An Up-Close View of Bacterial "Motors"

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

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

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

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

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

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

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

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

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

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

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

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

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Learning to Program Cellular Memory

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Experimental Economics: Results You Can Trust

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Studying Memory's 'Ripples'

Caltech neuroscientists have looked inside brain cells as they undergo the intense bursts of neural activity known as "ripples" that are thought to underlie memory formation. 

During ripples, a small fraction of brain cells, or neurons, fire synchronously in area CA1, a part of the hippocampus that is thought to be an important relay station for memories. "During a ripple, about 10 percent of the neurons in CA1 are activated, and these active neurons all fire within a tenth of a second," says Caltech graduate student Brad Hulse. "Two big questions have been: How do the remaining 90 percent of CA1 neurons stay quiet? And what is synchronizing the firing of the active neurons?"

In a new study, published online on February 17 in the journal Neuron, Hulse and his colleagues used a novel approach to show how the combination of excitatory and inhibitory inputs to CA1 work together to synchronize the firing of active neurons while keeping most neurons silent during ripples.

"For a long time, people studied these events by placing an electrode outside of a cluster of neurons. These extracellular recordings can tell you about the output of a group of brain cells, but they tell you very little about the inputs they're receiving," says study coauthor and Caltech research scientist Evgueniy Lubenov.

The Caltech scientists combined extracellular recording with a technique to look inside a neuron during ripples. They used fine glass pipettes with tips thinner than a tenth of the width of a human hair to measure directly the voltage difference, or "electrical potential," across the cellular membrane of individual neurons in awake mice.

Employing these two techniques in tandem allowed the scientists to monitor the activity inside a single neuron while still observing how the larger network was behaving. This in turn enabled them to piece together how excitatory inputs from CA3, a hippocampal region where memories are formed, affect the output of brain cells called pyramidal neurons in CA1. These neurons are important for transferring newly coded memories to other brain areas such as the neocortex for safekeeping and long-term storage. Ripples are thought to be the mechanism by which this transfer occurs.

The team discovered that the membrane potential of CA1 pyramidal cells increases during ripples. Surprisingly, this increase is relatively constant and independent of the strength of the input from CA3. For this to be the case, the direct excitation from CA3 must be balanced by proportional inhibition. The source of this inhibition is presumed to be a class of brain cells called feedforward interneurons, which receive direct inputs from CA3 and inhibit CA1 pyramidal cells.

"There seems to be a circuit mechanism that balances excitation and inhibition, so that for most neurons, these two forces cancel out," says study leader Thanos Siapas, professor of computation and neural systems at Caltech.

Without a balanced inhibition, all of the neurons in CA1 could fire when the excitatory input is large enough. "This could cause runaway excitation and possibly trigger a seizure," says Hulse, who is the first author of the new study.

The team's finding explains why most CA1 pyramidal neurons remain silent during ripples, but it raises two important questions: Why do any neurons fire at all? And what controls the precise timing of those that do fire?

The Caltech researchers found that active neurons receive a much stronger excitatory input from CA3 than silent neurons do—one that is large enough to overcome the balancing inhibition. This large excitation originates from CA3 neurons with particularly strong connections to the active CA1 neurons. These connections are believed to be modified during behavior to encode memories.

Hence, it is the specific identity of CA3 neurons, rather than their sheer number, that is responsible for making CA1 neurons fire, the researchers say. This system might seem overly complex and redundant, but the end result is a flexible circuit—an ever-changing mosaic of active and inactive pyramidal neurons. "It's a shifting mosaic, but it's one that is dependent on the organism's memories and experience," Siapas says.

How do ripples exert their influence on the rest of the brain? The membrane potential of each neuron oscillates very rapidly during ripples to synchronize the firing of cells to within a few thousandths of a second. "By coordinating their activities, the CA1 neurons are maximizing the impact of their output on downstream areas of the brain. The overall effect is more powerful than if each neuron fired independently," Lubenov says. "It is the difference between clapping independently or in unison with others at a concert. The effect in the latter case is stronger, even with the same number of people applauding."

Neuroscientists previously thought that these fast oscillations were generated by rhythmic firing of inhibitory neurons, but the Caltech team showed that this cannot be the whole story. "Our experiments suggest that it is the interplay between rhythmic excitation and inhibition that drives these fast oscillations," Hulse says.

The paper, "Membrane Potential Dynamics of CA1 Pyramidal Neurons during Hippocampal Ripples in Awake Mice," is also coauthored by Laurent C. Moreaux, a research scientist at Caltech. Funding for the work was provided by the Mathers Foundation, the Gordon and Betty Moore Foundation, the National Institutes of Health, and the National Science Foundation. 

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Neuroscientists look inside brain cells undergoing the bursts of activity, or "ripples," that underlie memory formation

Counting Molecules with an Ordinary Cell Phone

Diagnostic health care is often restricted in areas with limited resources, because the procedures required to detect many of the molecular markers that can diagnose diseases are too complex or expensive to be used outside of a central laboratory. Researchers in the lab of Rustem Ismagilov, Caltech's Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering and director of the Jacobs Institute for Molecular Engineering for Medicine, are inventing new technologies to help bring emerging diagnostic capabilities out of laboratories and to the point of care. Among the important requirements for such diagnostic devices is that the results—or readouts—be robust against a variety of environmental conditions and user errors.

To address the need for a robust readout system for quantitative diagnostics, researchers in the Ismagilov lab have invented a new visual readout method that uses analytical chemistries and image processing to provide unambiguous quantification of single nucleic-acid molecules that can be performed by any cell-phone camera.

The visual readout method is described and validated using RNA from the hepatitis C virus—HCV RNA—in a paper in the February 22 issue of the journal ACS Nano.

The work utilizes a microfluidic technology called SlipChip, which was invented in the Ismagilov lab several years ago. A SlipChip serves as a portable lab-on-a-chip and can be used to quantify concentrations of single molecules. Each SlipChip encodes a complex program for isolating single molecules (such as DNA or RNA) along with chemical reactants in nanoliter-sized wells. The program also controls the complex reactions in each well: the chip consists of two plates that move—or "slip"—relative to one another, with each "slip" joining or separating the hundreds or even thousands of tiny wells, either bringing reactants and molecules into contact or isolating them. The architecture of the chip enables the user to have complete control over these chemical reactions and can prevent contamination, making it an ideal platform for a user-friendly, robust diagnostic device.

The new visual readout method builds upon this SlipChip platform. Special indicator chemistries are integrated into the wells of the SlipChip device. After an amplification reaction—a reaction that multiplies nucleic-acid molecules—wells change color depending on whether the reaction in it was positive or negative. For example, if a SlipChip is being used to count HCV RNA molecules in a sample, a well containing an RNA molecule that amplified during the reaction would turn blue; whereas a well lacking an RNA molecule would remain purple.

To read the result, a user simply takes a picture of the entire SlipChip using any camera phone. Then the photo is processed using a ratiometric approach that transforms the colors detected by the camera's sensor into an unambiguous readout of positives and negatives.

Previous SlipChip technologies utilized a chemical that would fluoresce when a reaction took place within a well. But those readouts can be too subtle for detection by a common cell-phone camera or can require specific lighting conditions. The new method provides guidelines for selecting indicators that yield color changes compatible with the color sensitivities of phone cameras, and the ratiometric processing removes the need for a user to distinguish colors by sight.

"The readout process we developed can be used with any cell-phone camera," says Jesus Rodriguez-Manzano, a postdoctoral scholar in chemical engineering and one of two first authors on the paper. "It is rapid, automated, and doesn't require counting or visual interpretation, so the results can be read by anyone—even users who are color blind or working under poor lighting conditions. This robustness makes our visual readout method appropriate for integration with devices used in any setting, including at the point of care in limited-resource settings. This is critical because the need for highly sensitive diagnostics is greatest in such regions."

The paper is titled "Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones." In addition to Rodriguez-Manzano, Mikhail Karymov is also a first author. Other Caltech coauthors include Stefano Begolo, David Selck, Dmitriy Zhukov, and Erik Jue. The work was funded by grants from the Defense Advanced Research Projects Agency, the National Institutes of Health, and an Innovation in Regulatory Science Award from the Burroughs Wellcome Fund. Microfluidic technologies developed by Ismagilov's group have been licensed to Emerald BioStructures, RainDance Technologies, and SlipChip Corp., of which Ismagilov is a founder.

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The new visual readout method to count individual nucleic-acid molecules within a sample can be performed by any cell-phone camera.

A New Twist on the History of Life

The idea that the wholesale relocation of Earth's continents 520 million years ago, also known as "true polar wander," coincided with a burst of animal speciation in the fossil record dates back almost 20 years to an original hypothesis by Joseph Kirschvink (BS, MS '75), Caltech's Nico and Marilyn Van Wingen Professor of Geobiology, and his colleagues. For more than a century, paleontologists including Charles Darwin have debated whether the so-called Cambrian explosion—a rapid period of species diversification that began around 542 million years ago—was the equivalent of an evolutionary "big bang" of biological innovation, or just an artifact of the incomplete fossil record.

In a new study published in the December issue of the American Journal of Science, a team of researchers including Kirschvink and Ross Mitchell, a postdoctoral scholar in geology at Caltech, describes a new model showing that during the proposed Cambrian true polar wander event, most continents would have moved toward the equator instead of toward the poles.

"It's long been observed that biological diversity is highest in the tropics, where nutrients and energy tend to be abundant," says Kirschvink. "One of the side effects of true polar wander is that sea level rises near the equator but falls near the poles, so the equatorial migration of most Cambrian land masses would have enhanced diversification into previously lower-diversity environments."

Using a model they developed, the team simulated the pattern of continental migration during the Cambrian and found that their results can explain the distribution of Cambrian fossils.

"Our model provides an explanation for why the fossil record looks the way it does, with many Cambrian fossil groups on some continents but few on others," says study coauthor Tim Raub (BS, MS '02), a lecturer at the University of St. Andrews in Scotland.

"The same sea-level rise which flooded those continents that shifted to the tropics and opened new ecological niches for faster speciation also led to more fossil preservation," Mitchell says. "In contrast, the few areas that shifted to the poles became less biologically diverse and also lost rock volume to erosion following sea-level drops due to true polar wander."

The scientists say their new findings could help resolve the debate started so long ago by Darwin. If their theory is correct, the Cambrian explosion is both a true and dramatic pulse of biological innovation and an expression of preferentially preserved shells on selectively submerged continental margins capable of containing fossils.

Funding for the study was provided by the National Science Foundation.

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Caltech Biologists Identify Gene That Helps Regulate Sleep

Caltech biologists have performed the first large-scale screening in a vertebrate animal for genes that regulate sleep, and have identified a gene that when overactivated causes severe insomnia. Expression of the gene, neuromedin U (Nmu), also seems to serve as nature's stimulant—fish lacking the gene take longer to wake up in the morning and are less active during the day.

The findings improve our understanding of how sleep is regulated—a process that we know surprisingly little about despite its clear importance. In the long term, the results suggest Nmu as a potential candidate for new therapies to address sleep disorders.

A paper describing the new screening process and its results appears in the February 17, 2016, issue of the journal Neuron. David Prober, assistant professor of biology at Caltech, began the work as a postdoctoral fellow at Harvard University, and has continued the work at Caltech since 2009. The lead authors on the paper are Cindy Chiu (PhD '14), a former graduate student in Prober's lab, and Jason Rihel, who collaborated with Prober at Harvard and now has his own lab at University College London.

"Sleep is a mysterious process," says Prober. "We spend a third of our lives doing it, and every animal with a complex nervous system seems to do it, so it must be important. But we still don't understand why we do it or how it's regulated."

Genetic screens are a powerful method that can help identify the genetic basis of such behaviors. They typically involve mutating the DNA of thousands of animals, raising them, identifying any resulting physical or behavioral differences, and determining which altered gene produced each mutation. This approach works well for simple model organisms, such as fruit flies and worms, because their anatomy is relatively simple and it is easy to raise large numbers of them, but is far more difficult in vertebrates, such as rodents.

Recently, zebrafish have emerged as a valuable vertebrate model system for studying sleep. Compared to a mouse, the small, striped fish are much easier to work with. Many can be raised in a small space (a larva is about 4 millimeters long, about the same size as a fruit fly); they develop quickly, exhibiting complex behaviors, such as hunting, by the time they are five days old; and they are transparent during their embryonic and larval stages, making it simpler for researchers to track what is happening inside their brains. Like humans, zebrafish sleep for consolidated periods of time at night. Furthermore, Prober says, "anatomical and molecular similarities between zebrafish and mammalian brains suggest that the basic neural circuits regulating sleep in zebrafish are likely conserved in mammals."

Rather than mutating the DNA and testing which functions were lost, the researchers used a gain-of-function approach in the new study. Just after fertilization, when the zebrafish embryos were still single cells, the researchers injected them with a DNA molecule, called a plasmid, carrying a gene that was inserted into the genome of some of the cells in each fish. In particular, they wanted to test genes that are predicted to encode for secreted proteins—those, like neuropeptides, that cells make and then release. Many of the genes that have been identified as being involved in sleep encode neuropeptides.

Using a genetic switch called a heat-shock promoter, which turns on only when the fish are heated to about 37 degrees Celsius, the biologists were able to control when the fish expressed each inserted gene. They kept the switch on long enough for the fish to overexpress each gene, making many copies of the products. Then they used computerized video trackers to monitor the fish for several days to see which genes affected sleep.

Next, the researchers made transgenic zebrafish for each of the genes that had demonstrated strong effects on sleep in the genetic screen. That labor-intensive approach gave them zebrafish in which all cells overexpressed a particular gene in response to heat shock, providing more robust results.

In the end, the most significant change resulted from overexpression of Nmu, a gene that is also found in mammals and is expressed in a part of the brain called the hypothalamus.

"After heat shock, the fish that overexpress Nmu are much more active both during the day and at night," says Prober. "The fish almost don't sleep at all the night following the heat shock—so they display a very profound form of insomnia."

When the researchers mutated the zebrafish so that they did not have Nmu, the larvae were less active during the day. Adult fish without the gene were particularly sluggish first thing in the morning.

Like humans, zebrafish normally start to wake up at the end of the night and then become much more active when the lights turn on. "The fish without Nmu are defective in this anticipation of dawn," says Prober. "So it seems that this gene is particularly important for the transition from nighttime sleep to daytime wakefulness."

To explore how Nmu promotes wakefulness, the researchers first investigated the gene's role in a stress response pathway known as the hypothalamic-pituitary-adrenal (HPA) axis. Researchers had previously shown Nmu to be involved in arousal caused by stressful situations and hypothesized that it was involved in activating the HPA axis. However, Prober and his colleagues found that Nmu suppressed sleep to the same extent in zebrafish mutants lacking a protein called the glucocorticoid receptor, which is necessary for HPA axis signaling, as it did in fish with a functional glucocorticoid receptor, suggesting that the gene does not act through the HPA axis.

The researchers then went back to the drawing board and asked which neurons in the brain became activated as a result of Nmu overexpression. Using a technique that labels activated neurons, they saw strong activation of a handful of cells that express a gene called corticotrophin-releasing hormone (CRH) in the brainstem.

"That was surprising because CRH is the gene that initiates the HPA axis response, but the cells that do that are in the hypothalamus, a different part of the brain, and they aren't activated when we overexpress Nmu," says Prober. "It's another population of CRH cells in the brainstem that are activated by Nmu overexpression."

A low dose of a drug that blocks CRH signaling completely blocked the wake-promoting effect of Nmu overexpression in zebrafish, the researchers found, whereas a higher dose also reduced wakefulness in normal fish.

"So not only is CRH signaling required for the effects of Nmu on behavior, it's also required for normal levels of activity," explains Prober.

Several wake-promoting or sleep-promoting genes and neurons have been identified, he notes. However, scientists still do not know which are the relevant ones for causing sleep disorders in humans. "Our study suggests that Nmu could be a good gene to look into."

Additional Caltech authors on the paper, "A Zebrafish Genetic Screen Identifies Neuromedin U as a Regulator of Sleep/Wake States," are Daniel A. Lee, Chanpreet Singh, Eric A. Mosser, Shijia Chen, Viveca Sapin, Uyen Pham, Jae Engle, Brett J. Niles, Christin J. Montz, and Sridhara Chakravarthy. Steven Zimmerman and Alexander F. Schier are additional authors from Harvard University. Kourosh Salehi-Ashtiani and Marc Vidal are authors from Harvard Medical School. The work was supported by grants from the National Institutes of Health, the European Research Council, University College London, the High-Tech Fund of the Dana Farber Cancer Institute, the Ellison Foundation, the Edward Mallinckrodt, Jr. Foundation, the Rita Allen Foundation, and the Brain and Behavior Research Foundation.

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A Gene That Helps Regulate Sleep
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By conducting a genetic screen in zebrafish, biologists have identified a gene that seems to serve as nature's stimulant.

Gravitational Waves Detected 100 Years After Einstein’s Prediction

LIGO opens new window on the universe with observation of gravitational waves from colliding black holes

For the first time, scientists have observed ripples in the fabric of spacetime called gravitational waves, arriving at the earth from a cataclysmic event in the distant universe. This confirms a major prediction of Albert Einstein's 1915 general theory of relativity and opens an unprecedented new window onto the cosmos.

Gravitational waves carry information about their dramatic origins and about the nature of gravity that cannot otherwise be obtained. Physicists have concluded that the detected gravitational waves were produced during the final fraction of a second of the merger of two black holes to produce a single, more massive spinning black hole. This collision of two black holes had been predicted but never observed.

The gravitational waves were detected on September 14, 2015 at 5:51 a.m. Eastern Daylight Time (09:51 UTC) by both of the twin Laser Interferometer Gravitational-wave Observatory (LIGO) detectors, located in Livingston, Louisiana, and Hanford, Washington, USA. The LIGO Observatories are funded by the National Science Foundation (NSF), and were conceived, built, and are operated by Caltech and MIT. The discovery, accepted for publication in the journal Physical Review Letters, was made by the LIGO Scientific Collaboration (which includes the GEO Collaboration and the Australian Consortium for Interferometric Gravitational Astronomy) and the Virgo Collaboration using data from the two LIGO detectors.

Based on the observed signals, LIGO scientists estimate that the black holes for this event were about 29 and 36 times the mass of the sun, and the event took place 1.3 billion years ago. About 3 times the mass of the sun was converted into gravitational waves in a fraction of a second—with a peak power output about 50 times that of the whole visible universe. By looking at the time of arrival of the signals—the detector in Livingston recorded the event 7 milliseconds before the detector in Hanford—scientists can say that the source was located in the Southern Hemisphere.

According to general relativity, a pair of black holes orbiting around each other lose energy through the emission of gravitational waves, causing them to gradually approach each other over billions of years, and then much more quickly in the final minutes. During the final fraction of a second, the two black holes collide into each other at nearly one-half the speed of light and form a single more massive black hole, converting a portion of the combined black holes' mass to energy, according to Einstein's formula E=mc2. This energy is emitted as a final strong burst of gravitational waves. It is these gravitational waves that LIGO has observed.

LIGO was originally proposed as a means of detecting these gravitational waves in the 1980s by Rainer Weiss, professor of physics, emeritus, from MIT; Kip Thorne, Caltech's Richard P. Feynman Professor of Theoretical Physics, emeritus; and Ronald Drever, professor of physics, emeritus, also from Caltech.

"With this discovery, we humans are embarking on a marvelous new quest: the quest to explore the warped side of the universe—objects and phenomena that are made from warped spacetime. Colliding black holes and gravitational waves are our first beautiful examples," says Thorne.

 "The description of this observation is beautifully described in the Einstein theory of general relativity formulated 100 years ago and comprises the first test of the theory in strong gravitation. It would have been wonderful to watch Einstein's face had we been able to tell him," says Weiss.

"Caltech thrives on posing fundamental questions and inventing new instruments to answer them," says Caltech president Thomas Rosenbaum, the Sonja and William Davidow Presidential Chair and professor of physics. "LIGO represents an exhilarating example of how this approach can transform our knowledge of the universe. We are proud to partner with NSF and MIT and our other scientific collaborators to lead this decades-long effort."

"Our observation of gravitational waves accomplishes an ambitious goal set out over five decades ago to directly detect this elusive phenomenon and better understand the universe, and, fittingly, fulfills Einstein's legacy on the 100th anniversary of his general theory of relativity," says Caltech's David H. Reitze, executive director of the LIGO Laboratory.

"This discovery is just the beginning," says Fiona Harrison, the Benjamin M. Rosen Professor of Physics and holder of the Kent and Joyce Kresa Leadership Chair of the Division of Physics, Mathematics and Astronomy. "Over the next years, LIGO will be putting general relativity to its most stringent tests ever, it will be discovering new sources of gravitational waves, and we will be using telescopes on the ground and in space to search for light emitted by these catastrophic events."

The existence of gravitational waves was first demonstrated in the 1970s and 80s by Joseph Taylor, Jr., and colleagues. Taylor and Russell Hulse discovered in 1974 a binary system composed of a pulsar in orbit around a neutron star. Taylor and Joel M. Weisberg in 1982 found that the orbit of the pulsar was slowly shrinking over time because of the release of energy in the form of gravitational waves. For discovering the pulsar and showing that it would make possible this particular gravitational wave measurement, Hulse and Taylor were awarded the Nobel Prize in Physics in 1993.

The new LIGO discovery is the first observation of gravitational waves themselves, made by measuring the tiny disturbances the waves make to space and time as they pass through the earth.

LIGO research is carried out by the LIGO Scientific Collaboration (LSC), a group of more than 1000 scientists from universities around the United States and in 14 other countries. More than 90 universities and research institutes in the LSC develop detector technology and analyze data; approximately 250 students are strong contributing members of the collaboration. The LSC detector network includes the LIGO interferometers and the GEO600 detector. The GEO team includes scientists at the Max Planck Institute for Gravitational Physics (Albert Einstein Institute, AEI), Leibniz Universität Hannover, along with partners at the University of Glasgow, Cardiff University, the University of Birmingham, other universities in the United Kingdom, and the University of the Balearic Islands in Spain.

"This detection is the beginning of a new era: The field of gravitational wave astronomy is now a reality," says Gabriela González, LSC spokesperson and professor of physics and astronomy at Louisiana State University.

The discovery was made possible by the enhanced capabilities of Advanced LIGO, a major upgrade that increases the sensitivity of the instruments compared to the first generation LIGO detectors, enabling a large increase in the volume of the universe probed—and the discovery of gravitational waves during its first observation run. The US National Science Foundation leads in financial support for Advanced LIGO. Funding organizations in Germany (Max Planck Society), the U.K. (Science and Technology Facilities Council, STFC) and Australia (Australian Research Council) also have made significant commitments to the project. Several of the key technologies that made Advanced LIGO so much more sensitive have been developed and tested by the German UK GEO collaboration. Significant computer resources have been contributed by the AEI Hannover Atlas Cluster, the LIGO Laboratory, Syracuse University, and the University of Wisconsin-Milwaukee. Several universities designed, built, and tested key components for Advanced LIGO: The Australian National University, the University of Adelaide, the University of Florida, Stanford University, Columbia University of New York, and Louisiana State University.

"In 1992, when LIGO's initial funding was approved, it represented the biggest investment the NSF had ever made," says France Córdova, NSF director. "It was a big risk. But the National Science Foundation is the agency that takes these kinds of risks. We support fundamental science and engineering at a point in the road to discovery where that path is anything but clear. We fund trailblazers. It's why the U.S. continues to be a global leader in advancing knowledge."

"The Advanced LIGO detectors are a tour de force of science and technology, made possible by a truly exceptional international team of technicians, engineers, and scientists," says David Shoemaker of MIT, the project leader for Advanced LIGO. "We are very proud that we finished this NSF-funded project on time and on budget, and delighted Advanced LIGO delivered its groundbreaking detection so quickly."

At each observatory, the two-and-a-half-mile (4-km) long L-shaped LIGO interferometer uses laser light split into two beams that travel back and forth down the arms (four-foot diameter tubes kept under a near-perfect vacuum). The beams are used to monitor the distance between mirrors precisely positioned at the ends of the arms. According to Einstein's theory, the distance between the mirrors will change by an infinitesimal amount when a gravitational wave passes by the detector. A change in the lengths of the arms smaller than one-ten-thousandth the diameter of a proton (10-19 meter) can be detected.

Independent and widely separated observatories are necessary to determine the direction of the event causing the gravitational waves, and also to verify that the signals come from space and are not from some other local phenomenon.

A network of detectors will significantly help to localize the sources. The Virgo detector will be the first to join later this year.

The LIGO Laboratory also is working closely with scientists in India at the Inter-University Centre for Astronomy and Astrophysics, the Raja Ramanna Centre for Advanced Technology, and the Institute for Plasma to establish a third Advanced LIGO detector on the Indian subcontinent. Awaiting approval by the government of India, it could be operational early in the next decade. The additional detector will greatly improve the ability of the global detector network to localize gravitational-wave sources.

Virgo research is carried out by the Virgo Collaboration, consisting of more than 250 physicists and engineers belonging to 19 different European research groups: 6 from Centre National de la Recherche Scientifique (CNRS) in France; 8 from the Istituto Nazionale di Fisica Nucleare (INFN) in Italy; 2 in The Netherlands with Nikhef; the Wigner RCP in Hungary; the POLGRAW group in Poland, and the European Gravitational Observatory (EGO), the laboratory hosting the Virgo detector near Pisa in Italy.

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Einstein's Prediction Confirmed by LIGO
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Social Hormone Promotes Cooperation in Risky Situations

A hormone implicated in monogamy and aggression in animals also promotes trust and cooperation in humans in risky situations, Caltech researchers say.

The findings, published the week of February 8 in the online edition of the Proceedings of the National Academy of Sciences, could prove useful for helping groups cooperate beneficially.

Research in rodents shows the hormone arginine vasopressin (AVP) promotes monogamous pair bonding and parental behavior, but also aggression in males. "Part of the dark side of monogamy is that an AVP-pumped-up male is more likely to behave aggressively toward intruders," says study coauthor Colin Camerer, the Robert Kirby Professor of Behavioral Economics at Caltech.

In the new study, Camerer and his team tested the hypothesis that AVP might also play a role in social bonding in people and could help explain our species' cooperative tendencies. "One of the reasons humans rule the world rather than apes is that we do things that require a great deal of trust. We cooperate in large-scale groups," Camerer says. "Where does that come from? Is it something like pair bonding but just scaled up? And if it is, what role does AVP play?"

To investigate these questions, Camerer and his colleagues administered a nasal spray containing AVP or a hormone-free nasal spray (a placebo) to 59 male volunteers, aged 19 to 32 years old. Pairs of subjects then used computers to play a so-called assurance game in which they had to choose whether or not to cooperate with another player; "assurance" comes from the fact that subjects will take a risky action if they are sufficiently assured that others will, too. When they cooperated, both players received more points than they would have if they did not mutually cooperate. If one player chose not to cooperate but his partner made the opposite decision, the non-cooperative player received an intermediate payoff whereas the cooperative player received nothing.

"The game is designed to mimic situations in which people are willing to help, but only if everyone else helps too," Camerer says. "Think of pitching in on a team project, or of a group of soldiers rushing the enemy. If a critical mass cooperates, then everyone else should go along. Thus it is in your best interest to help only if enough others do."

To help ensure the players were engaged, the points they accumulated were converted into actual money at the end of the game (usually around $20).

The experiment showed that players who received AVP before the game were significantly more likely to cooperate than those who received the placebo. "By targeting a specific hormonal system in the human brain, we could manipulate people's willingness to cooperate and help them do better," says Gideon Nave, a graduate student in Camerer's lab and a coauthor on the study.

Using control experiments, the researchers were also able to rule out other explanations for why the subjects were cooperating. For example, one possibility is that AVP was increasing the subjects' appetite for risks. Alternatively, the administered hormone might be amplifying their altruistic tendencies, so that they just wanted to help other people regardless of the risk to themselves.

"We found that when we asked them, 'Do you want to just give some money to this stranger?' they don't do it," Camerer says. "So AVP seems to be quite specialized to this particular type of risky cooperation."

To better understand the neural mechanism underlying AVP's effect on risky cooperation, the researchers conducted the same experiment but this time had subjects—a separate group of 34 men—play the game while their brains were being imaged using a functional magnetic resonance imaging (fMRI) scanner. The scans indicated that after AVP administration, a part of the brain's reward system known as the ventral pallidum—a region that is known to have an abundance of AVP receptors—showed a change in neural activity when the players decided to cooperate.

"That was very encouraging, because it showed that the hormone is activating a part of the brain that is known to be rich in AVP receptors," Camerer says.

Could the discovery that AVP increases the likelihood of risky cooperation have practical applications and be used, for example, to engender trust and foster cooperation in groups? Perhaps.

"You could imagine a high-stakes situation, such as a military operation, in which people have to trust each other to all do something difficult and it fails if anyone chickens out," Camerer says. "In that case, you might want to administer AVP to help ensure that everyone is cooperative."

In addition to Camerer and Nave, other coauthors on the paper, "Vasopressin increases human risky cooperative behavior," include Claudia Brunnlieb, Stephan Schosser, and Bodo Vogt of the University of Magdeburg and Thomas Münte and Marcus Heldmann at the University of Lübeck in Germany. The research was funded by a special grant of the Center for Behavioral Brain Sciences and by the Gordon and Betty Moore Foundation. 

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Social Hormone Promotes Cooperation
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A hormone implicated in monogamy and aggression in animals also promotes trust and cooperation in humans in risky situations, Caltech researchers say.

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