Rerouting Cancer

Cancer is capable of rapidly developing resistance to therapeutic drugs, rendering those drugs harmless—often before they have a chance to work. Now, researchers at Caltech and their colleagues have identified how at least one brain cancer, called glioblastoma multiforme (GBM), adapts so fast—and they show that by formulating the right combination of drugs, doctors could potentially overcome this resistance and stop a tumor in its tracks.

The work appears in the April 12 issue of the journal Cancer Cell.

Some cancer drugs are designed to target a cell's chemical circuitry. This network of signaling pathways controls how a healthy cell functions, but in many cancers, the pathways are hyperactivated, directly leading to the aggressive nature of the disease. By blocking a key pathway, a drug can, in principle, stop the tumor from growing.

"The concept is that if you block a key node in the pathway, then the communication can't proceed and the cells can't get the signals to divide and multiply," explains Jim Heath, the Elizabeth W. Gilloon Professor of Chemistry and co-corresponding author on the paper.

In reality, however, tumors can become resistant to a drug even if the drug works exactly as designed. With GBM, such resistance develops in almost every patient. "In some patients, you can treat with a drug that does everything you could want it to do, but you would never know that the drug hit the target because the tumor adapts so quickly," Heath says.

Some scientists have suspected that the cancer becomes resistant through Darwinian-type evolution, in a process similar to how bacteria develop resistance to antibiotics. That is, the genetic differences of certain cancer cells may make those cells resistant to a drug. Nonresistant cells are killed by the drug and their death leaves room for the naturally resistant cells—and tumors—to grow and multiply.

However, this mechanism was not what Heath and his colleagues found in studies of tissue from glioblastoma patients. Instead, the researchers discovered that the cancer cells that developed resistance to a drug were the same cells that had responded to the drug. When the drug blocks a signaling pathway in a cancer cell, they realized, the cell simply finds a detour, like a GPS navigator that reroutes to avoid traffic.

"You can block a key part," Heath says, "and the cells will respond to route around that part you blocked."

This notion of shifting pathways is not new, but the work is the first to show that the process can happen in as little as two days. In particular, the researchers found that the changes occur with a specific drug (CC214‑2) that targets a central GBM signaling-protein called mTOR. When mTOR is inhibited, certain GBM signaling pathways are repressed, but others are activated.

To map the detours, the researchers separated individual GBM cells from patient tumors and measured the levels of several key proteins in the cells. These proteins—called phosphoproteins because they are activated by the addition of a phosphoryl group to a molecule—carry signals throughout the cell. Measurements of the abundance of the proteins showed that the drug was effective.

The story was different at the single-cell level, at which Heath and his colleagues not only measured the levels of proteins in individual cells, but also the signaling between those proteins. For example, if protein A signals protein B, then the levels of A, as measured across many single cells, will correlate with the levels of B.  By measuring the presence of several such proteins, the researchers could infer the structure of the protein signaling network.

They discovered that after the drug was introduced, the cell activated new pathways that previously had been dormant. This drug-induced pathway activation suggested several combination therapies that might halt the development of drug resistance, as well as drugging strategies that would have no effect.

In mice, Heath and his team tested seven therapies or therapy combinations that they predicted would—or would not—halt resistance development. The four that they predicted would not work were, indeed, ineffectual; the three they thought would work, did. The researchers then showed that they could see similar effects in GBM patient tissues, as well as in melanoma tumor models. This kind of rapid drug adaptation by tumors may occur in many cancer types, and helps explain how cancers can develop resistance to targeted drugs so quickly, Heath says.

The good news is that, by identifying the drug-activated signaling pathways, one may be able to find drug combinations that will suppress resistance, Heath says. Eventually, he says, clinicians may be able to analyze a patient's tumor at the single-cell level to determine the best therapy strategy.

These kinds of drug combinations would likely remain a secondary therapy against cancer—used when treatments like chemotherapy, radiation, and surgery fail. But, Heath says, they are essential for staving off the resistance that has severely limited the benefits that patients currently receive from targeted therapies.

The first authors of the Cancer Cell paper, titled "Single cell phosphoproteomics resolves adaptive signaling dynamics and informs targeted combination therapy in glioblastoma," are Wei Wei (PhD '14), a visitor in chemistry at Caltech and assistant professor at UCLA, and Young Shik Shin (MS '06, PhD '11), who now works at a biotech startup. Both are former graduate students of Heath's. A third key contributor to the work was Beatrice Gini, formerly a member of the UC San Diego (UCSD) laboratory of co-corresponding author Paul Mischel and now at UC San Francisco. Other Caltech authors include Min Xue and Kiwook Hwang (PhD '13) and graduate students Jungwoo Kim and Yapeng Su. Authors also include researchers from UCSD, the University of Verona in Italy, Northwestern University, and the Celgene Corporation. Heath is board member of and holds a financial interest in IsoPlexis, a company that is commercializing a microchip technology similar to what was used for single-cell analyses in the research described. 

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A new strategy may help overcome cancer cells' drug resistance.

Midnight Blue: A New System for Color Vision

The swirling skies of Vincent Van Gogh's Starry Night illustrate a mystery that has eluded biologists for more than a century—why do we perceive the color blue in the dimly lit night sky? A newly discovered mechanism of color vision in mice might help answer this question, Caltech researchers say.

The work, which was done in the laboratory of Markus Meister, Anne P. and Benjamin F. Biaggini Professor of Biological Sciences, will be published on April 14 in the print edition of the journal Nature.

In humans, vision is enabled by two types of light-sensitive photoreceptor cells called rods and cones. When these photoreceptors detect light, they send a signal to specialized neurons in the retina called retinal ganglion cells, or RGCs, which then transmit visual information to the brain by firing electrical pulses along the optic nerve.

A standard biology textbook would likely explain that vision in dim light is enabled by rods—sensitive light detectors that are only capable of producing black and white vision. Color vision, on the other hand, is enabled by cones, which are active in bright light. Humans have three types of cones, and each cone contains a different light-sensitive chemical, or pigment, that reacts to different colors, or wavelengths, of light. We have red-, green-, and blue-sensitive cones, and the brain perceives color by comparing the different signals it receives from nearby cones of each type.

To explore whether or not there were other modes of color vision, Meister and his team studied another mammal: the mouse. Previous behavioral studies indicated that mice are indeed capable of some form of color vision. As in humans, that vision is dependent on light signals picked up by cones. Mice have two types of cones—one that is sensitive to medium-wavelength green light and one that is sensitive to short-wavelength ultraviolet light (UV).

"The odd thing about the mouse is that these two kinds of cones are actually located in different parts of the retina," Meister says. "Mice look at the upper part of the visual field with their UV cones and the lower part with their green cones. We wanted to know how a mouse perceives color when any given part of the image is analyzed with only one cone or the other cone—meaning the brain can't compare the two cone signals to determine a color."

The researchers discovered that a certain type of neuron in the mouse retina, called a JAMB retinal ganglion cell (J-RGC), was critical. These J-RGCs can signal color to the brain because they fire faster in response to green light and stop firing in response to ultraviolet light. Curiously, the J-RGCs were turned on by green light even in the upper part of the visual field, which contained no green cones.

Through additional experiments, Meister and his team discovered how the J-RGC compares signals from the ultraviolet cones to signals from rods, which are also sensitive in the green part of the spectrum. This revealed, for the first time, an essential antagonistic relationship between the rods and the cones of the retina. Rods excite a neuron called a horizontal cell, which then inhibits the ultraviolet cones.

Meister and his colleague, first author Maximilian Joesch from Harvard University, wanted to determine how this color vision system would be helpful to a mouse in its natural environment. To find out, they fitted a camera with filters that would replicate the wavelengths detected by the mouse rods and cones and used it to take images of plants and materials that a mouse might encounter in nature.

Their photographic scavenger hunt yielded two materials—seeds and mouse urine—that were much more visible through the mouse's green and ultraviolet system than through human color vision. The researchers speculate that because mice need seeds for sustenance and use urine for social communication—via "urine posts," a form of territorial marking—they might use this mechanism to find food and spot neighbors.

Meister says there is reason to believe that this same pathway—from rods to horizontal cells to cones—is responsible for the human perception of the color blue in dim light. In the human retina, the horizontal cell preferentially inhibits the red and green cones, but not the blue cones.

"In really dim light, our cones don't receive enough photons to work, but they continue to emit a low-level baseline signal to the rest of the retina that is independent of light," he explains. "The rods are active, however, and through the horizontal cell they inhibit both the red and green cones. Because this baseline signal from the red and green cones is suppressed, it looks like the blue cones are more active. To the rest of the retina, it seems like everything in the field of vision is blue."  

So, perhaps Van Gogh's color choice for the night sky was a biological decision as well as an artistic one. "Color has intrigued scientists, artists, and poets throughout human civilization. Our paper adds to the understanding of how this quality of the world is perceived," Meister says.

Meister's work was published in a paper titled "A neuronal circuit for color vision based on rod-cone opponency." Funding for the work was provided by the National Institutes of Health and The International Human Frontier Science Program Organization.

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Midnight Blue: A New System for Color Vision
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A newly discovered mechanism of color vision in mice might help answer why the dimly lit night sky has a bluish cast.

Biochemists Solve the Structure of Cell's DNA Gatekeeper

Caltech scientists have produced the most detailed map yet of the massive protein machine that controls access to the DNA-containing heart of the cell.

In a new study, a team led by André Hoelz, an assistant professor of biochemistry, reports the successful mapping of the structure of the symmetric core of the nuclear pore complex (NPC), a cellular gatekeeper that determines what molecules can enter and exit the nucleus, where a cell's genetic information is stored.

The study appears in the April 15, 2016 issue of the journal Science, featured on the cover.

The findings are the culmination of more than a decade of work by Hoelz's research group and could lead to new classes of medicine against viruses that subvert the NPC in order to hijack infected cells and that could treat various diseases associated with NPC dysfunction.

"The methods that we have been developing for the last 12 years open the door for tackling other large and flexible structures like this," says Hoelz. "The cell is full of such machineries but they have resisted structural characterization at the atomic level."

The NPC is one of the largest and most complex structures inside the cells of eukaryotes, the group of organisms that includes humans and other mammals, and it is vital for the survival of cells. It is composed of approximately 10 million atoms that together form the symmetric core as well as surrounding asymmetric structures that attach to other cellular machineries. The NPC has about 50 times the number of atoms as the ribosome—a large cellular component whose structure was not solved until the year 2000. Because the NPC is so big, it jiggles like a large block of gelatin, and this constant motion makes it difficult to get a clear snapshot of its structure.

In 2004, Hoelz laid out an ambitious plan for mapping the structure of the NPC: Rather than trying to image the entire assembly at once, he and his group would determine the crystal structures of each of its 34 protein subunits and then piece them together like a three-dimensional jigsaw puzzle. "A lot of people told us we were really crazy, that it would never work, and that it could not be done," Hoelz says.

Last year, the team published two papers in Science that detailed the structures of key pieces of the NPC's inner and outer rings, which are the two primary components of the NPC's symmetric core. The donut-shaped core is embedded in the nuclear envelope, a double membrane that surrounds the nucleus, creating a selective barrier for molecules entering and leaving the nucleus.

By being able to piece these crystal structures into a reconstruction of the intact human NPC obtained through a technique called electron cryotomography—in which entire isolated nuclei are instantaneously frozen, with all of their structures and molecules locked into place, and then probed with a transmission electron microscope to produce 2-D images that can be reassembled into a 3-D structure—"we bridged for the first time the resolution gap between low-resolution electron microscopy reconstructions that provide overall shape and high-resolution crystal structures that provide the precise positioning of all atoms," Hoelz says.

With these structures known, the mapping of the rest of the NPC's symmetric core came quickly. "It is just like when solving a puzzle," he says. "By placing the first piece confidently, we knew that we would eventually be able to place all of them."

As described in the new paper, Hoelz's research group now has solved the crystal structures of the last remaining components of the symmetric core's inner ring and determined where all of the rings' pieces fit in the NPC's overall structure.

To do this, the team had to first generate a complete "biochemical interaction map" of the entire symmetric core. Akin to a blueprint, this map describes the interconnections and interactions of all of the proteins, as part of a larger cellular machine. The process involved genetically modifying bacteria to produce purified samples of each of the 19 different protein subunits of the NPC's symmetric core and then combining the fragments two at a time inside a test tube to see which adhered to each other.

The team then used the completed interaction map as a guide for identifying the inner ring's key proteins and employed X-ray crystallography to determine the size, shape, and position of all of their atoms. X-ray crystallography involves shining X-rays on a crystallized sample and analyzing the pattern of rays reflected off the atoms in the crystal. The team analyzed thousands of samples at Caltech's Molecular Observatory—a facility developed with support from the Gordon and Betty Moore Foundation that includes an automated X-ray beam line at the Stanford Synchrotron Radiation Laboratory that can be controlled remotely from Caltech—and the GM/CA beam line at the Advanced Photon Source at the Argonne National Laboratory.

"We now had a clear picture of what the key jigsaw pieces of the NPC looked like and how they fit together," says Daniel Lin, a graduate student in Hoelz's lab and one of two first authors on the study.

The next step was to determine how the individual pieces fit into the larger puzzle of the NPC's overall structure. To do this, the team took advantage of an electron microscopy reconstruction of the entire human NPC published in 2015 by Martin Beck's group at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. The images from Beck's group were relatively low resolution and revealed only a rough approximation of the NPC's shape, but they still provided a critical framework onto which Hoelz's team could overlay their atomic high-resolution images, captured using X-ray crystallography. The NPC is the largest cellular structure ever pieced together using such an approach.

"We were able to use the biochemical interaction map we created to solve the puzzle in an unbiased way," Hoelz says. "This not only ensured that our pieces fit in the electron microscopy reconstruction, but that they also fit together in a way that made sense in the context of the interaction map."

Hoelz said his team is committed to solving the remaining asymmetric parts of the NPC, which include filamentous structures that serve as docking sites for so-called transport factors that ferry molecules safely through the pore and other cellular machineries that are critical for the flow of genetic information from DNA to RNA to protein.

"I suspect that things are going to move very quickly now," Hoelz says. "We know exactly what we need to do. It's like we're climbing Mount Everest for the first time, and we've made it to Camp 4. All that's left is the sprint to the summit."

Along with Hoelz and Lin, additional Caltech authors on the paper, "Architecture of the symmetric core of the nuclear pore," include research technician Emily Rundlet; Thibaud Perriches, George Mobbs, and Karsten Thierbach, all postdoctoral scholars in chemistry working in the Hoelz lab; and graduate students Ferdinand Huber and Leslie Collins. Other coauthors on the paper include former Hoelz lab member Tobias Stuwe—the second cofirst author of the paper—as well as former lab members Sandra Schilbach, Yanbin Fan, Andrew Davenport (PhD '15), and Young Jeon.

The work was supported by the National Institute of General Medical Sciences; the Caltech-Amgen Research Collaboration; the German Research Foundation; the Boehringer Ingelheim Fonds; the China Scholarship Council; Caltech startup funds; an Albert Wyrick V Scholar Award from the V Foundation for Cancer Research; a Mallinckrodt Scholar Award from the Edward Mallinckrodt Jr. Foundation; a Kimmel Scholar Award from the Sidney Kimmel Foundation; and a Camille Dreyfus Teacher-Scholar Award from the Camille & Henry Dreyfus Foundation. Hoelz is also an inaugural Heritage Principal Investigator of the Heritage Medical Research Institute for the Advancement of Medicine and Science at Caltech.

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The detailed map is the first to determine the structure of a massive protein machine with near-atomic resolution.

Probing the Transforming World of Neutrinos

Every second, trillions of neutrinos travel through your body unnoticed. Neutrinos are among the most abundant particles in the universe, but they are difficult to study because they very rarely interact with matter. To find traces of these elusive particles, researchers from Caltech have collaborated with 39 other institutions to build a 14,000-ton detector the size of two basketball courts called NuMI Off-Axis Electron Neutrino Appearance, or NOvA. The experiment, located in northern Minnesota, began full operation in November 2014 and published its first results in Physical Review Letters this month.

The experiment aims to observe neutrino oscillations—or the conversion of one type of neutrino into another—to learn about the subatomic composition of the universe. There are three different types, or "flavors," of neutrinos—muon-, tau-, and electron-type. The NOvA experiment has made successful detections of the transformation of muon-type neutrinos into electron-type neutrinos. Discovering more about the frequency and nature of neutrino oscillations is an important step to determining the masses of different types of neutrinos, a crucial unknown component in every cosmological model of the universe.

Though neutrinos rarely interact with matter, one in every 10 billion neutrinos that passes through the detector will interact with an atom in the detector. To observe these collisions, a beam of neutrinos 500 miles away at Fermilab in Chicago is fired every 1.3 seconds in a 10-microsecond burst at the detector. The detector is made up of 344,000 cells, each like a pixel in a camera and each filled with a liquid scintillator, a chemical that emits light when electrically charged particles pass through it. When a neutrino smashes into an atom of this liquid—an event estimated to happen once for every 10 billion neutrinos that pass through—it produces a distinctive spray of particles, such as electrons, muons, or protons. When these particles pass through a cell, fluorescent chemicals light up the cell, allowing scientists can track the paths of the particles from the collision.


A muon-type neutrino interaction in the NOvA detector, as viewed by the vertically oriented cells (top panel) and horizontally oriented cells (bottom panel). By using cells oriented both ways, researchers can build a three-dimensional version of the event. The neutrino entered from the left in this image, from the direction of Fermilab. Each colored pixel represents an individual detector cell, with warmer colors corresponding to more observed light and thus more energy deposited by traversing particles. The muon produced in this collision left the long, tell-tale line of active cells along its path. Other particles emanating from the interaction point are also visible. Credit: NOvA Collaboration

"Each type of neutrino leaves a particular signature when it interacts in the detector," says Ryan Patterson (BS '00), an assistant professor of physics and the leader of NOvA's data-analysis team. "Fermilab makes a stream of almost exclusively muon-type neutrinos. If one of these hits something in our detector, we will see the signatures of a particle called a muon. However, if an electron-type neutrino interacts in our detector, we see the signatures of an electron."

Because the beam of neutrinos coming from Fermilab is designed to produce almost entirely muon-type neutrinos, there is a high probability that any signatures of electron-type neutrinos come from a muon-type neutrino that has undergone a transforming oscillation.

Researchers estimated that if oscillations were not occurring, 201 muon-type neutrinos would have been measured over the initial data-taking period, which ended in May 2015. But during this first data-collection run, NOvA saw the signatures of only 33 muon-type neutrinos—suggesting that muon-type neutrinos were disappearing because some had changed type. The detector also measured six electron-type neutrinos, when only one of this type would be expected if oscillations were not occurring.

"We see a large rate for this transition, much higher than it needed to be, given our current knowledge," Patterson says. "These initial data are giving us exciting clues already about the spectrum of neutrino masses."

The Caltech NOvA team led the research and development on the detector elements.  The goal was to make each detector cell sensitive enough to identify the faint particle signals over background noise. The team designed the individual detector elements to operate at -15 degrees Celsius to keep noise—aberrant vibrations and other signals in the data—at a minimum, and also built structures to remove the condensation that can occur at such low temperatures. By the end of construction in 2014, all 12,000 detector arrays, each serving 32 cells, had been built at Caltech.

"The spatial resolution on a detector of this size is unprecedented," Patterson says. "The whole detector is highly 'active'—which means that most of it is actually capable of detecting particles. We have tried to minimize the amount of 'dead' material, like support structures. Additionally, although the different types of neutrinos leave different signatures, these signatures can look similar—so we need as much discrimination power as we can get."

Discovering more about the nature of neutrino oscillations gives important insights into the subatomic world and the evolution of the universe.

"We know that two of the neutrinos are similar in mass, and that a third has a rather different mass from the other two. But we still do not know whether this separated mass is larger or smaller than the other two," Patterson says. Through precise study of neutrino oscillations with NOvA, researchers hope to solve this mass-ordering mystery. "The neutrino mass ordering has connections throughout physics, from the growth of structure in the universe to the behavior of particles at inaccessibly high energies," he says, with NOvA unique among operating experiments because of its sensitivity to this mass ordering.

In the future, researchers at NOvA plan to determine if antineutrinos oscillate at the same rate as neutrinos—that is, to see if neutrinos and antineutrinos behave symmetrically. If NOvA finds that they do not, this discovery could, in turn, help reveal why today the amount of matter in the universe is so much greater than the amount of antimatter, whereas in the early universe, the proportions of the two were balanced.

"These first results demonstrate that NOvA is operating beautifully and that we have a rich physics program ahead of us," Patterson says.

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Is Risk-Taking Behavior Contagious?

Why do we sometimes decide to take risks and other times choose to play it safe? In a new study, Caltech researchers explored the neural mechanisms of one possible explanation: a contagion effect.

The work is described in the March 21 online early edition of the Proceedings of the National Academy of Sciences.

In the study led by John O'Doherty, professor of psychology and director of the Caltech Brain Imaging Center, 24 volunteers repeatedly participated in three types of trials: a "Self" trial, in which the participants were asked to choose between taking a guaranteed $10 or making a risky gamble with a potentially higher payoff; an "Observe" trial, in which the participants observed the risk-taking behavior of a peer (in the trial, this meant a computer algorithm trained to behave like a peer), allowing the participants to learn how often the peer takes a risk; and a "Predict" trial, in which the participants were asked to predict the risk-taking tendencies of an observed peer, earning a cash prize for a correct prediction. Notably in these trials the participants did not observe gamble outcomes, preventing them from further learning about gambles.

O'Doherty and his colleagues found that the participants were much more likely to make the gamble for more money in the "Self" trial when they had previously observed a risk-taking peer in the "Observe" trial. The researchers noticed that after the subjects observed the actions of a peer, their preferences for risk-taking or risk-averse behaviors began to reflect those of the observed peer—a so-called contagion effect. "By observing others behaving in a risk-seeking or risk-averse fashion, we become in turn more or less prone to risky behavior," says Shinsuke Suzuki, a postdoctoral scholar in neuroscience and first author of the study.

To look for indications of risk-taking behavior in specific brain regions of subjects participating in the trials, the Caltech team used functional magnetic resonance imaging (fMRI), which detects brain activity.

By combining computational modeling of the data from the "Self" behavioral trials with the fMRI data, the researchers determined that a region of the brain called the caudate nucleus responds to the degree of risk in the gamble; for example, a riskier gamble resulted in a higher level of observed activity in the caudate nucleus, while a less risky gamble resulted in a lower level of activity. Additionally, the more likely the participants were to make a gamble, the more sensitively activity in the caudate nucleus responded to risk. "This showed that, in addition to the behavioral shift, the neural processing of risk in the caudate is also altered. Also, both the behavioral and neural responses to taking risks can be changed through passively observing the behavior of others," Suzuki says.

The "Predict" behavioral trials were designed to test whether a participant could also learn and predict the risk-taking preferences of an observed peer. Indeed, the researchers found that the participants could successfully predict these preferences—with the learning process occurring even faster if the participant's risk-taking preferences mirrored those of the peer. Furthermore, the fMRI data collected during the "Observe" trial showed that a part of the brain called the dorsolateral prefrontal cortex (dlPFC) was active when participants were learning about others' attitudes toward risk.

The researchers also found differences among participants in functional connectivity between the caudate nucleus and the dlPFC that were related to the strength of the contagion effect—meaning that these two brain regions somehow work together to make a person more or less susceptible to the contagiousness of risk-taking behavior. The work provides an explanation of how our own risk-taking behaviors can be influenced simply by observing the behaviors of others. This study, Suzuki says, is the first to demonstrate that a neural response to risk is altered in response to changes in risk-taking behavior.

"Our findings provide insight into how observation of others' risky behavior affects our own attitude toward risk," Suzuki says—which might help explain the susceptibility of people to risky behavior when observing others behaving in a risky manner, such as in adolescent peer groups. In addition, the findings might offer insight into the formation and collapse of financial bubbles. "The tendency of financial markets to collectively veer from bull markets to bear markets and back again could arise, in part, due to the contagion of observing the risk-seeking or risk-averse investment behaviors of other market participants," he says.

"The findings reported in this paper are part of a broader research goal at Caltech, in which we are trying to understand how the brain can learn from other people and make decisions in a social context," O'Doherty says. "Ultimately, if we can understand how our brains function in social situations, this should also enable us to better understand how brain circuits can go awry, shedding light on social anxiety, autism, and other social disorders."

The paper is titled, "Behavioral contagion during learning about another agent's risk-preferences acts on the neural representation of decision-risk." In addition to Suzuki and O'Doherty, other Caltech coauthors include instructional assistant Emily Jensen and visiting associate in finance Peter Bossaerts. The work was funded by a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad and the Caltech Conte Center for the Neurobiology of Social Decision Making, which is supported by the National Institute of Mental Health.

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The neural processing of risk in our brain is changed when we observe the risk-taking behaviors of others, Caltech study says.

Nanoparticle-Based Cancer Therapies Shown to Work in Humans

A team of researchers led by Caltech scientists has shown that nanoparticles can function to target tumors while avoiding adjacent healthy tissue in human cancer patients.

"Our work shows that this specificity, as previously demonstrated in preclinical animal studies, can in fact occur in humans," says study leader Mark E. Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering at Caltech. "The ability to target tumors is one of the primary reasons for using nanoparticles as therapeutics to treat solid tumors."

The findings, published online the week of March 21 in the journal Proceedings of the National Academy of Sciences, demonstrate that nanoparticle-based therapies can act as a "precision medicine" for targeting tumors while leaving healthy tissue intact.

In the study, Davis and his colleagues examined gastric tumors from nine human patients both before and after infusion with a drug—camptothecin—that was chemically bound to nanoparticles about 30 nanometers in size.

"Our nanoparticles are so small that if one were to increase the size to that of a soccer ball, the increase in size would be on the same order as going from a soccer ball to the planet Earth," says Davis, who is also a member of the City of Hope Comprehensive Cancer Center in Duarte, California, where the clinical trial was conducted.

The team found that 24 to 48 hours after the nanoparticles were administered, they had localized in the tumor tissues and released their drug cargo, and the drug had had the intended biological effects of inhibiting two proteins that are involved in the progression of the cancer. Equally important, both the nanoparticles and the drug were absent from healthy tissue adjacent to the tumors.

The nanoparticles are designed to be flexible delivery vehicles. "We can attach different drugs to the nanoparticles, and by changing the chemistry of the bond linking the drug to the nanoparticle, we can alter the release rate of the drug to be faster or slower," says Andrew Clark, a graduate student in Davis's lab and the study's first author.

Davis says his team's findings suggest that a phenomenon known as the enhanced permeability and retention (EPR) effect is at work in humans. In the EPR effect, abnormal blood vessels that are "leakier" than normal blood vessels in healthy tissue allow nanoparticles to preferentially concentrate in tumors. Until now, the existence of the EPR effect has been conclusively proven only in animal models of human cancers.

"Our results don't prove the EPR effect in humans, but they are completely consistent with it," Davis says.

The findings could also help pave the way toward more effective cancer drug cocktails that can be tailored to fight specific cancers and that leave patients with fewer side effects.

"Right now, if a doctor wants to use multiple drugs to treat a cancer, they often can't do it because the cumulative toxic effects of the drugs would not be tolerated by the patient," Davis says. "With targeted nanoparticles, you have far fewer side effects, so it is anticipated that a drug combination can be selected based on the biology and medicine rather than the limitations of the drugs."

These nanoparticles are currently being tested in a number of phase-II clinical trials. (Information about trials of the nanoparticles, denoted CRLX101, is available at www.clinicaltrials.gov).

In addition to Davis and Clark, other coauthors on the study, entitled "CRLX101 nanoparticles localize in human tumors and not in adjacent, nonneoplastic tissue after intravenous dosing," include Devin Wiley (MS '11, PhD '13) and Jonathan Zuckerman (PhD '12); Paul Webster of the Oak Crest Institute of Science; Joseph Chao and James Lin at City of Hope; and Yun Yen of Taipei Medical University, who was at City of Hope and a visitor in the Davis lab at the initiation of the clinical trial. The research was supported by grants from the National Cancer Institute and the National Institutes of Health and by Cerulean Pharma Inc. Davis is a consultant to and holds stock in Cerulean Pharma Inc. 

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An Up-Close View of Bacterial "Motors"

Bacteria are the most abundant form of life on Earth, and they are capable of living in diverse habitats ranging from the surface of rocks to the insides of our intestines. Over millennia, these adaptable little organisms have evolved a variety of specialized mechanisms to move themselves through their particular environments. In two recent Caltech studies, researchers used a state-of-the-art imaging technique to capture, for the first time, three-dimensional views of this tiny complicated machinery in bacteria.

"Bacteria are widely considered to be 'simple' cells; however, this assumption is a reflection of our limitations, not theirs," says Grant Jensen, a professor of biophysics and biology at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI). "In the past, we simply didn't have technology that could reveal the full glory of the nanomachines—huge complexes comprising many copies of a dozen or more unique proteins—that carry out sophisticated functions."

Jensen and his colleagues used a technique called electron cryotomography to study the complexity of these cell motility nanomachines. The technique allows them to capture 3-D images of intact cells at macromolecular resolution—specifically, with a resolution that ranges from 2 to 5 nanometers (for comparison, a whole cell can be several thousand nanometers in diameter). First, the cells are instantaneously frozen so that water molecules do not have time to rearrange to form ice crystals; this locks the cells in place without damaging their structure. Then, using a transmission electron microscope, the researchers image the cells from different angles, producing a series of 2-D images that—like a computed tomography, or CT, scan—can be digitally reconstructed into a 3-D picture of the cell's structures. Jensen's laboratory is one of only a few in the entire world that can do this type of imaging.

In a paper published in the March 11 issue of the journal Science, the Caltech team used this technique to analyze the cell motility machinery that involves a structure called the type IVa pilus machine (T4PM). This mechanism allows a bacterium to move through its environment in much the same way that Spider-Man travels between skyscrapers; the T4PM assembles a long fiber (the pilus) that attaches to a surface like a grappling hook and subsequently retracts, thus pulling the cell forward.

Although this method of movement is used by many types of bacteria, including several human pathogens, Jensen and his team used electron cryotomography to visualize this cell motility mechanism in intact Myxococcus xanthus—a type of soil bacterium. The researchers found that the structure is made up of several parts, including a pore on the outer membrane of the cell, four interconnected ring structures, and a stemlike structure. By systematically imaging mutants, each of which lacked one of the 10 T4PM core components, and comparing these mutants with normal M. xanthus cells, they mapped the locations of all 10 T4PM core components, providing insights into pilus assembly, structure, and function.

"In this study, we revealed the beautiful complexity of this machine that may be the strongest motor known in nature. The machine lets M. xanthus, a predatory bacterium, move across a field to form a 'wolf pack' with other M. xanthus cells, and hunt together for other bacteria on which to prey," Jensen says.

Another way that bacteria move about their environment is by employing a flagellum—a long whiplike structure that extends outward from the cell. The flagellum is spun by cellular machinery, creating a sort of propeller that motors the bacterium through a substrate. However, cells that must push through the thick mucus of the intestine, for example, need more powerful versions of these motors, compared to cells that only need enough propeller power to travel through a pool of water.

In a second paper, published in the online early edition of the Proceedings of the National Academy of Sciences (PNAS) on March 14, Jensen and his colleagues again used electron cryotomography to study the differences between these heavy-duty and light-duty versions of the bacterial propeller. The 3-D images they captured showed that the varying levels of propeller power among several different species of bacteria can be explained by structural differences in these tiny motors.

In order for the flagellum to act as a propeller, structures in the cell's motor must apply torque—the force needed to cause an object to rotate—to the flagellum. The researchers found that the high-power motors have additional torque-generating protein complexes that are found at a relatively wide radius from the flagellum. This extra distance provides greater leverage to rotate the flagellum, thus generating greater torque. The strength of the cell's motor was directly correlated with the number of these torque-generating complexes in the cell.

"These two studies establish a technique for solving the complete structures of large macromolecular complexes in situ, or inside intact cells," Jensen says. "Other structure determination methods, such as X-ray crystallography, require complexes to be purified out of cells, resulting in loss of components and possible contamination. On the other hand, traditional 2-D imaging alone doesn't let you see where individual protein pieces fit in the complete structure. Our electron cryotomography technique is a good solution because it can be used to look at the whole cell, providing a complete picture of the architecture and location of these structures."

The work involving the type IVa pilus machinery was published in a Science paper titled "Architecture of the type IVa pilus machine." First author Yi-Wei Chang is a research scientist at Caltech; additional coauthors include collaborators from the Max Planck Institute for Terrestrial Microbiology, in Marburg, Germany, and from the University of Utah. The study was funded by the National Institutes of Health (NIH), HHMI, the Max Planck Society, and the Deutsche Forschungsgemeinschaft.

Work involving the flagellum machinery was published in a PNAS paper titled "Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold." Additional coauthors include collaborators from Imperial College London; the University of Texas Southwestern Medical Center; and the University of Wisconsin–Madison. The study was supported by funding from the UK's Biotechnology and Biological Sciences Research Council and from HHMI and NIH.

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Learning to Program Cellular Memory

What if we could program living cells to do what we would like them to do in the body? Having such control—a major goal of synthetic biology—could allow for the development of cell-based therapies that might one day replace traditional drugs for diseases such as cancer. In order to reach this long-term goal, however, scientists must first learn to program many of the key things that cells do, such as communicate with one another, change their fate to become a particular cell type, and remember the chemical signals they have encountered.

Now a team of researchers led by Caltech biologists Michael Elowitz, Lacramioara Bintu, and John Yong (PhD '15) have taken an important step toward being able to program that kind of cellular memory using tools that cells have evolved naturally. By combining synthetic biology approaches with time-lapse movies that track the behaviors of individual cells, they determined how four members of a class of proteins known as chromatin regulators establish and control a cell's ability to maintain a particular state of gene expression—to remember it—even once the signal that established that state is gone.

The researchers reported their findings in the February 12 issue of the journal Science.

"We took some of the most important chromatin regulators for a test-drive to understand not just how they are used naturally, but also what special capabilities each one provides," says Elowitz, a professor of biology and bioengineering at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI). "We're playing with them to find out what we can get them to do for us."

Rather than relying on a single protein to program all "memories" of gene expression, animal cells use hundreds of different chromatin regulators. These proteins each do basically the same thing—they modify a region of DNA to alter gene expression. That raises the question, why does the cell need all of these different chromatin regulators? Either there is a lot of redundancy built into the system or each regulator actually does something unique. And if the latter is the case, synthetic biologists would like to know how best to use these regulators as tools—how to select the ideal protein to achieve a certain effect or a specific type of cellular memory.

Looking for answers, the researchers turned to an approach that Elowitz calls "build to understand." Rather than starting with a complex process and trying to pick apart its component pieces, the researchers build the targeted biological system in cells from the bottom up, giving themselves a chance to actually watch what happens with each change they introduce.

In this case, that meant sticking different chromatin regulators—four gene-silencing proteins—down onto a specific section of DNA and seeing how each behaved. In order to do that the researchers engineered cells so that adding a small molecule would cause one of the gene-silencing regulators to bind to DNA near a particular gene that codes for a fluorescent protein. By tracking fluorescence in individual cells, the researchers could readily determine whether the regulator had turned off the gene. The researchers could also release the regulator from the DNA and see how long the gene remembered its effect.

Although there are hundreds of chromatin regulators, they can be categorized into about a dozen broader classes. For this study, the researchers tested regulators from four biochemically diverse classes.

"We tried a variety to see if different ones give you different types of behavior," explains Bintu. "It turns out they do."

For a month at a time, the researchers used microscopy or flow cytometry to observe the living cells, using cell-tracking software that they wrote and time-lapse movies to watch individual cells grow and divide. In some cases, after a regulator was released, the cells and their daughter cells remained dark for days and then lit back up, indicating that they remembered the modification transiently. In other cases, the cells never lit back up, indicating more permanent memory.

After modification, the genes were always in one of three states—"awake" and actively making protein, "asleep" and inactive but able to wake up in a matter of days, or "in a coma" and unable to be awakened within 30 days. Within an individual cell, the genes were always either completely on or off.

That led the researchers to the surprising finding that the regulators control not the level or degree of expression of a particular gene in an individual cell, but rather how many cells in a population have that gene on or off.

"You're controlling the probability that something is on or off," says Elowitz. "We think that this is something that's very useful generally in a multicellular organism—that in many cases, the organism may want to tell cells, 'I just want 30 percent of you to differentiate. You don't all need to do it.' This chromatin regulation system seems ready-made for orders like those."

In addition, the researchers found that the type of memory imparted by each of the four chromatin regulators was different. One produced permanent memory, turning off the gene and putting a fraction of cells into a coma for the full 30 days. One yielded short-term memory, with the cells immediately waking up. "The really interesting thing we found is that some of the regulators give this type of hybrid memory where some of the cells awaken while a fraction of the cells remain in a deep coma," says Bintu. "How many are in the coma depends on how long you gave the signal—how long the chromatin regulator stayed attached."

Going forward, the group plans to study additional chromatin regulators in the same manner, developing a better sense of the many ways they are used in the cell and also how they might work in combination. In the longer term they want to put these proteins together with other cellular components and begin programming more complex developmental behavior in synthetic circuits.

"This is a step toward realizing this emerging vision of programmable cell-based therapies," says Elowitz. "But we are also answering more basic research questions. We see these as two sides of the same coin. We're not going to be able to program cells effectively until we understand what capabilities their core pathways provide. "

Additional Caltech authors on the paper, "Dynamics of epigenetic regulation at the single-cell level," include Yaron E. Antebi and Kayla McCue (BS '15). Yasuhiro Kazuki, Narumi Uno, and Mitsuo Oshimura of Tottori University in Japan are also coauthors. The work was supported by the Defense Advanced Research Projects Agency, the Human Frontier Science Program, the Jane Coffin Childs Memorial Fund for Medical Research, the Beckman Institute at Caltech, the Burroughs Wellcome Fund, and HHMI.

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Learning to Program Cellular Memory
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Combining synthetic biology approaches with time-lapse movies, biologists have determined how some proteins shape a cell's ability to remember signals.

Experimental Economics: Results You Can Trust

Reproducibility is an important measure of validity in all fields of experimental science. If researcher A publishes a particular scientific result from his laboratory, researcher B should be able to follow the same protocol and achieve the same result in her laboratory. However, in recent years many results in a variety of disciplines have been questioned for their lack of reproducibility. A new study suggests that published results from experimental economics—a field pioneered at Caltech—are better than average when it comes to reproducibility.

The work was published in the March 3 online issue of the journal Science.

"Trying to reproduce previous results is not glamorous or creative, so it is rarely done. But being able to get the same result over and over is part of the definition of what makes knowledge scientific," says Colin Camerer, the Robert Kirby Professor of Behavioral Economics at Caltech and lead author on the paper.

The study was based on a previous method used to assess the replication of psychology experiments. In the earlier technique, called the reproducibility project psychology (RPP), researchers replicated 100 original studies published in three of the top journals in psychology—and found that although 97 percent of the original studies reported so-called "positive findings" (meaning a significant change compared to control conditions), such positive findings were reliably reproduced only 36 percent of the time.

Using this same technique, Camerer and his colleagues reproduced 18 laboratory experimental papers published in two top-tier economics journals between 2011 and 2014. Eleven of the 18—roughly 61 percent—showed a "significant effect in the same direction as in the original study." The researchers also found that the sample size and p-values—a standard measure of statistical confidence—of the original studies were good predictors for the success of replication, meaning they could serve as good indicators for the reliability of results in future experiments.

"Replicability has become a major issue in many sciences over the past few years, with often low replication rates," says paper coauthor Juergen Huber of the University of Innsbruck. "The rate we report for experimental economics is the highest we are aware of for any field."

The authors suggest that there are some methodological research practices in laboratory experimental economics that contribute to the good replication success. "It seems that the culture established in experimental economics—incentivizing subjects, publication of the experimental procedure and instructions, no deception—ensures reliable results. This is very encouraging given that it is a very young discipline," says Michael Kirchler, another coauthor and collaborator from the University of Innsbruck.

"As a journal editor myself, we are always curious whether experimental results will replicate across populations and cultures, and these results from multiple countries are really reassuring," says coauthor Teck-Hua Ho from the National University of Singapore.

Coauthor Magnus Johannesson from the Stockholm School of Economics adds, "It is extremely important to investigate to what extent we can trust published scientific findings and to implement institutions that promote scientific reproducibility."

"For the past half century, Caltech has been a leader in the development of social science experimental methods. It is no surprise that Caltech scholars are part of a group that use replication studies to demonstrate the validity of these methods," says Jean-Laurent Rosenthal, the Rea A. and Lela G. Axline Professor of Business Economics and chair of the Division of the Humanities and Social Sciences at Caltech.

The work was published in a paper titled, "Evaluating Replicability of Laboratory Experiments in Economics." Other coauthors are: Taisuke Imai and Gideon Nave from Caltech; Johan Almenberg from Sveriges Riksbank in Stockholm; Anna Dreber, Eskil Forsell, Adam Altmejd, Emma Heikensten, and Siri Isaksson from the Stockholm School of Economics; Taizan Chan and Hang Wu from the National University of Singapore; Felix Holzmeister and Michael Razen from the University of Innsbruck; and Thomas Pfeiffer from the New Zealand Institute for Advanced Study.

The study was funded by the Austrian Science Fund, the Austrian National Bank, the Behavioral and Neuroeconomics Discovery Fund, the Jan Wallander and Tom Hedelius Foundation, the Knut and Alice Wallenberg Foundation, the Swedish Foundation For Humanities and Social Sciences, and the Sloan Foundation.

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A study led by Caltech's Colin Camerer reproducing experimental economics studies finds that published results in this field are actually quite reliable.

JPL News: Pulsar Web Could Detect Low-Frequency Gravitational Waves

The recent detection of gravitational waves by the Laser Interferometer Gravitational-Wave Observatory (LIGO) came from two black holes, each about 30 times the mass of our sun, merging into one. Gravitational waves span a wide range of frequencies that require different technologies to detect. A new study from the North American Nanohertz Observatory for Gravitational Waves (NANOGrav) has shown that low-frequency gravitational waves could soon be detectable by existing radio telescopes.

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