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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Caltech Scientists Discover Mechanism for Wind Detection in Fruit Flies

Researchers say flies' antennae use different populations of neurons to detect wind and sound

PASADENA, Calif.--Tiny, lightweight fruit flies need to know when it's windy out so they can steady themselves and avoid being knocked off their feet or blown off course. But how do they figure out that it's time to hunker down? According to a team led by California Institute of Technology (Caltech) scientists reporting in this week's issue of the journal Nature, the flies have evolved a specialized population of neurons in their antennae that let them know not only when the wind is blowing, but also the direction from which it is coming.

The behavior of fruit flies in the face of a stiff breeze is remarkable in and of itself, notes David J. Anderson, the Roger W. Sperry Professor of Biology at Caltech, and a Howard Hughes Medical Institute (HHMI) Investigator. "We discovered that you can stop a fly dead in its tracks by blowing a gentle stream of air over it," he explains, adding that the flies' immobility is so complete, you could pick one up with a pair of chopsticks as long as a steady stream of wind was passing over the insect. Once the wind stops blowing, however, the flies immediately start walking around again. [EDITORS: Video of this behavior in the flies is available on request.]

But the response is also of interest from a scientific point of view, because it represents a fairly simple, innate defensive response that scientists can begin to tease apart in order to understand just how such behaviors are programmed in our genes. "It's more than just stupid pet tricks with fruit flies," Anderson says.

"We quickly realized that it would be interesting to ask just how the wind acts on the flies to make them stop walking. How do they sense the wind? How do they transfer that message to their brain so they know to stop moving while the wind is blowing?"

As it turns out, fruit flies are unusual in how they sense wind. Other insects have sensory hairs that stand up from the cuticle--or outer body wall--and, when blown about by a passing wind, trigger a neural response. The fruit flies, on the other hand, use their antennae to detect a breeze and its general direction, based on how the antenna moves in the breeze.

"This posed a bit of a puzzle for us," Anderson explains. "It's been long assumed that the main function of the neurons in the antennae was hearing."

And that is at least one of the antennae's functions. The flies' antennae detect nearby sounds--like the male's courtship song--that cause vibrations in the air, a bit like ripples in a pond after a rock has been thrown. Those vibrations twist the antennae slightly, exciting the neurons within.

Wind, on the other hand, is not a regularly oscillating wave; instead, it's a steady stream of air particles moving past the fly from various directions. The antennae move in the wind, but they don't twist rapidly back and forth as they do in response to sound.

Says Anderson: "What we wanted to understand was, how can flies tell the difference between sound and wind using the same sensory organ?"

There were two possible answers to this question. The first was that a fly's antennae are equipped with a single, versatile type of neuron that changes its firing pattern depending on whether it's detecting sound or wind, and that the differences in that firing pattern are picked up and somehow decoded by the fly's brain.

The other possibility, says Anderson, was that a fly's antennae contain two distinctly different populations of neurons--one that responds to oscillating air to detect sound, and another that responds to flowing air particles to detect wind.

The right answer? Number two. By selectively knocking out subsets of neurons, Anderson's graduate student Suzuko Yorozu was able to show that Johnston's organ--an area in a fruit fly antenna where sound detection is known to occur--does indeed contain at least two entirely separate groups of neurons. She also showed that each neuron type detects only one type of stimulus (sound for one; wind for the other), and that each sends its message to a distinct and separate area of the brain.

"The sound-sensitive neurons are preferentially activated by small movements of the antenna that are oscillatory in nature, firing only when the antenna twists, and turn off quickly," says Anderson. "The neurons that respond to wind, on the other hand, turn on when the antenna is pushed by air flow, and they stay on until the wind stops blowing." In other words, says Yorozu, "the intrinsic properties of these neurons are very different."

The end result of these separate pathways is that the flies exhibit absolutely distinct types of behaviors, with the sound-detecting neurons leading to behaviors like copulation (in the case of the courtship song), while the wind-detecting neurons prompt flies to come to a dead stop for safety's sake when air is blowing past with any real speed.

In addition to Anderson and Yorozu, other authors on the Nature paper, "Distinct sensory representations of wind and near-field sound in the Drosophila brain," include Caltech and HHMI postdoctoral scholar Allan Wong, Caltech visiting associate Brian Fischer, Caltech postdoctoral scholar Heiko Dankert, Maurice Kernan from SUNY Stony Brook, Azusa Kamikouchi from the University of Tokyo and the University of Cologne, and Kei Ito from the University of Cologne.

This work was supported by a grant from the National Science Foundation.

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Caltech Biologists Find Optimistic Worms Are Ready for Rapid Recovery

PASADENA, Calif.-- For the tiny soil-dwelling nematode worm Caenorhabditis elegans, life is usually a situation of feast or famine. Researchers at the California Institute of Technology (Caltech) have found that this worm has evolved a surprisingly optimistic genetic strategy to cope with these disparate conditions--one that could eventually point the way to new treatments for a host of human diseases caused by parasitic worms.

As reported in a paper published in the February 26 issue of Science Express, Paul W. Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute, along with postdoctoral scholar L. Ryan Baugh, looked at the worms' genetic response to conditions of scarcity and plenty.

In dozens of batches of the worms, consisting of tens of millions of individuals, Baugh, now an assistant professor at Duke University, synchronized hatching, so that all of the animals in each batch emerged from their eggs at the same time.

Some of the hatched worms were allowed to develop under conditions with scarce nutrients, and others with plentiful nutrients. At precise time intervals (3, 6, 9, 12, and 15 hours after hatching), subsets of both populations were killed en masse and ground up. Their messenger RNA--the genetic material that is produced upon the activation of genes and then translated to produce proteins--was harvested and analyzed at Caltech's Jacobs Genetics and Genomics Laboratory, a specialized facility designed to conduct large-scale genetic analyses.

In this way, the researchers measured the expression of every one of the worms' approximately 20,000 genes, to determine how that expression differed depending on food availability.

"We also did an experiment in which we took the starved worms and refed them, and took the fed worms and starved them, to see how rapid their response was to the changing conditions," Sternberg says.

The researchers found that the worms responded far more rapidly to being fed than being starved. Being fed also caused the activation of a far greater number of genes than did starvation. For example, three hours of feeding worm larvae that had previously been starved caused the activation of 381 genes, while starving formerly fed worm larvae for three hours caused the activation of only 56 genes.

In addition, the research revealed that as many genes are involved in the worms' response to nutrition as are involved in their overall development. Many of the genes that play a role in that nutritional response have to do with energy metabolism, and in changing the way the animals utilize and store energy.

"It looks like C. elegans is primed to respond faster to better conditions. It is optimistic," Sternberg says. "These worms live, most of the time, in scarcity. They are facing bad conditions--that is, no food--most of the time. Probably they've evolved to take advantage when times get better for a brief period. They grow and reproduce."

The worms' quick response to food appears to be controlled by a vital cellular protein called RNA Polymerase II (RNA Pol II), which is responsible for transcribing DNA into mRNA. In a separate experiment, Sternberg and his colleagues found that RNA Pol II accumulates on genes that respond rapidly to being fed, but in advance of that feeding.

"We speculate that this polymerase accumulation is part of the way in which they can respond so quickly. It's already engaged, ready to go, ready to send out the message. It's like having Paul Revere on the North Shore, ready to ride, when the food comes," Sternberg says.

"It is kind of interesting in hard economic times to think whether we can learn anything from this organism, in terms of being optimistic or pessimistic. Maybe the take-home message is that sometimes when you are faced with scarcity, you should still be optimistic."

Sternberg speculates that other nematodes, including the parasitic worms that cause elephantiasis in humans, and other lymphatic filarial diseases, may also go through similar transitions in nutrition as they transition from one host (say, a mosquito) to another (a human). Those transitions may be mediated by a similar accumulation of RNA Pol II on particular genes. Identifying those genes could provide potential targets for new types of therapeutic drugs.

The paper, "RNA Pol II Accumulates at Promoters of Growth Genes During Developmental Arrest," was coauthored by Baugh, Sternberg, and John DeModena, a member of the biology research staff at Caltech. The work was supported by the Howard Hughes Medical Institute.

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Caltech Scientists Find Evidence for Precise Communication Across Brain Areas During Sleep

PASADENA, Calif.--By listening in on the chatter between neurons in various parts of the brain, researchers from the California Institute of Technology (Caltech) have taken steps toward fully understanding just how memories are formed, transferred, and ultimately stored in the brain--and how that process varies throughout the various stages of sleep.

Their findings, published in the February 26 issue of the journal Neuron, may someday even help scientists understand why dreams are so difficult to remember.

Scientists have long known that memories are formed in the brain's hippocampus, but are stored elsewhere--most likely in the neocortex, the outer layer of the brain. Transferring memories from one part of the brain to the other requires changing the strength of the connections between neurons and is thought to depend on the precise timing of the firing of brain cells.

"We know that if neuron A in the hippocampus fires consistently right before neuron B in the neocortex, and if there is a connection from A to B, then that connection will be strengthened," explains Casimir Wierzynski, a Caltech graduate student in computation and neural systems, and first author on the Neuron paper. "And so we wanted to understand the timing relationships between neurons in the hippocampus and the prefrontal cortex, which is the front portion of the neocortex."

The research team--led by Athanassios Siapas, a Bren Scholar in the Caltech Division of Biology and an associate professor of computation and neural systems--used high-tech recording and computational techniques to listen in on the firing of neurons in the brains of rats. These techniques helped them pinpoint a number of neuron pairs that had precisely the kind of synchronous relationship they were looking for--one in which a hippocampal neuron's firing was followed within milliseconds by the firing of a neuron in the prefrontal cortex.

"This is exactly the kind of relationship that would be needed for the hippocampus to effect changes in the neocortex--such as the consolidation, or laying down, of memories," adds Wierzynski.

Once these spike-timing relationships between the hippocampal and prefrontal cortex neurons had been established, the team used their high-tech eavesdropping techniques to hear what goes on in the brains of sleeping rats--since sleep, as Siapas points out, has long been thought to be the optimal time for the memory consolidation.

As it turns out, those thoughts were right--but only part of the time.

The team did indeed hear "bursts" of neuronal chatter during sleep--but only during a phase of sleep known as slow-wave sleep (SWS), the deep, dreamless periods of sleep. "It turns out that during slow-wave sleep there are these episodes where a lot of the cells in the hippocampus will all fire very close to the same time," says Wierzynski. In response, some cells in the prefrontal cortex will fire in near unison as well, just milliseconds later. "What's interesting is that the bulk of the precise spike timing happens during these bursts, and not outside of these bursts," he adds.

On the other hand, during rapid-eye-movement (REM) sleep, the previously chatty neuron pairs seemed to talk right past each other, firing at the same rates as before but no longer in concert.

"It was surprising," says Wierzynski, "to find that the timing relationship almost completely went away during REM sleep."

Since REM sleep is the phase during which dreaming occurs, the scientists speculate that this absence of memory-consolidating chatter may eventually help to explain why dreams can be so difficult to remember.

As intriguing as that idea may be, the researchers caution that these findings only raise possibilities, providing avenues for further research in the field.

"Now that we've shown this link," says Siapas, "we have a framework we can use to study these questions further. This is just a step toward our goal of some day fully understanding the relationship between memory and sleep."

Other coauthors on the paper, entitled "State-dependent spike timing relationships between hippocampal and prefrontal circuits during sleep," included Evgueniy Lubenov, a postdoctoral scholar in biology at Caltech, and Caltech graduate student Ming Gu.

This work was supported by a National Defense Science and Engineering Graduate Fellowship, the Caltech Information Science and Technology Center for Biological Circuits Design, the James S. McDonnell Foundation, the Bren Foundation, the McKnight Foundation, the Whitehall Foundation, and the National Institutes of Health.

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Caltech Researchers Help Unlock the Secrets of Gene Regulatory Networks

PASADENA, Calif.-- A quartet of studies by researchers at the California Institute of Technology (Caltech) highlight a special feature on gene regulatory networks recently published in the Proceedings of the National Academy of Sciences (PNAS).

The collection of papers, "Gene Networks in Development and Evolution Special Feature, Sackler Colloquium," was coedited by Caltech's Eric H. Davidson, the Norman Chandler Professor of Cell Biology. His coeditor was Michael Levine, professor of genetics, genomics and development at the University of California, Berkeley. 

"The control system that determines how development of an animal occurs in each species is encoded in the genome, and the physical location of the sequences where this code is resident is being revealed in a new area of systems biology--the study of gene regulatory networks," says Davidson. Gene regulatory networks are the complex networks of gene interactions that direct the development of any given species.

The papers in the collection focus on the gene regulatory networks of a variety of organisms, including fruit flies, soil-dwelling nematodes, sea urchins, lampreys, and mice.

"These networks lie at the heart of the regulatory apparatus, and they consist of genes that encode proteins that regulate other genes, and the DNA sequences which control when and where they are expressed," says Davidson, who authored a paper in the special feature about a gene regulatory network found in sea urchin embryos. He and Levine also coauthored a perspective in the same issue of the journal on the properties of gene regulatory networks.

In one paper, Ellen V. Rothenberg, one of the two Albert Billings Ruddock Professors of Biology at Caltech, examines, in mice, the intricate developmental pathway that causes blood stem cells to differentiate into T cells, a varied class of immune system cells that help the body fight off infection. 

The paper, Rothenberg says, represents a "codification of everything we know about T cell development. We've found that getting the right balances of the various regulatory signals is absolutely crucial for the T cells to come out right. It gives one a sense of how subtle and sophisticated the regulation can be."

Another study in the special feature by Marianne Bronner-Fraser, the second Albert Billings Ruddock Professor of Biology, focuses on the gene regulatory network underlying neural crest formation in the lamprey, the most primitive living vertebrate. The neural crest is a group of embryonic cells that are pinched off during the formation of the neural tube--the precursor to the spinal cord--and then migrate throughout the developing body to form other nervous system structures. 

The study "reveals order and linkages within the network at early stages," Bronner-Fraser says. "Because the neural crest cell type represents a vertebrate innovation, our work in lampreys shows that this network is ancient and tightly conserved to the base of vertebrates," she says.

The fourth of the Caltech papers, by Paul W. Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI), and his colleagues, looks at a postembryonic gene regulatory network in Caenorhabditis elegans, a soil-dwelling worm commonly studied by developmental biologists. The gene regulatory network studied by Sternberg and his colleagues controls the formation of the worm's vulva, which connects the uterus with the outside and allows the passage of sperm and eggs. 

All of the papers in the special feature arise out of presentations at a Sackler Colloquium held at the National Science Foundation's Beckman Center in Irvine, California, in February 2008. 

Davidson's paper, "Gene regulatory network subcircuit controlling a dynamic spatial pattern of signaling in the sea urchin embryo," coauthored with Caltech postdoctoral scholar Joel Smith, was funded by the National Institutes of Health's (NIH) Institute of Child Health and Development and General Medical Sciences Institute and a California Institute of Regenerative Medicine (CIRM) fellowship to Smith.

Rothenberg's paper, "A gene regulatory network armature for T lymphocyte specification," represents a collaboration between Rothenberg and Hamid Bolouri, a visiting associate at Caltech, with support from the NIH, the Albert Billings Ruddock Professorship, the Louis A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, and the DNA Sequencer Royalty Fund. The paper was coauthored by Caltech senior postdoctoral research scholar Constantin Georgescu, and William Longabaugh of the Institute for Systems Biology in Seattle.

Bronner-Fraser's paper, "Gene regulatory networks in neural crest development and evolution," was coauthored by Caltech postdoctoral research scholars Natalya Nikitina and Tatjana Sauka-Spengler.

Sternberg's paper, "The Caenorhabditis elegans vulva: A post-embryonic gene regulatory network controlling organogenesis," was funded by the NIH and the HHMI.

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Three Caltech Scientists Receive Ellison Medical Foundation Awards

The Senior Scholar Awards are aimed at promoting basic research into the underlying processes that control aging

PASADENA, Calif.--The Ellison Medical Foundation (EMF) has awarded Senior Scholar Awards of nearly $1 million each to three California Institute of Technology (Caltech) researchers for exploratory projects in the molecular biology of aging processes and age-related diseases.

The brainchild of Laurence J. Ellison, Oracle cofounder and CEO, and Nobel Prize-winning biologist Joshua Lederberg, the EMF supports basic research that integrates molecular biology and the biomedicine of aging. Its Senior Scholar Awards fund exploratory work by acclaimed researchers, many new to the study of aging. Over the past decade, a board of six distinguished scientists has selected awardees by adhering to Lederberg and Ellison's belief that the way to get positive scientific results was to "look for smart people who had track records of creative, productive work and who had a good idea," according to EMF's website. The foundation would then "give them money and stand back. It would favor basic research that was too risky or speculative to attract mainstream funding."

Caltech's awardees take that mandate seriously. For instance, Jacqueline Barton, the Arthur and Marian Hanisch Memorial Professor and professor of chemistry, plans to use her Senior Scholar Award to explore novel ways the body can defend itself against oxidative damage, a major contributor to aging. Barton is known for her work in understanding charge transport in DNA--examining the way in which electrical charges are moved along a DNA strand, and what role charge transport plays in creating DNA damage. But now she is beginning to consider ways in which charge transport might actually be protecting DNA as well. For instance, Barton believes that DNA charge transport may provide a way for the DNA to send out a long-range signal when it undergoes oxidative damage, alerting DNA-bound proteins such as p53--known as "the guardian of the genome" because of its role in cancer prevention via DNA repair--to set into motion the processes that will eventually lead to the mending of damaged strands. "This would be a paradigm shift with respect to current biological mechanisms for cellular activation," says Barton.

Judith Campbell, professor of chemistry and biology at Caltech, is exploring the ways in which a yeast protein her lab discovered--a DNA-synthesizing enzyme called Dna2--might work to safeguard the bits of DNA at the end of chromosomes, called telomeres. Telomeres are made of repeated sequences of DNA and act to protect the ends of the chromosome from damage, much like the plastic wrapped around the end of a shoelace. Each time a cell divides, however, its telomeres get a little bit shorter; eventually, this aging process leads to the cell's death. But what Campbell has found is that, in yeast at least, Dna2 seems to help maintain the length of the telomeres, slowing down the aging process. She intends to use her Senior Scholar Award to begin studying Dna2 in humans, rather than yeast. "Extending our work to human cells will allow me to contribute to the application of fundamental biology to the improvement of human health," she says. "This has been a burgeoning but frustratingly slow field. We hope this award will allow us to identify new targets--including but not limited to Dna2--whose manipulation can lead to telomere stability. This can, in turn, be expected to have an effect on the life span of the organism as a whole, by keeping at least some of the diseases of aging at bay."

David Baltimore, Caltech President Emeritus and Robert Andrews Millikan Professor of Biology, has been researching the role of tiny bits of RNA--called micro-RNAs or miRs--in the process of aging. First discovered in the 1990s, micro-RNAs appear to control gene expression and seem to play a role in the development and inhibition of cancer, in the development of immune cells, and in the body's response to inflammation. According to Baltimore, miRs can influence a wide variety of behaviors in cells--everything from differentiation to proliferation to functional behavior. Baltimore's group will use the Senior Scholar Award to compare the micro-RNA profiles in the cells of young mice to the profiles found in the cells of old mice. They will focus particular attention on specific miRs they have already discovered, which they have found to play a role in inflammation--a process that seems to increase as we age. "When we find an miR that is affected by aging, we will examine its targets, its cellular specificity, the effects from its overproduction, and the consequences of a knockout," says Baltimore.

"This award spotlights three of Caltech's most prominent researchers in the field," says Caltech president Jean-Lou Chameau. "It recognizes not only the promise of their research efforts, but also the originality of the ideas which they are pursuing. It is from these sorts of programs--programs that are aimed at allowing researchers to venture into new research arenas--that real creativity is nurtured."

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About Caltech: Caltech is recognized for its highly select student body of 900 undergraduates and 1,200 graduate students, and for its outstanding faculty. Since 1923, Caltech faculty and alumni have garnered 32 Nobel Prizes and five Crafoord Prizes.

In addition to its prestigious on-campus research programs, Caltech operates the W. M. Keck Observatory in Mauna Kea, the Palomar Observatory, the Laser Interferometer Gravitational-Wave Observatory (LIGO) and the Jet Propulsion Laboratory. Caltech is a private university in Pasadena, California. For more information, visit http://www.caltech.edu.

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Caltech Researchers Get First Look at How Groups of Cells Coordinate Their Movements

Findings also provide new and detailed insights into what happens during the early stages of embryonic development

PASADENA, Calif.--Using novel imaging, labeling, and data-analysis techniques, scientists from the California Institute of Technology (Caltech) have been able to visualize, for the first time, large numbers of cells moving en masse during some of the earliest stages of embryonic development.

The findings not only provide insight into this stage of development--called gastrulation--but give a more general glimpse at how a living organism choreographs the motions of thousands of cells at one time. Previous research has been generally limited to imaging the movements of single cells.

The work was published in the December 5 issue of the journal Science.

The Caltech team observed the vast reorganization and migration of cells that occurs during gastrulation in Drosophila, the fruit fly. During gastrulation, a single-layered tube of cells collapses and then begins to spread. The scientists focused their attention on the movement of cells from two cross sections of that tube, paying particular attention to the movements of cells in the mesoderm--the inner layer of cells in the early embryo--relative to the cells in the ectoderm, or outer layer of cells. (The endoderm, the innermost of the three primary germ cell layers, has not yet formed at this stage of development.)

The researchers used a technique called two-photon excited fluorescence imaging to get a glimpse at what goes on during gastrulation in a living embryo. "This is the first time we've been able to see mesoderm cells within a developing Drosophila embryo," says Angelike Stathopoulos, assistant professor of biology at Caltech and the paper's principal investigator. "It was important not only because we were able to watch many cells move at one time, but because we were able to take that data and make sense of it."

Making sense of the data meant reducing the movement of each cell to a vector, and then analyzing that vector not just along its x, y, and z axes, but by looking at its cylindrical coordinates. "The spreading has both an angular and a radial component," Stathopoulos notes.

What the data showed was that the mesodermal cells move in a directed manner, traveling down and moving outward--diverging--at the same time. The ectoderm, on the other hand, moves down but converges as it goes. Thus, the mesoderm--which sits atop the ectoderm--passively rides the downward wave of ectodermal movement, but has to actively swim against the tide to spread outward.

Credit: Science; McMahon, Supatto, Fraser, Stathopoulos/Caltech

"It's as if the mesodermal cells are on a moving sidewalk," says Stathopoulos, "but as they're being moved along, they keep taking steps to the side."

The researchers found, in addition, that this choreography is anything but chaotic. The cells stayed in more or less the same order throughout their travels, following a set of "leader" cells and rarely if ever crossing over the midline of the pack.

"If you look at movies of the motion," says Stathopoulos, "you see all this jiggling, and you'd think the cells are mixing all around. And yet, they're not."

"We were able to follow the whole process," adds Scott Fraser, the Anna L. Rosen Professor of Biology, director of the Beckman Institute's Biological Imaging Center, and a paper coauthor. "We were able to label and watch the cells doing the motion, and the events that guide the motion."

In addition to looking at normal gastrulation, the scientists also looked at what happens to gastrulation when mutations occur. "We watched normal behavior, and the cues that guided it," says Fraser. "Then we could use the power of genetics to break one of the cues and analyze what was different, to determine how that cue was involved in the process. Being able to visualize groups of cells lets us do this in a more complex and powerful way. Before, you could say, 'It's broken.' Now, we can say how it is broken."

The findings turned up some other surprising tidbits about fruit-fly embryology, as well. For instance, while scientists had known that the cells in the mesoderm tended to divide twice during gastrulation, they didn't know if this was applicable only to a particular subset of cells, or if it was a hard-and-fast rule. Turns out, it was a rule. "We saw that each and every cell divides twice," says Stathopoulos. "Even more surprising, we saw that the timing of those divisions is based on the cells' original position in the tube--even though, by the time they divide, they've traveled from where they began. They actually remember where they came from."

While Stathopoulos says she's not yet sure what this timed division means, it's a clear sign that there are many more layers of order in embryonic development than had been previously suspected.

"What's fun about this," says Fraser, "is that all the different parts came together at the same time--genetics, labeling, imaging, analysis. It was like a perfect storm . . . in a good way."

The work detailed in the paper, "Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration," was supported by funding from the National Institutes of Health, the Searle Scholars Program, the March of Dimes, and Caltech's Beckman Institute.

Caltech graduate student Amy McMahon and postdoctoral scholar Willy Supatto shared primary authorship of this paper.

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Caltech Scientists Show Function of Helical Band in Heart

MRI technique provides evidence that could both settle a 50-plus-year debate and create a roadmap for future cardiac surgical techniques

PASADENA, Calif.--Scientists from the California Institute of Technology (Caltech) have created images of the heart's muscular layer that show, for the first time, the connection between the configuration of those muscles and the way the human heart contracts.

More precisely, they showed that the muscular band--which wraps around the inner chambers of the heart in a helix--is actually a sort of twisting highway along which each contraction of the heart travels.

Their findings were published in the December issue of the American Physiological Society journal, Heart and Circulatory Physiology.

Since the days of Leonardo da Vinci, observers of the human body have known that the heart's beat is not a simple in-and-out movement--that it has more than a little bit of a twist to it. "The heart twists to push blood out the same way you twist a wet towel to wring water out of it," explains Morteza Gharib, the principal investigator on the study, and the Hans W. Liepmann Professor of Aeronautics and professor of bioengineering in the Division of Engineering and Applied Science at Caltech.

Some 50 years ago, anatomist Francisco Torrent-Guasp was the first to show the helical configuration of the heart's myocardium--its muscular middle layer, the one that contracts with each heart beat.

But what he and subsequent generations of scientists were unable to do was to connect that myocardial band to the heart's function--to prove that the helical shape is important to the effective beating of the heart. Without that connection, physicians and scientists have tended to look at the heart as "just a piece of meat," says Gharib.

Until now, that is. Using a technique pioneered by Han Wen and his team at the National Institutes of Health, Gharib and his colleague Abbas Nasiraei Moghaddam, a Caltech graduate and visitor in bioengineering, were able to create some of the first dynamic images of normal myocardium in action at the tissue level. "We tagged and traced small tissue elements in the heart, and looked at them in space, so we could see how they moved when the heart contracts," Gharib explains. "In this way, we were able to see where the maximum physical contraction occurs in the heart and when--and to show that it follows this intriguing helical loop."

With each beat of the heart, a wave of contraction starts at the heart's apex--which, despite its name, is actually at the very bottom of the heart--and then travels up through the myocardium. "The only time the whole helix shows up in the images is at the end of systole, which is when the heart is contracting," says Gharib. "This simple band structure is akin to an engine behind the heart pumping action."

In addition to going a long way toward settling the decades-long structure/function debate surrounding Torrent-Guasp's work, this finding also has major implications for the surgical treatment of heart disease, Gharib says. "It's going to change the way we repair the heart," he explains. Knowing that the contractile wave travels along the helical pathway--instead of occurring throughout the heart all at once--has implications for which parts of the heart will be most vulnerable to a surgeon's scalpel, for instance. "Seventy-five percent of the function of the heart depends on this muscle," Gharib says. "Surgeons now know what to cut and what not to cut. This will help them to come up with new and more effective surgical procedures."

The work detailed in the paper, "Evidence for the existence of a functional helical myocardial band," was supported by funding from the Caltech MRI Center through a Discovery grant.

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Caltech Scientists Develop "Barcode Chip" for Cheap, Fast Blood Tests

PASADENA, Calif.-- A new "barcode chip" developed by researchers at the California Institute of Technology (Caltech) promises to revolutionize diagnostic medical testing. In less than 10 minutes, and using just a pinprick's worth of blood, the chip can measure the concentrations of dozens of proteins, including those that herald the presence of diseases like cancer and heart disease.

The device, known as the Integrated Blood-Barcode Chip, or IBBC, was developed by a group of Caltech researchers led by James R. Heath, the Elizabeth W. Gilloon Professor and professor of chemistry, along with postdoctoral scholar Rong Fan and graduate student Ophir Vermesh, and by Leroy Hood, president of the Institute for Systems Biology in Seattle, Washington.

An IBBC, described in a paper in the advance online edition of Nature Biotechnology, is about the size of a microscope slide and is made out of a glass substrate covered with silicone rubber. The chip's surface is molded to contain a microfluidics circuit--a system of microscopic channels through which the pinprick of blood is introduced, protein-rich blood plasma is separated from whole blood, and a panel of protein biomarkers is measured from the plasma.

The chip offers a significant improvement over the cost and speed of standard laboratory tests to analyze proteins in the blood. In traditional tests, one or more vials of blood are removed from a patient's arm and taken to a laboratory, where the blood is centrifuged to separate whole blood cells from the plasma. The plasma is then assayed for specific proteins. "The process is labor intensive, and even if the person doing the testing hurries, the tests will still take a few hours to complete," says Heath. A kit to test for a single diagnostic protein costs about $50.

"We wanted to dramatically lower the cost of such measurements, by orders of magnitude," he says. "We measure many proteins for the cost of one. Furthermore, if you reduce the time it takes for the test, the test is cheaper, since time is money. With our barcode chip, we can go from pinprick to results in less than 10 minutes."

A single chip can simultaneously test the blood from eight patients, and each test measures many proteins at once. The researchers reported on devices that could measure a dozen proteins from a fingerprick of blood, and their current assays are designed for significantly more proteins. "We are aiming to measure 100 proteins per fingerprick within a year or so. It's a pretty enabling technology," Heath says.

To perform the assay, a drop of blood is added to the IBBC's inlet, and then a slight pressure is applied, which forces the blood through a channel. As the blood flows, plasma is skimmed into narrow channels that branch off from the main channel. This part of the chip is designed as if it were a network of resistors, which optimizes plasma separation.

The plasma then flows across the "barcodes." The barcodes consist of a series of lines, each 20 micrometers across and patterned with a different antibody that allows it to capture a specific protein from the plasma passing over. When the barcode is "developed," the individual bars emit a red fluorescent glow, whose brightness depends upon the amount of protein captured.

In the Nature Biotechnology paper, the researchers used the chip to measure variations in the concentration of human chorionic gonadotropin (hCG), the hormone produced during pregnancy. "The concentration of this protein increases by about 100,000-fold as a woman goes through the pregnancy cycle, and we wanted to show that we could capture that whole concentration range through a single test," Heath says.

The scientists also used the barcode chip to analyze the blood of breast and prostate cancer patients for a number of proteins that serve as biomarkers for disease. The types and concentrations of the proteins vary from disease to disease and between different individuals. A woman with breast cancer, for example, will produce a different suite of biomarkers than will a man with prostate cancer, while a woman with an aggressive form of cancer may produce proteins that are different from a woman with a less-deadly cancer.

Those proteins can also change as a patient receives therapy. Thus, determining these biomarker profiles can allow doctors to create individualized treatment plans for their patients and improve outcomes. The ease and the speed with which results can be obtained using the IBBC also will potentially allow doctors to assess their patients' responses to drugs and to monitor how those responses evolve with time, much as a diabetic patient might use a blood glucose test to monitor insulin delivery.

The barcode chip is now being tested in human clinical trials on patients with glioblastoma, a common and aggressive form of brain tumor. The researchers are also using the chips in studies of healthy individuals, to determine how diet and exercise change the composition of the proteins in the blood.

Currently, the barcoded information is "read" with a common laboratory scanner that is also used for gene and protein expression studies. "But it should be very easy to design something like a supermarket UPC scanner to read the information," making the process even more user-friendly, says Fan, the first author on the paper.

"As personalized medicine develops, measurements of large panels of protein biomarkers are going to become important, but they are also going to have to be done very cheaply," Heath says. "It is our hope that these IBBCs will enable such inexpensive and multiplexed measurements."

The paper, "Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood," will be featured as the cover story in the December print edition of Nature Biotechnology. The work was funded by the National Cancer Institute and by the Institute for Collaborative Biotechnologies through a grant from the United States Army Research Office.

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Caltech's David Baltimore and Fiona Harrison Named among America's Best Leaders for 2008

U.S. News Media Group and Harvard's Center for Public Leadership recognize 24 of the country's foremost professionals

PASADENA, Calif.--Two prominent researchers from the California Institute of Technology (Caltech) have been named among the country's 24 top leaders by U.S. News Media Group in association with the Center for Public Leadership (CPL) at Harvard Kennedy School. The 2008 edition of America's Best Leaders--available online at www.usnews.com/leaders and on newsstands Monday, November 24--includes honors for Caltech's David Baltimore and Fiona Harrison.

According to U.S. News, the Best Leaders issue features "some of the country's most visionary individuals," highlighting those professionals "who continue to offer optimism and hope through their work."

Nobel Laureate David Baltimore, the Robert Andrews Millikan Professor of Biology and former president of Caltech, was lauded by the magazine for the way he has "profoundly influenced national science policy on such issues as recombinant DNA research and the AIDS epidemic. He is an accomplished researcher, educator, administrator and public advocate for science and engineering, and is considered one of the world's most influential biologists."

Baltimore was awarded the Nobel Prize in Physiology or Medicine in 1975, at the age of 37. He served as president of Caltech for nine years and was named president emeritus in 2006. He had previously spent almost 30 years on the faculty of the Massachusetts Institute of Technology, where he contributed widely to the understanding of cancer, AIDS, and the molecular basis of the immune response, and where he served as founding director of the Whitehead Institute for Biomedical Research from 1982 until 1990. He served as president of the American Association for the Advancement of Science from 2007 to 2008, and is currently its chair.

Baltimore says he is "very honored to be named among this exceptional group of leaders."

Leadership, he says, "involves a vision of the future, and a generosity of spirit that allows the leader to take pleasure in the accomplishments of others." Leadership can't be about micromanaging every aspect of a large organization, he notes. "It's about picking the right people, motivating them, and encouraging them. You have to make sure those people are pulling in the same direction. You have to catalyze interaction between people so that they see a commonality of interests--so that they follow a common line."

Fiona Harrison, a professor of physics and astronomy at Caltech, was chosen for her work as principal investigator of the NuSTAR (Nuclear Spectroscopic Telescope Array) Mission, a pathfinder mission that will "open the high energy X-ray sky for sensitive study for the first time," she says. She started developing the technologies necessary to realize NuSTAR more than a decade ago, and assembled and led a team of scientists and engineers to design and implement the mission. NuSTAR was selected by NASA through a competitive process and will launch in mid-2011.

Harrison "has devoted her career to studying energetic phenomena in the Universe, including massive black holes and stellar explosions, and developing advanced instrumentation for focusing and detection of X-rays and gamma-rays," notes U.S. News. She is a recipient of a NASA Graduate Student Research Fellowship and winner of the Robert A. Millikan Prize Fellowship in Experimental Physics, and was awarded a Presidential Early Career Award for Scientists and Engineers by President Clinton. She joined the faculty of Caltech in 1995.

"It is an honor to be recognized among such a diverse and distinguished group of people," says Harrison.

"Most aspiring scientists don't focus on leadership as a quality necessary to accomplish their research," she adds. "As experiments and projects get larger and more complex, however, good leadership is often necessary in order to make progress. For me, creating and motivating an effective team were skills I learned in order to make the science I am passionate about happen."

In a collaborative effort between U.S. News and Harvard's CPL, the leaders were selected by a nonpartisan and independent committee, convened and organized by the center, without the participation of U.S. News editors. The selection criteria used by the committee in choosing the honorees included the ability to set direction, achieve results, and cultivate a culture of growth.

"Even though Americans have lost confidence in current leadership, over the past year they have had unique opportunities to observe and debate the qualities of strong leaders," says Brian Kelly, editor of U.S. News & World Report. "With our Best Leaders issue, we widen the lens to examine people who are showing leadership in unexpected ways across a wide variety of fields."

Other honorees include Lance Armstrong, founder of the Lance Armstrong Foundation; Herbie Hancock, chairman of the Thelonious Monk Institute of Jazz Performance Arts; Marian Wright Edelman, founder and president of the Children's Defense Fund; Anthony Fauci, director of the National Institute for Allergy and Infectious Disease; Robert Gates, U.S. Secretary of Defense; and Steven Spielberg, director and producer, and founder of the Shoah Foundation.

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