they are unable to reach their targets in the body.

Robert Prud’homme, professor of chemical and biological engineering, addresses this problem by putting the drugs inside of nanoparticles, each about one-thousandth of the width of a human hair, which are then covered with a polymer called polyethylene glycol. The small size enables dissolution of the drug to increase their bioavailability.

Nanoparticles can also tackle a different delivery challenge presented by a growing class of drugs called biologics that, as the name implies, are made from cellular or genetic components. The problem is that they are all water-soluble, which make them easy prey for patrolling proteins that identify them as foreign and degrade them. The solution is similar: nanoparticles protect the biologics long enough for them to carry out their mission.

In addition to increasing the time that drugs spend flowing through the body, Prud’homme’s nanoparticles are capable of targeted delivery. This is necessary in the case of toxic drugs, including many cancer treatments, which would damage healthy cells if allowed to roam freely throughout the body. Prud’homme puts appendages, called ligands, on the outside of his nanoparticles that, due to their specific shape and chemical properties, attach to their target and only to their target. In collaboration with Patrick Sinko at Rutgers University, Prud’homme’s group discovered the optimum density of an appendage called a mannose ligand, used to target cells harboring tuberculosis microbes. The unexpected result was that attaching more appendages to a nanoparticle decreased its targeting effectiveness. This demonstration of the complex interplay between engineered nanoparticles and how the human body responds to them is a major theme of the Prud’homme research team. In addition to tuberculosis, the researchers are using these principles to attack cancer, inflammation, and bacterial infections.

The project receives funding from the National Institutes of Health, the National Science Foundation, Princeton’s IP Accelerator Fund and the School of Engineering and Applied Sciences Old Guard Grant.

–By Takim Williams

Imaging system tracks brain activity of a freely moving worm

TO EXPLORE HOW THE BRAIN controls behavior, researchers have for the first time captured the whole-brain activity of a freely moving animal, in this case a nematode worm called Caenorhabditis elegans.

Using an imaging system they designed, Andrew Leifer, a Lewis-Sigler Fellow, and Joshua Shaevitz, an associate professor of physics and the Lewis-Sigler Institute for Integrative Genomics, measured the activity of 78 of the worm brain’s 125-plus neurons, which they engineered to turn green when active.

The setup consists of cameras that monitor the worm’s position and a motorized stage that adjusts to track the worm as it roams freely. The researchers were able to show significant correlations between neuron activity patterns and behaviors such as moving backward or forward, and turning. The team included Jeffrey Nguyen, a postdoctoral research associate and first author on the study, and colleagues at the Lewis-Sigler Institute and the Princeton Neuroscience Institute.

The study was posted on the preprint server arXiv.org and was funded by Princeton’s Dean for Research Innovation Fund for New Ideas in the Natural Sciences, the Simons Foundation and the National Institutes of Health.

–By Catherine Zandonella

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Energy boost: Study sheds light on mitochondrial disease

Ileana Cristea

Ileana Cristea PHOTO BY FRANK WOJCIECHOWSKI

INSIDE OUR CELLS, TINY FACTORIES convert nutrients from food into a form of energy that cells can use. Failure of these factories, known as mitochondria, can lead to metabolic disorders that are difficult to diagnose and even harder to treat.

Now researchers have identified an important regulator of cellular energy production that could aid in the diagnosis and treatment of a range of conditions. In a study published on Dec. 18, 2014, in the journal Cell, the researchers demonstrated that an enzyme known as Sirtuin 4 acts as a guardian of cellular energy production.

“The finding has broad implications in human health,” said Ileana Cristea, associate professor of molecular biology, who led the study. “Stress, nutritional deficiencies and viral infections can impact Sirtuin 4 functions and trigger dysfunction in energy metabolism,” Cristea said. “With this knowledge, we now have a new regulatory point that can be targeted in therapeutic interventions.”

Cristea’s team discovered that Sirtuin 4 turns off energy production by removing certain protein modifications, called lipoylation, from a key part of the energy-making machinery, called the pyruvate dehydrogenase complex.

The research team included former Postdoctoral Researcher Rommel Mathias and Associate Research Scholar Todd Greco in the Cristea laboratory, as well as collaborators Thomas Shenk, the James A. Elkins Jr. Professor in the Life Sciences, and Yibin Kang, the Warner-Lambert/Parke-Davis Professor of Molecular Biology.

Cristea’s research is supported by the National Institute on Drug Abuse, the National Institute of Allergy and Infectious Disease, the National Institute of General Medical Sciences, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

–By Catherine Zandonella

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Listening in on bacterial communications

Leah Bushin and Mohammad Syedsayamdost

While an undergraduate, Leah Bushin (left) co-authored an article on the structure of a signaling molecule involved in bacterial communication with co-first author Kelsey Schramma and adviser Mohammad Seyedsayamdost (right), assistant professor of chemistry, PHOTO BY C. TODD REICHART

BACTERIA SPEAK TO ONE ANOTHER using a soundless language known as quorum sensing. In a step toward translating bacterial communications, researchers have revealed the structure and biosynthesis of streptide, a signaling molecule involved in the quorum sensing system common to many diseasecausing streptococci bacteria.

The research team included undergraduate Leah Bushin, who was the co-first author on an article published on April 20, 2015, in Nature Chemistry. Bushin helped determine the structure of streptide as part of her undergraduate senior thesis project.

To explore how bacteria communicate, first she had to grow them, a challenging process in which oxygen had to be rigorously excluded. Next, she isolated the streptide and analyzed it using two-dimensional nuclear magnetic resonance (NMR) spectroscopy, a technique that allows scientists to deduce the connections between atoms.

The experiments revealed that streptide contains an unprecedented crosslink between two unactivated carbons on the amino acids lysine and tryptophan. To figure out how this novel bond was being formed, the researchers took a closer look at the gene cluster that produces streptide. Within the gene cluster, they suspected that a radical S-adenosyl methionine (SAM) enzyme, which they dubbed StrB, could be responsible for this unusual modification.

“Radical SAM enzymes catalyze absolutely amazing chemistries,” said Kelsey Schramma, a graduate student and the other co-first author on the article. The team showed that one of the iron-sulfur clusters reductively activated one molecule of SAM, kicking off a chain of one-electron (radical) reactions that gave rise to the novel carbon-carbon bond.

Kelsey Schramma is a graduate student in chemistry working on a project to study bacterial communication. Disrupting communication could lead to novel strategies to fight infections. PHOTO CREDIT: C. TODD REICHART

Kelsey Schramma is a graduate student in chemistry working on a project to study bacterial communication. Disrupting communication could lead to novel strategies to fight infections. PHOTO CREDIT: C. TODD REICHART

“The synergy between Leah and Kelsey was great,” said Mohammad Seyedsayamdost, an assistant professor of chemistry who led the research, which was supported by the National Institutes of Health. “They expressed interest in complementary aspects of the project, and the whole ended up being greater than the sum of its parts,” he said.

Future work will target streptide’s biological function — its meaning in the bacterial language — as well as confirming its production by other streptococcal bacteria strains.

–By Tien Nguyen

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Measles may weaken immune system up to three years

Measles vaccination

Measles may weaken immune system up to three years. PHOTO BY SHUTTERSTOCK

THE MEASLES VIRUS can lead to serious disease in children by suppressing their immune systems for up to three years, according to a study published in the journal Science on May 8, 2015. The study provides evidence that measles may throw the body into a much longer-term state of “immune amnesia,” where essential memory cells that protect the body against infectious diseases are partially wiped out. This vulnerability was previously thought to last a month or two.

“We already knew that measles attacks immune memory, and that it was immunosuppressive for a short amount of time. But this paper suggests that immune suppression lasts much longer than previously suspected,” said C. Jessica Metcalf, co-author and assistant professor of ecology and evolutionary biology and public affairs, who is affiliated with Princeton’s Woodrow Wilson School of Public and International Affairs.

The research findings suggest that — apart from the major direct benefits — measles vaccination may also provide indirect immunological protection against other infectious diseases.

The work was funded by the Bill & Melinda Gates Foundation, the Science and Technology Directorate of the Department of Homeland Security, and the Research and Policy for Infectious Disease Dynamics (RAPIDD) Program of the National Institutes of Health’s Fogarty International Center.

–By B. Rose Huber

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Bioengineering: Unlocking the secrets of human health

Bioengineering cover imageBy Takim Williams

RED-HOT RIVERS OF MOLTEN COPPER and aluminum alloys streamed from one receptacle to another. As an undergraduate watching the demonstration in a materials science class, Clifford Brangwynne was reminded of cells migrating through the bloodstream. He realized at that moment that he could mold his interest in materials science and engineering into work that might ultimately have implications for human health.

Bioengineering blood vessel imageScientists like Brangwynne, now an assistant professor of chemical and biological engineering at Princeton, recognize the natural connection between engineering and the life sciences. Their research is setting the groundwork for future applications in health and medicine, including curing diseases such as Alzheimer’s, growing replacement organs and preventing developmental abnormalities. Each of these pursuits hinges on the understanding that living matter obeys the same principles as nonliving matter.

Discovering the relevant principles — and using them to manipulate biological systems to meet our needs — is the goal of the growing field of bioengineering. “The thing that we do that’s different from other scientists who are looking at states of matter and their properties is that we are doing it in the context of living cells,” Brangwynne said. “What is the state of matter inside of a cell and how does that enable biological function?”

Brangwynne gestures toward a can of Gillette shaving foam next to a cylinder of silly putty on his desk and explains that the familiar grade school schema of three states of matter — solid, liquid, gas — is not entirely accurate. There are phases in between, and combinations with their own surprising properties.

“A mound of foam is essentially a solid,” Brangwynne said. “You can push on it and it deforms, and when you take your finger away it springs back into shape. You’ve taken something that is 95 percent gas — it’s mostly air — and 5 percent liquid, and you’ve combined those in such a way that you get a solid.”

Correct biological function depends on transitions between these phases of matter. For example, your blood — typically a free-flowing liquid — clots to form a protective scab. However, these transitions can cause problems, for instance when an internal blood clot causes a stroke.

Likewise, the liquid inside each cell — the cytoplasm — regularly goes through local phase transitions, some of which are disruptive. This issue is linked to neurodegenerative diseases, including Alzheimer’s disease and amyotrophic lateral sclerosis (Lou Gehrig’s disease). In these cases, proteins aggregate and spontaneously transition from a liquid phase into a sticky, solid-like state, prohibiting normal function in the brain or nervous system. These phase transitions are also thought to be involved in controlling cell size and growth, and thus diseases such as cancer.

Nucleoli from amphibian egg cells

Researchers in the Brangwynne lab use nucleoli from amphibian egg cells to study the role of gravity in determining the size of living cells. IMAGE COURTESY OF CLIFFORD BRANGWYNNE LAB

Brangwynne’s research focuses on gaining a better understanding of these living states of matter, and how they can be manipulated. Over the past few years, his group has published several studies exploring the molecules that control intracellular phase transitions and how the living matter within a cell affects gene regulation and cell size.

In one particularly exciting study, graduate student Marina Feric discovered that liquid-phase droplets of RNA and protein are biophysically linked to cell size through the force of gravity. Brangwynne, who receives support from the National Science Foundation (NSF) and the National Institutes of Health (NIH), is optimistic that these fundamental studies will lead to medical applications. “We’re certainly hoping to use our findings to perturb these systems and keep cells in a healthier state,” he said.

Swimming upstream

Like Brangwynne, Professor Howard Stone followed the flow of ideas from an area of engineering — fluid mechanics — to biology. “The living world involves flow almost by definition,” said Stone, who is the Donald R. Dixon ’69 and Elizabeth W. Dixon Professor of Mechanical and Aerospace Engineering. “You circulate blood, you breathe streams of air in and out, you sweat to regulate temperature. If you study fluid mechanics, and if you’re somewhat open-minded, it’s easy to stumble across biological problems.”

One of the biological problems that caught Stone’s attention is how bacteria move in a fluid. Moving through water is far more difficult for bacteria than it is for a human. For single-celled creatures a millionth of a meter long, the force of friction dominates their ability to swim in a given direction. Instead, bacteria are usually just carried along for the ride.

Image of lungs

“The living world involves flow almost by definition.” –Howard Stone, the Donald R. Dixon ’69 and Elizabeth W. Dixon Professor of Mechanical and Aerospace Engineering

The dominance of friction led to a discovery in Stone’s lab four years ago by visiting graduate student Yi Shen, who found that P. aeruginosa, a dangerous pathogen sometimes found in hospitals, can move against a current. The bacterium loses its flagellum — a long tail for swimming — when it adheres to a surface, which for many cells dictates the end of mobility. Yet P. aeruginosa can drag itself along a wall of, for example, a branched medical tube, by its small, tentacle-like pili, which are strong enough to resist the force of friction.

After observing this phenomenon, Stone and his collaborators — Professor of Molecular Biology Zemer Gitai; Albert Siryaporn, an associate research scholar in molecular biology; and Kevin Minyoun Kim, a graduate student in chemistry — began researching this behavior in systems mimicking the human body.

“Your blood vessels have branches. Your lungs are branched. We’ve made model branched systems and used a pump — like a heart — to drive fluid through it,” Stone said. “Bacteria inoculated into this flow sometimes end up in places you wouldn’t expect, and that, at this time, a simulation would never predict.” This basic research, which is supported by NSF, may allow us to better anticipate the movements of pathogens — in our bodies or our environments — in order to prevent infection and contamination.

Fluid environment

Bacteria are single-celled organisms that can form colonies, but a more sophisticated arrangement occurs in our own bodies, where huge communities of cells organize into tissues and organs. Celeste Nelson, an associate professor of chemical and biological engineering, studies the fetal development of these organs, which depends on the fluid environment in which they form.

One of the organs that forms in a fluid environment is the lung. A fetus’s lungs are filled with fluid during gestation. Nelson uses tissue cultures — parts of organs grown in laboratory dishes — and manipulates the speed and pressure of tiny streams of liquid that are directed onto the growing lung cells by small tubes.

Nelson has discovered that the higher the pressure of this fluid in the fetal lung, the more quickly the lungs develop, whereas lower pressure leads to slower development. Several congenital disorders can derail lung development, and Nelson’s work — which is supported by NIH, NSF, the David and Lucile Packard Foundation, the Camille and Henry Dreyfus Foundation, the Burroughs Wellcome Fund, the Essig Enright Family Foundation, and Princeton’s Project X, which provides seed funding for unconventional research — may improve our ability to diagnose such problems early.

The lungs, kidneys, mammary glands and other organs develop through a branched structure, which is an efficient space-filling strategy for functions that require maximum surface area. This exponential branching pattern is a highly reproducible selfassembly process, and in Nelson’s opinion, the forest of alveoli in the lungs is the most beautiful example. “The 23 generations of branches means several hundred million paths,” said Nelson. “Every one of those paths is needed for efficient diffusion of oxygen into the infant blood stream immediately after birth. “What’s amazing,” she said, “is that all of the branches in my lungs look exactly like the branches in your lungs.”

Lung image

Lung tissue extracted from a reptile embryo helps the Nelson lab study the effect of the fluid environment on lung development.

Nelson’s lab also studies a behavior in cancer cells called reversion, which — if it could be induced — would turn many cancers into benign, treatable illnesses. She collaborates with Derek Radisky, a researcher at the Mayo Clinic in Jacksonville, Florida. For Nelson, who started studying breast cancer while a postdoctoral fellow, the body’s organ systems have a mechanical elegance.

Timing is everything

Stanislav Shvartsman is as fascinated by the chemical aspects of development as Nelson is by the mechanical aspects. His research focuses on embryogenesis, the very early stages of fetal development.

“When you want to bake a cake, it’s not enough to say that you need eggs and milk and flour,” said Shvartsman, a professor of chemical and biological engineering and the Lewis-Sigler Institute for Integrative Genomics. “Knowing the ingredients, and even knowing the sequence in which you add these ingredients — which is what we know from genetics — is not enough to bake a cake that tastes good.”

When the recipe — the proper quantities of chemicals released by the cells of the embryo at the proper times — isn’t followed exactly, there are consequences for the developing organism. For example, a large class of developmental abnormalities, known as RAS-opathies, is associated with asymmetry in the craniofacial complex, stunted height, congenital heart defects, developmental delays and other issues. Such defects are observed in one in every thousand births and are believed to be caused by mutations in genes of the Ras-MAPK pathway.

Biologists know which genes are mutated, and even where to find these genes on our DNA. What they don’t know is why these particular mutations lead to a distinct set of clinical features. To find out, researchers turn to organisms that macroscopically look very different from us — such as bacteria and worms — but are very similar at the cellular level. Shvartsman’s research group, which is supported by NIH and NSF, uses the fruit fly to study embryogenesis.

Initially, the handful of cells that make up an embryo are all identical. By the time of birth, that homogenous handful will have given rise to brain, nerve, heart, blood and every other kind of cell required for a living, breathing organism. In order to differentiate into the right kind of cell at the right time, and to arrange into the correct three-dimensional shape, the embryonic cells have to communicate. They speak to each other through a language of chemical signals.

The signals are actually protein molecules, Shvartsman said. A protein released by one cell attaches to a receptor protein embedded in the surface of a neighboring cell. That surface protein reacts by changing shape, and in turn changing the internal environment of its cell. In this way cells “hear” each other. At any given time multiple cells are releasing various proteins, and the combination of signals floating through the embryonic environment tells a cell what to become, or when to divide to make more of itself.

To crack the code, Shvartsman is looking at one signal at a time, beginning with a set of proteins that is well understood genetically thanks to the work of such Princeton biologists as Gertrud Schüpbach, the Henry Fairfield Osborn Professor of Biology. By controlling the amount of these proteins released in the fly embryo, Bomyi Lim, a former graduate student in Shvartsman’s lab and now a postdoctoral research associate at the Lewis-Sigler Institute for Integrative Genomics, has discovered the minimum dosage necessary for proper structural development. This is the first step in a long process, but it is a milestone, and Shvartsman is excited about continuing the process. “It’s very exciting to work in a field where there’s no risk of ever saying, ‘This is the end of the times-table. There is no more material to learn,’” he said.

Image of fruit fly embryo structure

The image shows thin slices of a part of fruit fly embryos where stem cells turn into mature eggs. Created by graduate students Yogesh Goyal and Bomyi Lim and postdoctoral researcher Miriam Osterfield in the laboratory of Stanislav Shvartzman, the image was selected for display in Princeton’s 2014 Art of Science competition.

Some assembly required

Brangwynne views embryogenesis as the epitome of self-assembly, the process by which small, disorganized components interact based on simple rules to form complex structures without human intervention. A classic example is the snowflake, a delicate crystalline jewel formed in midair as water molecules freeze. Engineers have been trying to take advantage of self-assembly for some time — often in Brangwynne’s field of materials science — where time and money could be saved if certain synthetic materials would form on their own in a solution, rather than being painstakingly put together atom by atom.

“The embryo of an organism like C. elegans essentially starts out as just a bag of molecules,” Brangwynne said. Once the egg is fertilized, it begins to organize, and the unstructured soup of molecules turns into a wriggling worm. “There’s absolutely nothing that human engineering can do that comes anywhere close to what I just described takes place in embryos all the time,” Brangwynne said.

While Princeton scientists are importing methods and paradigms from engineering disciplines to biology, they see a two-way street, recognizing that biology itself has methods to share.

“We would like to learn how nature, through hundreds of millions of years of evolution, has generated these systems that are just completely unbelievable in their level of sophistication,” Brangwynne said. “It’s as if we’ve been visited by an alien civilization that was millions of years more advanced than us. The first thing we would do would be to take a really close look at that spaceship. We’d try to figure out what it is made of, what are the principles that govern its flight and its control systems. That’s what we are doing with biological systems.”

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Striking resemblance: A physical law may govern very different biological activities

Starlings

FLOCKS OF BIRDS FLY ACROSS THE SKY in shifting configurations. In the retina of an eye, millions of neurons ignite in ever-changing combinations, translating light into meaningful images. Yet both of these seemingly random behaviors have an underlying order that can be described by mathematics.

Like these cells and birds, when atoms and molecules come together they can display coordinated behaviors that are more than the sum of their parts. At a critical point, such as the boundary between liquid and gas, local interactions between molecules propagate through an entire material, changing its essential properties.

Princeton biophysicist William Bialek thinks criticality may also underlie collective behaviors in living organisms, and he’s using real-world data to test this hypothesis. Recently, Bialek and his colleagues have analyzed the flocking behaviors of birds, the genetic networks of fruit fly embryos and the activation patterns of salamander neurons.

“In physics, we use the same mathematical language to describe many seemingly different behaviors,” said Bialek, the John Archibald Wheeler/Battelle Professor in Physics and the Lewis-Sigler Institute for Integrative Genomics. “So we understand that the emergence of collective behavior from all the individual interactions has a kind of universality.”

To explore the possibility that this universality might extend to living systems, Bialek made use of a large dataset on the changing positions and velocities of thousands of individual birds in a flock of starlings. A group of Italian physicists used multiple cameras to record the birds and calculate their exact locations over time in three dimensions — “a technical triumph,” according to Bialek.

The researchers, including former Princeton postdoctoral fellows Thierry Mora and Aleksandra Walczak, analyzed the deviations of each bird from the flock’s average speed and direction of movement. They found not only that these variations were correlated between nearby birds, but also that the fluctuations from the average propagated through the group over long distances. This pattern of rapid, remote signal transmission echoes the changes that occur among molecules during a phase change from solid to liquid or liquid to gas. At a critical point, this could allow information to spread swiftly through the group, enabling the whole flock to nimbly change direction.

“The model you build just by keeping track of what each bird does relative to its neighbors predicts what happens throughout the entire flock,” Bialek said. “And it does so with an accuracy that is beyond what we had any reason to expect. It’s really a very precise prediction.”

Other biological examples of criticality play out on a microscopic scale. Bialek has an ongoing collaboration with Princeton’s Squibb Professor in Molecular Biology Eric Wieschaus, a Howard Hughes Medical Institute researcher and Nobel Prize winner, who has uncovered many of the genes involved in the embryonic development of the fruit fly — a model biological system.

Bialek has found signatures of criticality in gene activation patterns during the first few hours of fly embryo development. The synchronized actions of “gap genes” establish the fly’s 14-segment body plan. Mutations in these genes lead to gaps between segments, whose effects are reflected in the names of the genes: two examples are “hunchback” and “giant.”

Recently, Thomas Gregor, an assistant professor of physics and also a member of the Lewis-Sigler Institute, has developed experimental tools to precisely measure the activity of many gap genes at once, all along the halfmillimeter length of the fly embryo. These measurements allowed Bialek and physics graduate student Dmitry Krotov to test whether the patterns of gene activity across the embryo fit a model of criticality. Indeed, using data from 24 embryos, they found that fluctuations from the average level of gene activity at each point along the embryo were correlated between certain pairs of gap genes, which regulate one another’s activity like on/off switches. They mapped the locations of these switch points, which appear to act like signals that spread over long distances, just as changes in velocity are correlated in a flock of birds.

Bialek has also looked for signatures of criticality among the activation patterns in a patch of 160 nerve cells from a salamander retina, a model system for studying this light-sensing layer of the eye. In collaboration with Michael Berry, an associate professor of molecular biology and the Princeton Neuroscience Institute, Bialek and his colleagues showed how the coordinated activity of the neurons could be tuned to a critical state.

Bialek thinks critical systems may be common features of life that have repeatedly evolved in different organisms and at different levels — both molecular and behavioral. This could explain why, though systems of cells or groups of organisms could be organized in any number of possible ways, networks with similar properties continue to emerge.

“Is there anything special about the way nature has organized things in living systems?” Bialek wondered. He said much more work is necessary to claim criticality as a general biological principle. “But I do think we’re seeing in the data, somehow, signs of that specialness — things that it seems you can only get if the system has been set up in particular ways and not in others,” he said. “That I find very appealing.”

This work was supported in part by the National Science Foundation, the National Institutes of Health, the Howard Hughes Medical Institute, the W.M. Keck Foundation and the Swartz Foundation.

–By Molly Sharlach

No more mirrors: a new way of making molecules for tracking disease

Abigail Doyle

Abigail Doyle, associate professor of chemistry (Photo by C. Todd Reichart)

RADIOACTIVITY IS USUALLY ASSOCIATED with nuclear fallout or comic-book spider bites, but in very small amounts it can be a useful tool for diagnosing diseases.

Small molecules containing a radioactive isotope of fluorine, called 18F, allow doctors to track tumors using a scanning procedure known as positron emission tomography (PET). But existing methods of making 18F radiotracers tend to produce molecules that are identical in every way but one — the molecules are oppositely oriented, like a person’s right and left hand, or like mirror images. Due to their distinct 3-D structures, only one of the mirror images — known as enantiomers — is useful for tracking tumors.

Now, researchers at Princeton University led by Abigail Doyle, an associate professor of chemistry, report a route that can selectively produce just one type of enantiomer — either right-handed or left-handed — which could aid researchers in making more potent radiotracers. The work was published online in March 2014 in the Journal of the American Chemical Society.

“We know that in biology, small-molecule interactions with enzymes often depend on the 3-D properties of the molecule,” Doyle said. “Being able to prepare the enantiomers of a given tracer, in order to optimize which tracer has the best binding and imaging properties, could be really useful.”

Molecules

Molecules such as the one pictured can occur as two varieties that are identical in every way but one — they are mirror images of each other. Due to their distinct 3-D structures, these mirror images, known as enantiomers, interact with the body differently. Abigail Doyle’s group report a route that can selectively produce just one type of enantiomer, a result that could help researchers make more potent radiotracers for use in disease diagnosis.

Doyle’s research team developed a cobalt fluoride catalyst that causes radioactive fluoride to react with epoxides — triangleshaped molecules that contain an oxygen atom. The researchers’ method demonstrated excellent ability to select single enantiomers for 11 substrates, five of which are known pre-clinical PET tracers.

With this new method, researchers can test single enantiomers of existing or new PET radiotracers and evaluate if these compounds offer any advantage over the enantiomeric mixtures. Ultimately, the goal is to use this chemistry to identify a completely novel PET radiotracer for imaging.

Currently, there are only four FDA-approved 18F radiotracers. One of the major limitations to discovering PET tracers is the source of 18F. Existing 18F sources are strongly basic and, during the process of making the 18F radiotracer, can cause the elimination of alcohol and amine groups and rearrange the groups into mixtures of enantiomers in a process called racemization.

Under Doyle’s less basic reaction conditions, however, even alcohols and secondary amines are tolerated and no racemization is observed. The research was supported by the National Institutes of Health, the National Science Foundation and the Pennsylvania Department of Health.

First author Thomas Graham, who earned his Ph.D. in spring 2014, and graduate student Frederick Lambert commuted to the University of Pennsylvania, where they conducted the radiolabeling experiments in the laboratory of collaborator Hank Kung, an emeritus professor of radiology.

“We demonstrated that the radioactivity is high enough that we could actually use it for imaging. That’s an exciting next step,” Doyle said.

–By Tien Nguyen