Caltech Researchers Pinpoint Neurons that Control Obesity in Fruit Flies

Research could lead to the development of a new model for the study of human obesity and its treatment

PASADENA, Calif.—A team of scientists from the California Institute of Technology (Caltech) have pinpointed two groups of neurons in fruit fly brains that have the ability to sense and manipulate the fly's fat stores in much the same way as do neurons in the mammalian brain. The existence of this sort of control over fat deposition and metabolic rates makes the flies a potentially useful model for the study of human obesity, the researchers note.

Their findings were published in the August 13 issue of the journal Neuron.

By manipulating neural activity in fruit fly brains using transgenic techniques, the researchers found that, "just as in mammals, fly fat-store levels are measured and controlled by specific neurons in the brain," says Caltech postdoctoral scholar Bader Al-Anzi, the Neuron paper's first author. "Silencing these neurons created obese flies, while overactivating them produced lean flies."

Mammalian brains are given information about the body's fat stores by hormones such as leptin and insulin, and respond to that information by inducing changes in food intake and metabolism to maintain a constant body weight. The researchers found that similar behavioral and metabolic changes occurred in the fruit flies, though which changes occurred depended on which of the two sets of newly identified neurons was silenced.

For instance, silencing one group of neurons led to an increase in food intake, a decrease in metabolism, and an increase in the synthesis of fatty acids (the building blocks of fat). Silencing the other group led to a similar decrease in metabolism and increase in fatty-acid synthesis, as well as to a defect in the flies' ability to utilize their fat stores.

Increasing activity in either of the groups of neurons, on the other hand, resulted in depletion of fat stores by increasing the flies' metabolism and decreasing their synthesis of fatty acids.

The next step is to "see exactly how neurons regulate fat storage, and how the two different groups of neurons identified in this study work," says Kai Zinn, professor of biology at Caltech, who led the research group. "They clearly regulate fat storage using different mechanisms."

The paper is the result of research originally led by Caltech biologist Seymour Benzer, a pioneer in the study of genes and behavior. Zinn continued this research after Benzer's death in late 2007.

"The goal was to establish a model system for obesity in humans," Zinn explains. "This could, at some point, eventually define new drug targets."

The search for a model system is critical, adds Al-Anzi. With obesity on the rise—statistics say that more than a third of adults in Western society are overweight—efforts to find its roots in human brains or human genes have similarly increased. Unfortunately, Al-Anzi notes, these efforts "have not been extremely successful."

In addition, says Al-Anzi, "While mammalian models such as the mouse have provided progress in the field, they tend to be difficult and expensive research subjects."

Thus, he notes, "The obesity research field would benefit greatly if another model organism could be used, one that is accessible for easy, fast, and affordable biomedical research methods. We believe the fruit fly can be such an organism.

"There is a surprising amount of overlap between the simple fruit fly and more complex mammals in many basic biological processes," Al-Anzi adds. "This is why it's an excellent model system for exploring such medically relevant issues as Alzheimer's disease, alcoholism, and addiction. Our results thus far suggest that body-weight regulation will be no different."

Having now established that fruit flies are indeed similar to mammals in the way they control fat deposition via the brain, researchers can begin to test antiobesity dietary or drug treatments on flies whose fat-regulating neurons have been silenced. "Treatments that cause these flies to return to normal body weight could then be retested for their effectiveness in a mammalian obesity model," Al-Anzi notes.

Knowing the neurons involved in the regulation of fat storage could also lead to identifying the genes that allow for the critical communications between the brain and the fat stores. "This can be done by identifying the genes that are selectively expressed only in those neurons," he explains.

In addition, this research should help researchers determine if the mechanisms behind appetite and body-weight regulation in fruit flies have been conserved over evolutionary time and throughout the animal kingdom. "This has been shown to be the case for genes that regulate behavioral phenomena like learning and circadian rhythms," notes Al-Anzi, "and we hope that body-weight and appetite regulation will be no different."

In addition to Al-Anzi, Zinn, and Benzer, other authors on the Neuron paper, "Obesity-blocking neurons in Drosophila," include Caltech research technician Viveca Sapin; Christopher Waters, formerly of Caltech; and biologist Robert Wyman from Yale University.

Their research was supported by a Life Sciences Research Foundation grant provided by Bristol-Myers Squibb to Al-Anzi, and by a National Institutes of Health RO1 grant to Benzer.

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Caltech Scientists Help Launch the First Standard Graphical Notation for Biology

Pasadena, Calif.—Researchers at the California Institute of Technology (Caltech) and their colleagues in 30 laboratories worldwide have released a new set of standards for graphically representing biological information—the biology equivalent of the circuit diagram in electronics. This visual language should make it easier to exchange complex information, so that biological models are depicted more accurately, consistently, and in a more readily understandable way.

The new standard, called the Systems Biology Graphical Notation (SBGN), was published in the August 8 issue of the journal Nature Biotechnology

Researchers use standardized visual languages to communicate complex information in many scientific and engineering fields.  A well-known example is the circuit diagram in electrical engineering. However, until now, biology lacked a standardized notation for describing biological interactions, pathways, and networks, even though the discipline is dominated by graphical information.

The SBGN project was launched in 2005 as a united effort to specifically develop a new graphical standard for molecular and systems-biology applications. The project, which was initiated by Hiroaki Kitano of the Systems Biology Institute in Tokyo, Japan, is coordinated by Nicolas Le Novère of the European Molecular Biology Laboratory's European Bioinformatics Institute in Cambridge, England, and senior research fellow Michael Hucka, codirector of the Biological Network Modeling Center at Caltech's Beckman Institute. The international team of researchers that created SBGN is composed of biochemists, modelers, and computer scientists, who developed the notation in collaboration with a broader community of researchers constituting the target user community.

"Engineers, architects, physicists, and software developers all have standard graphical notations for depicting the things they work on, which makes it possible for everyone in those fields to be on the same page, as it were," says Hucka. "I think SBGN represents the first truly broad-based attempt at establishing the same kind of standardization in biology."

SBGN will make it easier for biologists to understand each other's models and share network diagrams more easily, which, Hucka says, has never been more important than in today's era of high-throughput technologies and large-scale network reconstruction efforts. A standard graphical notation will help researchers share this mass of data more efficiently and accurately, which will benefit systems biologists working on a variety of biochemical processes, including gene regulation, metabolism, and cellular signaling.

"Finally, and perhaps most excitingly," adds Hucka, "I believe that, just as happened with the engineering fields, SBGN will act as an enabler for the emergence of new industries devoted to the creation of software tools for working with SBGN, as well as its teaching and publication."

Previous graphical notations in biology have tended to be ambiguous, used in different ways by different researchers, and only suited to specific needs—for example, to represent metabolic networks or signaling pathways. Past efforts to create a more rigid notation failed to become accepted as a standard by the community. Hucka and his collaborators believe that SBGN should be more successful because it represents a more concerted effort to establish a standard by engaging many biologists, modelers, and software-tool developers. In fact, many of those involved in the SBGN effort are the same pioneers who proposed previous notations, demonstrating the degree to which they endorse SBGN as a new standard.

To ensure that this new visual language does not become too vast and complicated, the researchers decided to define three separate types of diagram, which describe molecular process, relationships between entities, and links among biochemical activities. These different types of diagrams complement each other by representing different "views" of the same information, presented in different ways for different purposes, but reusing most of the same graphical symbols. This approach reduces the complexity of any one type of diagram while broadening the range of what can be expressed about a given biological system.

"As biology focuses more on managing complexity with quantitative and systematic methods, standards such as SBGN play an essential role. SBGN combines an intuitive notation with the rigorous style of engineering and math," says John Doyle, the John G. Braun Professor of Control and Dynamical Systems, Bioengineering, and Electrical Engineering at Caltech.

"As with SBML (the Systems Biology Markup Language), Mike and his collaborators have provided the kind of solid foundation that the whole community can build on. SBML has been a highly successful standardization effort for software interoperability, and SBGN is sure to have the same kind of impact on human communication in biology," Doyle adds.

The work at Caltech in the paper, "The Systems Biology Graphical Notation," was supported by the New Energy and Industrial Technology Development Organization and a Beckman Institute grant funding the Biological Network Modeling Center.

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Caltech-led Team Shows How Evolution Can Allow for Large Developmental Leaps

Researchers demonstrate how genetic mutations and natural variations combine to produce twin spores in bacteria

PASADENA, Calif.—How evolution acts to bridge the chasm between two discrete physiological states is a question that's long puzzled scientists. Most evolutionary changes, after all, happen in tiny increments: an elephant grows a little larger, a giraffe's neck a little longer. If those tiny changes prove advantageous, there's a better chance of passing them to the next generation, which might then add its own mutations. And so on, and so on, until you have a huge pachyderm or the characteristic stretched neck of a giraffe.

But when it comes to traits like the number of wings on an insect, or limbs on a primate, there is no middle ground. How are these sorts of large evolutionary leaps made?

According to a team led by scientists at the California Institute of Technology (Caltech), in close collaboration with Patrick Piggot and colleagues from the Temple University School of Medicine, such changes may at least sometimes be the result of random fluctuations, or noise (nongenetic variations), working alongside a phenomenon known as partial penetrance. Their findings were recently published online in the journal Nature.

"Our work shows how partial penetrance can play a role in evolution by allowing a species to gradually evolve from producing 100 percent of one form to developing 100 percent of another, qualitatively different, form," says Michael Elowitz, the Caltech assistant professor of biology and applied physics, Bren Scholar, and Howard Hughes Medical Institute investigator who led the team. "The intermediate states that occur along the way are not intermediate forms, but rather changes in the fraction of individuals that develop one way or the other."

Partial penetrance is the name given by evolutionary biologists to the degree to which a single genetic mutation may have different effects on different organisms in a population.

"If you take a bunch of cells and grow them in exactly the same environment, they'll be identical twin brothers in terms of the genes they have, but they may still show substantial differences in their behavior," says Avigdor Eldar, a postdoctoral scholar in biology at Caltech and the paper's first author. These sorts of variations—or noise, as the researchers call it—can actually allow a mutation to have an effect in some organisms but not in others. For example, while some genetically variable cells will show the expected effect of the mutation, others may still behave like a normal, or wild type, cell. And still others may do something else entirely.

"These mutant cells don't only show a different morphology," Eldar notes. "They show more variability in their behavior. In a population, you can see a mixture of several different behaviors, with some cells doing one thing and others doing something else."

In their Nature paper, Elowitz and Eldar, along with their colleagues, studied partial penetrance in a species of bacterium known as Bacillus subtilis. Specifically, they looked at the spores B. subtilis produces as a survival mechanism when times get tough. These spores are smaller, dormant clones of their so-called "mother cell." They're attached to the mother, but are separate entities with their own DNA.

A bacterial spore is designed specifically to do nothing but survive. "It doesn't grow, it doesn't do anything," says Eldar. "It just waits for the good times to return."

The wild-type B. subtilis bacterium always sporulates the same way: it creates a single spore, smaller than the mother cell, but with an exact single copy of the mother's chromosome.

What the scientists looked at was a "mutant in which the sporulation process was altered," Eldar explains. "Usually, these cells talk with each other, with the small spore telling the large mother cell, 'I'm here, and I'm doing OK.' In the wild-type cell, this chatter is loud; in the mutant, it's just a whisper, and the mother can't always hear."

When this whispering sort of mutation occurs, the researchers discovered, there are four possible outcomes:

  • The bacterium sporulates normally, like the wild type.
  • The bacterium makes two copies of its chromosome instead of one, so that there are three chromosomes but creates only a single spore. In this case, the mother cell retains two of the chromosomes and gives the spore one.
  • The bacterium makes only one copy of its chromosome, but creates two spores instead of one. In this case, each spore will have a chromosome, and the mother cell will have none. (This is a lethal mutation; neither the mother nor its spores will survive.)
  • The bacterium makes two copies of its chromosome instead of one, so that there are three chromosomes. It then creates two spores. In this case, the mother and each of the twin spores will have a single chromosome.

This last possibility, notes Eldar, is something that had never been seen before in B. subtilis. But that doesn't mean this twinning behavior doesn't have its advantages. "In some environments, it might be better for the cell," he says. "We know that because there are other species whose wild types do the same thing that our mutant was doing only once in a while."

The scientists soon realized that this variability was their way in to understanding how evolution makes the leap from one to another phenotype. "You can't switch from 1 to 1.1 spores," Eldar points out. "But it's easy to find a mutation that simply changes the frequency of the behavior. If 10 percent of the population makes 2 spores and the rest makes 1, that works. It solves the need for a quantum jump between 1 and 2 spores."

Once they had seen this rare behavior in a small minority of the bacteria, the researchers took the process one step further, tweaking other players in the sporulation system. For instance, they looked at what would happen if, in addition to dampening the communication between mother and spore—making the mother think she hadn't yet successfully produced a spore—you also increased the volume of the signals that tell the mother to replicate its chromosome.

Perhaps not surprisingly, they found that these sorts of changes increase the percentage of B. subtilis individuals that decide to produce two spores rather than one. In fact, by combining mutations, Eldar says, they were able to up the percentage of bacteria that create twin spores from about 1 percent (in singly mutated bacteria) to as high as 40 percent (in multiply mutated bacteria).

"When you have only a single mutation, twinning shows very low penetrance," Eldar says. "But when you add more and more mutations, you can build up the penetrance to very high levels."

"We showed that some mutations cause a low frequency of twin spores to develop in the same cell, rather than a single spore per cell, as occurs normally," Elowitz says. "The relative frequency of this form could be tuned up to high levels by other mutations."

This study provides a concrete example of a particular scenario to explain developmental evolution. "It illustrates a somewhat unfamiliar mode in which developmental evolution might work," Elowitz adds. "Qualitative changes from one form to another can proceed through changes in the relative frequencies—or penetrance—of those forms. 

"It's interesting that noise—these random fluctuations of proteins in the cell—is critical for this to work," he continues. "Noise is not just a nuisance in this system; it's a key part of the process that allows genetically identical cells to do very different things."

In addition, Elowitz notes, the work shows that "bacterial development can be a good system to enable further study of these general issues in developmental evolution."

Other researchers involved in the work included Caltech staff member Michelle Fontes and graduate student Oliver Loson; Piggot, Vasant Chary, and Panagiotis Xenopoulos from Temple University School of Medicine; and Jonathan Dworkin from the College of Physicians and Surgeons at Columbia University.

The work described in the Nature paper, "Partial penetrance facilitates developmental evolution in bacteria," was funded by grants from the Howard Hughes Medical Institute, the National Institutes of Health, the National Science Foundation, the International Human Frontier Science Organization, and the European Molecular Biology Organization.

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Maple Seeds and Animals Exploit the Same Trick to Fly

PASADENA, Calif.—The twirling seeds of maple trees spin like miniature helicopters as they fall to the ground. Because the seeds descend slowly as they swirl, they can be carried aloft by the wind and dispersed over great distances. Just how the seeds manage to fall so slowly, however, has mystified scientists.

In research published in the June 12 issue of the journal Science, researchers from Wageningen University in the Netherlands and the California Institute of Technology (Caltech) describe the aerodynamic secret of the enchanting swirling seeds.

The research, led by David Lentink, an assistant professor at Wageningen, and Michael H. Dickinson, the Zarem Professor of Bioengineering at Caltech, revealed that, by swirling, maple seeds generate a tornado-like vortex that sits atop the front leading edge of the seeds as they spin slowly to the ground. This leading-edge vortex lowers the air pressure over the upper surface of the maple seed, effectively sucking the wing upward to oppose gravity, giving it a boost. The vortex doubles the lift generated by the seeds compared to nonswirling seeds.

This use of a leading-edge vortex to increase lift is remarkably similar to the trick employed by insects, bats, and hummingbirds when they sweep their wings back and forth to hover. The finding means that plants and animals have converged evolutionarily on an identical aerodynamic solution for improving their flight performance.

To measure the flow of air created by swirling seeds, the scientists built plastic models of the seeds with radii of about five inches, or 5 to 10 times larger than a maple seed. The seeds were spun through a large tank of mineral oil using a specially designed robot, modified from a device at Caltech called "Robofly." Previously, Robofly helped to determine the aerodynamic forces that keep insects aloft.

The size of the model seed, the speed at which it spun through the tank, and the viscosity of the oil were chosen so that the characteristics of the fluid flow generated by the model were identical to those produced by real maple seeds—just flowing through oil instead of air.

Next, the scientists used a powerful laser to create a sheet of light that illuminated tiny glass beads added to the oil. They then used a camera to capture images of the motion of the beads as the model seed spun through the tank. The images revealed the presence of a tornado-like vortex lying near the front leading edge of the spinning seed. Force measurements attached to the model showed that the swirling vortex created extra lift that would act to slow the descent of a seed as it spun to the ground.

To verify the results from the robot seed models, the team built a wind tunnel at the Wageningen University to examine the flow created by real maple seeds as they spin freely. Smoke was used to visualize the flow of air around the spinning seeds. These studies of 32 specimens confirmed that real seeds do indeed produce a vortex that generates exceptionally high lift, and that the vortex is aerodynamically similar in structure to the vortex made by the flapping wings of insects, bats, and hummingbirds when they hover.

The research might have implications for the design of swirling parachutes—which have been designed by space agencies to slow the descent of future planetary probes exploring the atmospheres of planets such as Mars—and of micro-helicopters.

"Maple seeds could represent the most basic and simple design for a miniature helicopter, if the swirling wing could be powered by a micromotor," says Lentink. Single-rotor helicopters have been built and flown successfully with wing spans of roughly a meter, but never at the scale of a maple seed.

"There is enormous interest in the development of micro air vehicles, which, because of their size, must function using the same physical principles employed by small, natural flying devices such as insects and maple seeds," says Dickinson. For example, Lockheed Martin attempted to develop inexpensive "maple seed drone cameras" that could be deployed in large numbers for surveillance, "although the project is no longer funded," Lentink says.

"This is still an open challenge for future aerospace engineers, and our aerodynamic study of maple seeds could help design the first successful powered 'maple' helicopters," he adds. Over the past four years, Lentink, an aerospace engineer, has designed operational flying, flapping, and morphing micro air vehicles, inspired by his insect and bird flight research.

The other coauthors of the paper, "Leading-Edge Vortices Elevate Lift of Autorotating Plant Seeds," are W. B. Dickson of Caltech and J. L. van Leeuwen of Wageningen University. The research at Caltech was supported by the Netherlands Organization for Scientific Research and the National Science Foundation.

Go to http://mr.caltech.edu/assets/619-mapleseed.mp4 for a video showing the vortex on top of a flying maple seed.

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David Lentink
david.lentink@wur.nl
(617) 606-0576 (cell)
(781) 275-1725 ext 108

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Caltech Scientists Reveal How Neuronal Activity is Timed in Brain's Memory-Making Circuits

Study shows theta oscillations move across the hippocampus as traveling waves

PASADENA, Calif.-Theta oscillations are a type of prominent brain rhythm that orchestrates neuronal activity in the hippocampus, a brain area critical for the formation of new memories. For several decades these oscillations were believed to be "in sync" across the hippocampus, timing the firing of neurons like a sort of central pacemaker. A new study conducted by researchers at the California Institute of Technology (Caltech) argues that this long-held assumption needs to be revised. In a paper published in this week's issue of the journal Nature, the researchers showed that instead of being in sync, theta oscillations actually sweep along the length of the hippocampus as traveling waves.

"It was assumed that activity in the hippocampus is synchronized throughout," says Evgueniy Lubenov, a postdoctoral scholar at the Center for Biological Circuit Design at Caltech. "But when we looked simultaneously at many different anatomical locations across the hippocampus, we found instead a systematic delay in neuronal activity from site to site. Instead of the whole structure oscillating at once, we see traveling waves that propagate across the hippocampus in a consistent direction, along its long axis."

"In other words, the hippocampus has a series of local time zones, just like we have on Earth," adds Athanassios Siapas, associate professor of computation and neural systems and Bren Scholar at Caltech.

The hippocampus has long been known to be critical for the formation and maintenance of episodic memories-i.e., memories of experiences.  In the rat, hippocampal neurons also function as "place cells," only firing when the animal is in a particular spot in its environment.  Lubenov and Siapas began to analyze the theta oscillations generated when rats move around and explore their environment.  They watched how-and when-the rat's neurons fired relative to the rat's position and to the phase of the theta oscillations. They did these studies using multiple tetrodes-electrodes with four recording sites-that allowed them to simultaneously isolate the spiking of many individual neurons.

"Each of these neurons fires only in a restricted region of space," Lubenov says. "Furthermore, the spikes don't just happen any time-they pay attention to the phase of the ongoing theta oscillation. If you have access to the phase at which the neuron fired, you have additional information about where the rat was in space."

When the data about neuronal firing, oscillation phase, and rat location were combined, the researchers were able to show that neuronal activity indeed sweeps across the hippocampus in a wave, with its peak appearing in one region, then another, then another, rather than hitting the entire hippocampus in one synchronized pulse.

"This changes our notion of how spatial information is represented in the rat brain," notes Lubenov. "It was believed that the firing of hippocampal neurons encodes the physical location of the rat in its environment-in other words, a point of physical space. Our findings suggest that what is encoded is actually a portion of the rat's trajectory-that is, a segment of physical space."

"Such segments may be the elementary unit of hippocampal computation," adds Siapas. "Assume the path a rat takes in an environment is represented and stored as a sequence of point locations. If the rat visits the same location more than once, the representation becomes ambiguous. Representing the rat trajectory as a sequence of segments oriented in space resolves such ambiguities."

This finding may also have significant implications for understanding how information is transmitted from the hippocampus to other areas of the brain. "Different portions of the hippocampus are connected to different areas in other parts of the brain. The fact that hippocampal activity forms a traveling wave means that these target areas receive inputs from the hippocampus in a specific sequence rather than all at once," explains Siapas.

In addition, Siapas notes, it's unlikely that this behavior is found only in rat brains; after all, theta oscillations are ubiquitous in mammalian brains. "I would expect the traveling-wave nature of theta oscillations to be a general finding, applicable to humans as well," he says.

And while it is not known whether human hippocampal cells function as place cells, as they do in rats, "it may turn out to be the case that the human hippocampus plays a role in providing spatial cues that are important to episodic memory," Lubenov speculates. "We don't know yet."

What we do know is that, by showing that theta oscillations travel across the hippocampus, the Caltech team will likely change the way neuroscientists think about how the hippocampus works.

The work described in the Nature paper, "Hippocampal theta oscillations are travelling waves," was supported by the Caltech Information Science and Technology Center for Biological Circuit Design, a 21st Century McDonnell Foundation Award, the Bren Foundation, and the McKnight Foundation.

The paper's abstract can be accessed at http://dx.doi.org/10.1038/nature08010.

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Caltech, UCSF Scientists Determine How Body Differentiates Between a Scorch and a Scratch

Different types of painful stimuli are detected by different subsets of pain-sensing neurons in skin

PASADENA, Calif.--You can tell without looking whether you've been stuck by a pin or burnt by a match. But how? In research that overturns conventional wisdom, a team of scientists from the California Institute of Technology (Caltech) and the University of California, San Francisco (UCSF), have shown that this sensory discrimination begins in the skin at the very earliest stages of neuronal information processing, with different populations of sensory neurons--called nociceptors--responding to different kinds of painful stimuli.

Their findings were published this week in the early online edition of the Proceedings of the National Academy of Sciences (PNAS).

"Conventional wisdom was that the nociceptive neurons in the skin can't tell the difference between heat and mechanical pain, like a pin prick," says David Anderson, Seymour Benzer Professor of Biology, a Howard Hughes Medical Institute (HHMI) Investigator, and one of the paper's lead authors. "The idea was that the skin is a dumb sensor of anything unpleasant, and that higher brain areas disentangle one pain modality from another, to tell you if you've been scorched or scratched."

This conventional wisdom came from recording the electrical responses of nociceptive neurons, where it was shown that these neurons are capable of sensing pretty much every kind of painful stimulus--from pin pricks to heat to cold. But this, Anderson notes, was not sufficient to understand the control of pain-avoidance behavior. "We were asking the cells what the cells can sense, not asking the animal what the cells can sense," he explained.

And so Anderson and coprincipal investigator Allan Basbaum, chair of the Department of Anatomy at UCSF, decided to ask the animal. To do so, they created a genetically engineered mouse in which specific populations of pain-sensing neurons can be selectively destroyed. They were then able to see if the mouse continued to respond to different types of stimuli by pulling its paw away when exposed to a relatively gentle heat source or poked with a nylon fishing line.

What the researchers found was that, when they killed off a certain population of nociceptor neurons, the mice stopped responding to being poked, but still responded to heat. Conversely, when the researchers injected a toxin to destroy a different population of neurons, the mice stopped responding to heat, but their sense of poke remained intact.

"This tells us that the fibers that mediate the response to being poked are neither necessary nor sufficient for a behavioral response to heat," Anderson explains, "and vice versa for the fibers that mediate the response to heat."

In addition, Anderson notes, neither of these two classes of sensory neurons seem to be required for responding to a painful cold stimulus, like dry ice. Research into pinpointing that population of cells is ongoing.

"This tells us that the discernment of different types of painful stimuli doesn't happen only in the brain--it starts in the skin, which is therefore much smarter than we thought," says Anderson. "That's a pretty heretical point of view."

It's also a potentially useful point of view, as Anderson points out. "If doctors want to repair or replace damaged nerve fibers in conditions such as diabetic neuropathy," he explains, "they need to make sure they're replacing the right kind of nerve fibers."

In addition to Anderson, the paper's coauthors include graduate student Daniel Cavanaugh from UCSF, postdoctoral scholar Hyosang Lee and HHMI Research Specialist Liching Lo from Caltech, Shannon Shields from UCSF (now at the Hospital Nacional de Paraplejicos in Toledo, Spain), and Mark Zylka, a former postdoctoral fellow at Caltech now on the faculty at the University of North Carolina, Chapel Hill.

Work on the PNAS paper, "Distinct subsets of unmyelinated primary sensory fibers mediate behavioral responses to noxious thermal and mechanical stimuli," was funded by grants from the National Institutes of Health, the National Alliance for Research on Schizophrenia and Affective Disorders, the Searle Scholars Program, the Whitehall, Klingenstein, Sloan and Rita Allen Foundations, the Christopher and Dana Reeve Foundation, and the Howard Hughes Medical Institute.

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Caltech Scientists Show Why Anti-HIV Antibodies are Ineffective at Blocking Infection

Findings provide possible explanation for failure of decades-long AIDS vaccine search

Their findings were published last week in the online early edition of the Proceedings of the National Academy of Sciences (PNAS).

"This study helps to clarify the obstacles that antibodies face in blocking infection," says Pamela Bjorkman, the Max Delbrück Professor of Biology at Caltech and a Howard Hughes Medical Institute Investigator, "and will hopefully shed more light on why developing an effective vaccine for HIV has proven so elusive."

Y-shaped antibodies are best at neutralizing viruses--i.e., blocking their entry into cells and preventing infection--when both arms of the Y are able to reach out and bind to their target proteins at more or less the same time. In the case of HIV, antibodies that can block infection target the proteins that stud the surface of the virus, which stick out like spikes from the viral membrane. But an antibody can only bind to two spikes at the same time if those spikes fall within its span--the distance the antibody's structure allows it to stretch its two arms.

"When both arms of an antibody are able to bind to a virus at the same time," says Joshua Klein, a Caltech graduate student in biochemistry and molecular biophysics and the PNAS paper's first author, "there can be a hundred- to thousandfold increase in the strength of the interaction, which can sometimes translate into an equally dramatic increase in its ability to neutralize a virus. Having antibodies with two arms is nature's way of ensuring a strong binding interaction."

As it turns out, this sort of double-armed binding is easier said than done--at least in the case of HIV.

In their PNAS paper, Bjorkman and Klein looked at the neutralization capabilities of two different monoclonal antibodies isolated from HIV-infected individuals. One, called b12, binds a protein known as gp120, which forms the upper portion of an HIV's protein spike. The other, 4E10, binds to gp41, which is found on a lower portion of the spike known as the stalk.

The researchers broke each of the antibodies down into their component parts and compared their abilities to bind and neutralize the virus. They found, as expected, that one-armed versions of the b12 antibody were less effective at neutralizing HIV than two-armed versions. When they looked at the 4E10 antibody, by comparison, they found that having two arms conferred almost no advantage over having only one arm. In addition, they found that larger versions of 4E10 were less effective than smaller ones. These results highlight potential obstacles that vaccines designed to elicit antibodies similar to 4E10 might face.

But b12 has its own obstacles to overcome as well. In fact, when the researchers looked more closely at their data, they realized that the benefits of having two arms--even for b12--were much smaller than those seen for antibodies against viruses like influenza. In other words, the body's natural anti-HIV antibodies are much less effective at neutralizing HIV than they should be.

But why?

"The story really starts to get interesting when we think about what the human immunodeficiency virus actually looks like," says Klein. Whereas a single influenza virus's surface is studded with approximately 450 spikes, he explains, the similarly sized HIV may have fewer than 15 spikes.

With spikes so few and far between, finding two that both fall within the reach of a b12 or 4E10 antibody--the spans of which generally measure between 12 and 15 nanometers--becomes much more of a challenge.

"HIV may have evolved a way to escape one of the main strategies our immune system uses to defeat infections," says Klein. "Based on these data, it seems that the virus is circumventing the bivalent effect that is so key to the potency of antibodies."

"I consider this a very important paper because it changes the focus of the discussion about why anti-HIV antibodies are so poor," adds virologist David Baltimore, the Robert Andrews Millikan Professor of Biology and a Nobel Prize winner. "It brings attention to a long-recognized but often forgotten aspect of antibody attack--that they attack with two heads. What this paper shows is that anti-HIV antibodies are restricted to using one head at a time and that makes them bind much less well. Responding to this newly recognized challenge will be difficult because it identifies an intrinsic limitation on the effectiveness of almost any natural anti-HIV antibodies."

In addition to Bjorkman and Klein, the authors on the PNAS paper, "Examination of the contributions of size and avidity to the neutralization mechanisms of the anti-HIV antibodies b12 and 4E10," are Caltech research technicians Priyanthi Gnanapragasam, Rachel Galimidi, and Christopher Foglesong, and senior research specialist Anthony West, Jr.

The work described in the paper was supported by a Bill and Melinda Gates Foundation Grant through the Grand Challenges in Global Health Initiative and the Collaboration for AIDS Vaccine Discovery.

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Writer: 
Lori Oliwenstein
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Caltech Scientists Control Complex Nucleation Processes using DNA Origami Seeds

PASADENA, Calif.--The construction of complex man-made objects--a car, for example, or even a pizza--almost invariably entails what are known as "top-down" processes, in which the structure and order of the thing being built is imposed from the outside (say, by an automobile assembly line, or the hands of the pizza maker).

"Top-down approaches have been extremely successful," says Erik Winfree of the California Institute of Technology (Caltech). "But as the object being manufactured requires higher and higher precision--such as silicon chips with smaller and smaller transistors--they require enormously expensive factories to be built."

The alternative to top-down manufacturing is a "bottom-up" approach, in which the order is imposed from within the object being made, so that it "grows" according to some built-in design.

"Flowers, dogs, and just about all biological objects are created from the bottom up," says Winfree, an associate professor of computer science, computation and neural systems, and bioengineering at Caltech. Along with his coworkers, Winfree is seeking to integrate bottom-up construction approaches with molecular fabrication processes to construct objects from parts that are just a few billionths of a meter in size that essentially assemble themselves.

In a recent paper in the Proceedings of the National Academy of Sciences (PNAS), Winfree and his colleagues describe the development of an information-containing DNA "seed" that can direct the self-assembled bottom-up growth of tiles of DNA in a precisely controlled fashion. In some ways, the process is similar to how the fertilized seeds of plants or animals contain information that directs the growth and development of those organisms.

"The big potential advantage of bottom-up construction is that it can be cheap"--just as the mold that grows in your kitchen does so for free--"and can be massively parallel, because the objects construct themselves," says Winfree.

But, he adds, while bottom-up approaches have been extremely useful in biology, they haven't played as significant a role in technology, "because we don't have a great grasp on how to design systems that build themselves. Most examples of bottom-up technologies are specific chemical processes that work great for a particular task, but don’t easily generalize for constructing more complex structures."

To understand how complexity can be programmed into bottom-up molecular fabrication processes, Winfree and his colleagues study and understand the processes--or algorithms--that generate organization not just in computers but also in the natural world.

"Tasks can be solved by carrying out well-defined rules, and these rules can be carried out by a mindless mechanism such as a computer," he says. "The same set of rules can perform different tasks when given different inputs, and there exist 'universal programs' that can perform any task required of it, as specified in its input. Your laptop is such a universal computer; it can run any software that you download, and in principle, any feasible task could be programmed."

These principles also have been exploited by natural evolution, Winfree says: "Every cell, it appears, is a kind of universal computer that can be instructed in seemingly limitless ways by a DNA genome that specifies what chemical processes to execute, thus building an active organism. The aim of my lab has been to understand algorithms and information within molecular systems."

Winfree's investigations into algorithmic self-assembly earned him a MacArthur "genius" prize in 2000; his collaborator, Paul W. K. Rothemund, a senior research associate at Caltech and a coauthor of the PNAS paper, was awarded the same no-strings-attached grant in 2007 for his work designing scaffolded "DNA origami" structures that self-assemble into nearly arbitrary shapes (such as a smiley face and a map of the Western Hemisphere).

The structures designed by Rothemund, which could eventually be used in smaller, faster computers, were used as the seeds for the programmed self-assembly of DNA tiles described in the current paper.

In the work, the researchers designed several different versions of a DNA origami rectangle, 95 by 75 nanometers, which served as the seeds for the growth of different types of ribbon-like crystals of DNA. The seeds were combined in a test tube with other bits of DNA, called "tiles," heated, and then cooled slowly.

"As it cools, the first origami seed and the individual tiles form, as their component DNA molecules begin sticking to each other and folding into shape--but the tiles and origami don't stick to each other yet," Winfree explains.

"Then, at a lower temperature, the tiles start to stick to each other and to the origami. The critical concept here is that the DNA tiles will only form crystals if the process gets started by a seed, upon which they can grow," he says.

In this way, the DNA ribbons self-assemble themselves, but only into forms such as ribbons with particular widths and ribbons with stripe patterns prescribed by the original seed.

The work, Winfree says, "exhibits a degree of control over information-directed molecular self-assembly that is unprecedented in accuracy and complexity, which makes me feel that we are finally beginning to understand how to program information into molecules and have that information direct algorithmic processes."

The paper, "An information-bearing seed for nucleating algorithmic self-assembly," was published in the March 24 issue of the Proceedings of the National Academy of Sciences.

The other authors of the paper are undergraduate Robert D. Barish and visiting scholar Rebecca Schulman. The work was supported by grants from the National Aeronautics and Space Administration's astrobiology program, the National Science Foundation, and the Focus Center Research Program, and a gift from Microsoft Research.

Writer: 
Kathy Svitil
Writer: 

Caltech Researchers Train Computers to Analyze Fruit-Fly Behavior

Program will make it possible to link genes to behaviors, scientists say

PASADENA, Calif.--Scientists at the California Institute of Technology (Caltech) have trained computers to automatically analyze aggression and courtship in fruit flies, opening the way for researchers to perform large-scale, high-throughput screens for genes that control these innate behaviors. The program allows computers to examine half an hour of video footage of pairs of interacting flies in what is almost real time; characterizing the behavior of a new line of flies "by hand" might take a biologist more than 100 hours.

This work--led by Pietro Perona, the Allen E. Puckett Professor of Electrical Engineering at Caltech, and David J. Anderson, the Roger W. Sperry Professor of Biology at Caltech, and a Howard Hughes Medical Institute Investigator--is detailed in the April issue of Nature Methods.

"Everyone wants to know how genes control behavior," notes Anderson. "But in order to apply powerful genetic analyses to complicated social behaviors like aggression and courtship, you need accurate ways of measuring--of scoring--those behaviors."

Previously, the only way to do this was to have students "watch video tapes over and over to record one particular type of behavior at a time," says Anderson. Using this method to measure a number of different types of behaviors--like lunging, tussling, chasing, circling, and copulating--or even to determine the way the flies orient their bodies or set their wings when they encounter another fly, requires the student to watch the same bit of video repeatedly, each time looking at the behavior of a single pair of flies. "In order to screen for mutations affecting aggressive behavior, we would have to analyze something like 2,000 pairs of flies," says Anderson. "It's been virtually impossible to do this without a small army of graduate students."

Enter Perona and Heiko Dankert, a postdoctoral scholar in electrical engineering. Using the techniques of machine vision and combining them with other engineering advancements, the two began training computers to see and recognize aggression and courtship behaviors. The result? An automated system that can monitor a wide variety of behaviors in videos of interacting fruit-fly pairs in a matter of minutes.

"This is a coming-of-age moment in this field," says Perona. "By choosing among existing machine vision techniques, we were able to put together a system that is much more capable than anything that had been demonstrated before."

The team fed the computer the characteristic details of what each individual behavior looks like on video. A lunge, for instance, begins with a shortening of the fly's body as the fly rears up; the fly then makes a quick darting movement, closing to within a few centimeters of another fly.

Once the computer had mastered these details, the researchers then compared the computer's analysis of a piece of video to the analysis produced by a human. "We looked at how many instances the computer caught, and how many it missed," says Anderson. "By looking at the errors the computer made, we were able to further refine our descriptions to create an even more accurate system."

In the end, Anderson notes, this back-and-forth resulted in a program that is "actually better than humans at detecting some of the instances of the various behaviors."

"Where previous experiments had been carried out on 100 to 1,000 frames of video, we carried out our experiments on 100,000 frames of video," Perona adds. "And while previous experiments showed numerous errors in tracking, we get very few. We are able to give accurate performance figures."

The next step, says Anderson, is to try to extend this automatic behavior-detection system to mice--a more difficult task when you're dealing with a fuzzy-edged creature like a mammal, but one that is important if we hope to some day link the genes behind fruit-fly behaviors with the genes that may cause similar behaviors in humans.

"Our visual system tells us a lot about what other people are doing--who is eating, who is beating someone else up, who is blushing, who got the guy or girl," Perona notes. "One goal of my field, computational vision, is designing machines that can detect and interpret human intentions, actions and activities. To do that, we need to start with organisms that are simpler and easier to study. David Anderson showed me how interesting and rich fly behaviors are, and so we started collaborating."

"There's a lot of information in these videos that we can now squeeze out in order to understand what controls these social interactions in flies," Anderson adds. "It makes it possible for us to study what we were not capable of studying before."

In addition to Anderson, Perona, and Dankert, other authors on the Nature Methods paper, "Automated Monitoring and Analysis of Social Behavior in Drosophila," include Caltech graduate student Liming Wang, and Eric Hoopfer, a Caltech postdoctoral scholar in biology.

This work was supported by the Howard Hughes Medical Institute, the National Science Foundation, and a Feodor Lynen fellowship awarded to Dankert by the Alexander von Humboldt Foundation.

Writer: 
Lori Oliwenstein
Writer: 

Caltech Researchers Find Tiny Genetic Change Keeps Nicotine from Binding to Muscle Cells

Research provides insight into the way nicotine works in the brain

PASADENA, Calif.--A tiny genetic mutation is the key to understanding why nicotine--which binds to brain receptors with such addictive potency--is virtually powerless in muscle cells that are studded with the same type of receptor. That's according to California Institute of Technology (Caltech) researchers, who report their findings in the March 26 issue of the journal Nature.

By all rights, nicotine ought to paralyze or even kill us, explains Dennis Dougherty, the George Grant Hoag Professor of Chemistry at Caltech and one of the leaders of the research team. After all, the receptor it binds to in the brain's neurons--a type of acetylcholine receptor, which also binds the neurotransmitter acetylcholine--is found in large numbers in muscle cells. Were nicotine to bind with those cells, it would cause muscles to contract with such force that the response would likely prove lethal.

Obviously, considering the data on smoking, that is not what happens. The question has long been: Why not?

"It's a chemical mystery," Dougherty admits. "We knew something subtle had to be going on here, but we didn't know exactly what."

That subtlety, it turns out, lies in the slight tweaking of the structure of the acetylcholine receptor in muscle cells versus its structure in brain cells.

The shape of the acetylcholine receptor, and the way the chemicals that bind with it contort themselves to fit into that receptor, is determined by a number of different weak chemical interactions. Perhaps most important is an interaction that Dougherty calls "underappreciated"--the cation-π interaction, in which a positively charged ion and an electron-rich π system come together.

Back in the late 1990s, Dougherty and colleagues had shown that the cation-π interaction is indeed a key part of acetylcholine's ability to bind to the acetylcholine receptors in muscles. "We assumed that nicotine's charge would cause it to do the same thing, to have the same sort of strong interaction that acetylcholine has," says Dougherty. "But we found that it didn't."

This would explain why smoking doesn't paralyze us; if the nicotine can't get into the muscle's acetylcholine receptors, it can't cause the muscles to contract.

But how, then, does nicotine work its addictive magic on the brain?

It took another decade for the scientists to be able to peek at what happens in brain cells' acetylcholine receptors when nicotine arrives on the scene. Turns out that in brain cells, unlike in muscle cells, nicotine makes the exact same kind of strong cation-π interaction that acetylcholine makes in both brain and muscle cells.

"In addition," Dougherty notes, "we found that nicotine makes a strong hydrogen bond in the brain's acetylcholine receptors. This same hydrogen bond, in the receptors in muscle cells, is weak."

The cause of this difference in binding potency, says Dougherty, is a single point mutation that occurs in the receptor near the key tryptophan amino acid that makes the cation-π interaction. "This one mutation means that, in the brain, nicotine can cozy up to this one particular tryptophan much more closely than it can in muscle cells," he explains. "And that is what allows the nicotine to make the strong cation-π interaction."

Dougherty says the best way to visualize this change is to think of the receptor as a box with one open side. "In muscle cells, this box is slightly distorted, so that the nicotine can't get to the tryptophan," he says. "But in the brain, the box is subtly reshaped. That's the thing: It's the shape, not the composition, of the box that changes. This allows the nicotine to make strong interactions, to become very potent. In other words, it's what allows nicotine to be addictive in the brain."

"Several projects in our labs are converging on the molecular and cellular mechanisms of the changes that occur when the brain is repeatedly exposed to nicotine," adds study coauthor Henry Lester, the Bren Professor of Biology at Caltech. "We think that the important events begin with the rather tight and selective interaction between nicotine and certain receptors in the brain. This Nature paper teaches us how this interaction occurs, at an unprecedented level of resolution."

Dougherty notes that these findings might one day lead to better drugs to combat nicotine addiction and other neurological disorders. "The receptor we describe in this paper is an important drug target," he says. "It might help pharmaceutical companies develop a better drug than nicotine to do the good things nicotine does--enhance cognition, increase attention--without being addictive and toxic."

The research described in the Nature paper, "Nicotine binding to brain receptors requires a strong cation-π interaction," was supported by the National Institutes of Health and the California Tobacco-Related Disease Research Program of the University of California. In addition to Dougherty and Lester, the paper's coauthors include Xinan Xiu, a former Caltech graduate student, and current graduate students Nyssa Puskar and Jai Shanata. Shanata's work on this research was partially supported by a National Research Service Award training grant.

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Writer: 
Lori Oliwenstein
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