How an RNA Gene Silences a Whole Chromosome

Researchers at Caltech have discovered how an abundant class of RNA genes, called long non-coding RNAs (lncRNAs, pronounced link RNAs) can regulate key genes. By studying an important lncRNA, called Xist, the scientists identified how this RNA gathers a group of proteins and ultimately prevents women from having an extra functional X-chromosome—a condition in female embryos that leads to death in early development. These findings mark the first time that researchers have uncovered the detailed mechanism of action for lncRNA genes.

"For years, we thought about genes as just DNA sequences that encode proteins, but those genes only make up about 1 percent of the genome. Mammalian genomes also encode many thousands of lncRNAs," says Assistant Professor of Biology Mitch Guttman, who led the study published online in the April 27 issue of the journal Nature. These lncRNAs such as Xist play a structural role, acting to scaffold—or bring together and organize—the key proteins involved in cellular and molecular processes, such as gene expression and stem cell differentiation.

Guttman, who helped to discover an entire class of lncRNAs as a graduate student at MIT in 2009, says that although most of these genes encoded in our genomes have only recently been appreciated, there are several specific examples of lncRNA genes that have been known for decades. One well-studied example is Xist, which is important for a process called X chromosome inactivation.

All females are born with two X chromosomes in every cell, one inherited from their mother and one from their father. In contrast, males only contain one X chromosome (along with a Y chromosome). However, like males, females only need one copy of each X-chromosome gene—having two copies is an abnormality that will lead to death early during development. The genome skirts these problems by essentially "turning off" one X chromosome in every cell.

Previous research showed that Xist is essential to this process and does this by somehow preventing transcription, the initial step of the expression of genes on the X chromosome. However, because Xist is not a traditional protein-coding gene, until now researchers have had trouble figuring out exactly how Xist stops transcription and shuts down an entire chromosome.

"To start to make sense of what makes lncRNAs special and how they can control all of these different cellular processes, we need to be able to understand the mechanism of how any lncRNA gene can work. Because Xist is such an important molecule and because so much is known about what it does, it seemed like a great system to try to dissect the mechanisms of how it and other lncRNAs work," Guttman says.

lncRNAs are known to corral and organize the proteins that are necessary for cellular processes, so Guttman and his colleagues began their study of the function of Xist by first developing a technique to find out what proteins it naturally interacts with in the cell. With a new method, called RNA antisense purification with mass spectrometry (RAP-MS), the researchers extracted and purified Xist lncRNA molecules, as well as the proteins that directly interact with Xist, from mouse embryonic stem cells. Then, collaborators at the Proteome Exploration Laboratory at Caltech applied a technique called quantitative mass spectrometry to identify those interacting proteins.

"RNA usually only obeys one rule: binding to proteins. RAP-MS is like a molecular microscope into identifying RNA-protein interactions," says John Rinn, associate professor of stem cell and regenerative biology at Harvard University, who was not involved in the study. "RAP-MS will provide critically needed insights into how lncRNAs function to organize proteins and in turn regulate gene expression."

Applying this to Xist uncovered 10 specific proteins that interact with Xist. Of these, three—SAF-A (Scaffold attachment factor-A), LBR (Lamin B Receptor), and SHARP (SMRT and HDAC associated repressor protein)—are essential for X chromosome inactivation. "Before this experiment," Guttman says, "no one knew a single protein that was required by Xist for silencing transcription on the X chromosome, but with this method we immediately found three that are essential. If you lose any one of them, Xist doesn't work—it will not silence the X chromosome during development."

The new findings provide the first detailed picture of how lncRNAs work within a cellular process. Through further analysis, the researchers found that these three proteins performed three distinct, but essential, roles. SAF-A helps to tether Xist and all of its hitchhiking proteins to the DNA of the X chromosome, at which point LBR remodels the chromosome so that it is less likely to be expressed. The actual "silencing," Guttman and his colleagues discovered, is done by the third protein of the trio: SHARP.

To produce functional proteins from the DNA (genes) of a chromosome, the genes must first be transcribed into RNA by an enzyme called RNA polymerase II. Guttman and his team found that SHARP leads to the exclusion of polymerase from the DNA, thus preventing transcription and gene expression.

This information soon may have clinical applications. The Xist lncRNA silences the X chromosome simply because it is located on the X chromosome. However, previous studies have demonstrated that this RNA and its silencing machinery can be used to inactivate other chromosomes—for example, the third copy of chromosome 21 that is present in individuals with Downs' syndrome.

"We are starting to pick apart how lncRNAs work. We now know, for example, how Xist localizes to sites on X, how it silences transcription, and how it can change DNA structure," Guttman says. "One of the things that is really exciting for me is that we can potentially leverage the principles used by lncRNAs, move them around in the genome, and use them as therapeutic agents to target specific defective pathways in disease."

"But I think the real reason why this is so important for our field and even beyond is because this is a different type of regulation than we've seen before in the cell—it is a vast world that we previously knew nothing about," he adds.

This work was published in a recent paper titled: "The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3." The co-first authors of the paper are Caltech postdoctoral scholar Colleen A. McHugh and graduate student Chun-Kan Chen. Other coauthors from Caltech are Amy Chow, Christine F. Surka, Christina Tran, Mario Blanco, Christina Burghard, Annie Moradian, Alexander A. Shishkin, Julia Su, Michael J. Sweredoski, and Sonja Hess from the Proteome Exploration Laboratory. Additional authors include Amy Pandya-Jones and Kathrin Plath from UCLA and Patrick McDonel from MIT.

The study was supported by funding from the Gordon and Betty Moore Foundation, the Beckman Institute, the National Institutes of Health, the Rose Hills Foundation, the Edward Mallinckrodt Foundation, the Sontag Foundation, and the Searle Scholars Program.

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Oka Awarded Grant for "Exceptional Young Scientists"

Yuki Oka, an assistant professor of biology, has been named a 2015 Searle Scholar. The Searle Scholars Program provides grants to young faculty to support research in the biomedical sciences and chemistry. Fifteen scholars are named annually, each receiving $100,000 per year for three years.

"I'm very excited and honored by this award," says Oka, who studies how the brain compiles both internal and external sensory information in order to maintain homeostasis, or internal stability of the body. In particular, Oka's group studies how the brain controls the feeling of thirst, and how that feeling drives us to drink water. There are multiple processes involved in regulating thirst in the brain.  

"Our research group aims to understand how these thirst signals are processed in the brain and how they ultimately drive specific behavioral outputs," Oka says. "We recently identified two distinct neural populations controlling drinking behavior in two opposite directions: driving and suppressing thirst." By manipulating these neural populations in animals, the group found that it could artificially create or suppress the desire to drink water.

Before joining the faculty at Caltech, Oka was a postdoctoral scholar at Columbia University. He received his PhD from the University of Tokyo. He is the 18th current Caltech faculty member to be named a Searle Scholar.

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Microbes Help Produce Serotonin in Gut

Although serotonin is well known as a brain neurotransmitter, it is estimated that 90 percent of the body's serotonin is made in the digestive tract. In fact, altered levels of this peripheral serotonin have been linked to diseases such as irritable bowel syndrome, cardiovascular disease, and osteoporosis. New research at Caltech, published in the April 9 issue of the journal Cell, shows that certain bacteria in the gut are important for the production of peripheral serotonin.

"More and more studies are showing that mice or other model organisms with changes in their gut microbes exhibit altered behaviors," explains Elaine Hsiao, research assistant professor of biology and biological engineering and senior author of the study. "We are interested in how microbes communicate with the nervous system. To start, we explored the idea that normal gut microbes could influence levels of neurotransmitters in their hosts."

Peripheral serotonin is produced in the digestive tract by enterochromaffin (EC) cells and also by particular types of immune cells and neurons. Hsiao and her colleagues first wanted to know if gut microbes have any effect on serotonin production in the gut and, if so, in which types of cells. They began by measuring peripheral serotonin levels in mice with normal populations of gut bacteria and also in germ-free mice that lack these resident microbes.

The researchers found that the EC cells from germ-free mice produced approximately 60 percent less serotonin than did their peers with conventional bacterial colonies. When these germ-free mice were recolonized with normal gut microbes, the serotonin levels went back up—showing that the deficit in serotonin can be reversed.

"EC cells are rich sources of serotonin in the gut. What we saw in this experiment is that they appear to depend on microbes to make serotonin—or at least a large portion of it," says Jessica Yano, first author on the paper and a research technician working with Hsiao.

The researchers next wanted to find out whether specific species of bacteria, out of the diverse pool of microbes that inhabit the gut, are interacting with EC cells to make serotonin.

After testing several different single species and groups of known gut microbes, Yano, Hsiao, and colleagues observed that one condition—the presence of a group of approximately 20 species of spore-forming bacteria—elevated serotonin levels in germ-free mice. The mice treated with this group also showed an increase in gastrointestinal motility compared to their germ-free counterparts, and changes in the activation of blood platelets, which are known to use serotonin to promote clotting.

Wanting to home in on mechanisms that could be involved in this interesting collaboration between microbe and host, the researchers began looking for molecules that might be key. They identified several particular metabolites—products of the microbes' metabolism—that were regulated by spore-forming bacteria and that elevated serotonin from EC cells in culture. Furthermore, increasing these metabolites in germ-free mice increased their serotonin levels.

Previous work in the field indicated that some bacteria can make serotonin all by themselves. However, this new study suggests that much of the body's serotonin relies on particular bacteria that interact with the host to produce serotonin, says Yano. "Our work demonstrates that microbes normally present in the gut stimulate host intestinal cells to produce serotonin," she explains.

"While the connections between the microbiome and the immune and metabolic systems are well appreciated, research into the role gut microbes play in shaping the nervous system is an exciting frontier in the biological sciences," says Sarkis K. Mazmanian, Luis B. and Nelly Soux Professor of Microbiology and a coauthor on the study. "This work elegantly extends previous seminal research from Caltech in this emerging field".

Additional coauthor Rustem Ismagilov, the Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering, adds, "This work illustrates both the richness of chemical interactions between the hosts and their microbial communities, and Dr. Hsiao's scientific breadth and acumen in leading this work."

Serotonin is important for many aspects of human health, but Hsiao cautions that much more research is needed before any of these findings can be translated to the clinic.

"We identified a group of bacteria that, aside from increasing serotonin, likely has other effects yet to be explored," she says. "Also, there are conditions where an excess of peripheral serotonin appears to be detrimental."

Although this study was limited to serotonin in the gut, Hsiao and her team are now investigating how this mechanism might also be important for the developing brain. "Serotonin is an important neurotransmitter and hormone that is involved in a variety of biological processes. The finding that gut microbes modulate serotonin levels raises the interesting prospect of using them to drive changes in biology," says Hsiao.

The work was published in an article titled "Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis." In addition to Hsiao, Yano, Mazmanian, and Ismagilov, other Caltech coauthors include undergraduates Kristie Yu, Gauri Shastri, and Phoebe Ann; graduate student Gregory Donaldson; postdoctoral scholar Liang Ma. Additional coauthor Cathryn Nagler is from the University of Chicago.

This work was funded by an NIH Director's Early Independence Award and a Caltech Center for Environmental Microbial Interactions Award, both to Hsiao. The study was also supported by NSF, NIDDK, and NIMH grants to Mazmanian, NSF EFRI and NHGRI grants to Ismagilov, and grants from the NIAID and Food Allergy Research and Education and University of Chicago Digestive Diseases Center Core to Nagler.

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A Molecular Arms Race: The Immune System Versus HIV

Watson Lecture Preview

It is now more than 30 years after the first AIDS epidemic, and an effective vaccine against HIV does not yet exist—partly because the virus quickly mutates to evade the vaccine's antibodies. On Wednesday, April 1, at 8 p.m. in Caltech's Beckman Auditorium, Pamela J. Bjorkman, Caltech's Max Delbrück Professor of Biology and an investigator with the Howard Hughes Medical Institute, will describe ways to neutralize that mutational advantage. Admission is free.

 

What do you do?

We are structural biologists who use various imaging techniques to look at biological macromolecules and assemblies, sometimes in purified forms and sometimes in tissues. For example, we study HIV proteins alone, on viruses, and on viruses in tissues during an infection. Utilizing high-resolution structures of individual proteins, we are trying to apply our knowledge of the chemistry of protein-protein interactions to understanding what makes some antibodies produced by HIV-infected people good at neutralizing viruses and other antibodies less effective. We then try to reengineer good antibodies to make them even better in hopes that they could be used therapeutically to prevent or treat HIV infection.

 

What's the neatest thing about what you do?

Using imaging techniques such as X-ray crystallography and electron microscopy, we can visualize structures in three dimensions, sometimes even localizing all of the atoms in a protein structure. This feels a bit like spying on nature—forcing her to reveal secrets that we can hopefully use to combat HIV/AIDS.

 

How did you get into this line of work?

I was hooked after taking chemistry in high school. I knew then that I wanted to use chemistry to understand biology. I became interested in HIV about 10 years ago when I started teaching the Caltech freshman biology class and used HIV as a model system to understand basic principles of biology, especially evolution. HIV is an amazing example of successful evolution against which the human immune system loses, but I hope that we can win the war against HIV through a fundamental understanding of how it works.

 

Named for the late Caltech professor Earnest C. Watson, who founded the series in 1922, the Watson Lectures present Caltech and JPL researchers describing their work to the public. Many past Watson Lectures are available online at Caltech's iTunes U site.

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Research Suggests Brain's Melatonin May Trigger Sleep

If you walk into your local drug store and ask for a supplement to help you sleep, you might be directed to a bottle labeled "melatonin." The hormone supplement's use as a sleep aid is supported by anecdotal evidence and even some reputable research studies. However, our bodies also make melatonin naturally, and until a recent Caltech study using zebrafish, no one knew how—or even if—this melatonin contributed to our natural sleep. The new work suggests that even in the absence of a supplement, naturally occurring melatonin may help us fall and stay asleep.

The study was published online in the March 5 issue of the journal Neuron.

"When we first tell people that we're testing whether melatonin is involved in sleep, the response is often, 'Don't we already know that?'" says Assistant Professor of Biology David Prober. "This is a reasonable response based on articles in newspapers and melatonin products available on the Internet. However, while some scientific studies show that supplemental melatonin can help to promote sleep, many studies failed to observe this, so the effectiveness of melatonin supplements is controversial. More importantly, these studies don't tell you anything about what naturally occurring melatonin normally does in the body."

There are several factors at play when you are starting to feel tired. Sleep is thought to be regulated by two mechanisms: a homeostatic mechanism, which responds to the body's internal cues for sleep, and a circadian mechanism that responds to external cues such as darkness and light, signaling appropriate times for sleep and wakefulness.

For years, researchers have known that melatonin production is regulated by the circadian clock, and that animals produce more of the hormone at night than they do during the day. However, this fact alone is not enough to prove that melatonin promotes sleep. For example, although nocturnal animals sleep during the day and are active at night, they also produce the most melatonin at night.

In the hopes of determining, once and for all, what role the hormone actually plays in sleep, Prober and his team at Caltech designed an experiment using the larvae of zebrafish, an organism commonly used in research studies because of its small size and well-characterized genome. Like humans, zebrafish are also diurnal—awake during the day and asleep at night—and produce melatonin at night.

But how exactly can you tell if a young zebrafish has fallen asleep? There are behavioral criteria—including how long a zebrafish takes to respond to a stimulus, like a knock on the tank, for example. "Based on these criteria, we found that if the zebrafish larvae don't move for one or more minutes, they are in a sleep-like state," Prober says.

To test the effect of naturally occurring melatonin on sleep, the researchers first compared the sleep patterns of normal, or "wild-type," zebrafish larvae to those of zebrafish larvae that are unable to produce the hormone because of a mutation in a gene called aanat2. They found that fish with the mutation slept only half as long as normal fish. And although a normal zebrafish begins to fall asleep about 10 minutes after "lights out"—about the same amount of time it takes a human to fall asleep—it took the aanat2 mutant fish about twice as long.

"This result was surprising because it suggests that almost half of the sleep that the larvae are getting at night is due to the effects of melatonin," Prober says. "That suggests that melatonin normally plays an important role in sleep and that you need this natural melatonin both to fall asleep and to stay asleep."

In both humans and zebrafish, melatonin is produced in a part of the brain called the pineal gland. To confirm that the mutation-induced reduction in sleep was actually due to a lack of melatonin, the researchers next used a drug to specifically kill the cells of the pineal gland, thus halting the hormone's production. The drug-treated fish showed the same reduction in sleep as fish with mutated aanat2. When the drug treatment stopped, allowing pineal gland cells to regenerate, the fish returned to a normal sleep pattern.

Sleep patterns, like many other biological and behavioral processes, are known to be regulated by the circadian clock. In an organism, the circadian clock aligns these processes with daily changes in the environment, such as daylight and darkness at night. However, while a great deal is known about how the circadian clock works, it was not known how the clock regulates sleep. Because the researchers had determined that melatonin is involved in promoting natural sleep, they next asked whether melatonin mediates the circadian regulation of sleep.

They first raised both wild-type and aanat2 mutant zebrafish larvae in a normal light/dark cycle—14 hours of light followed by 10 hours of darkness—to entrain their circadian clocks. Then, when the larvae were 5 days old, they switched both populations to an environment of constant darkness. In this "free running" condition, the circadian clock continues to function in the absence of daily light and dark signals from the environment. As expected, the wild-type fish maintained their regular circadian sleep cycle. The melatonin-lacking aanat2 mutants, however, showed no cyclical sleep patterns.  

"This was really surprising," says Prober. "For years, people have been looking in rodents for a factor that's required for the circadian regulation of sleep and have found a few other candidate molecules that, like melatonin, are regulated by the circadian clock and can induce sleep when given as supplements. However, mutants that lack these factors had normal circadian sleep cycles," says Prober. "One thought was that maybe all of these molecules work together and that you'd have to make mutations in multiple genes to see an effect. But we found that eliminating one molecule, melatonin, is the whole show. It's one of those rare and surprisingly clear results."

After finding that melatonin is necessary for the circadian regulation of sleep, Prober next wanted to ask how it does this. To find out, Prober and his colleagues looked to a neuromodulator called adenosine—part of the homeostatic mechanism that promotes sleep. As an animal expends energy throughout the day, adenosine accumulates in the brain causing the animal to feel more and more tired—a pressure that is relieved through sleep.

The researchers treated both wild-type and melatonin-deficient aanat2 mutant fish with drugs that activate adenosine signaling. They found that although the drugs had no effect on the wild-type fish, they restored normal sleep amounts in aanat2 mutants. This result suggests that melatonin may be promoting sleep, in part, by turning on adenosine—providing a long sought-after link between the homeostatic and circadian processes that regulate sleep.

Prober and his colleagues hypothesize that the circadian clock drives the production of melatonin, which then promotes sleep through yet-to-be-determined mechanisms while also stimulating adenosine production, thus promoting sleep through the homeostatic pathway. Although more experiments are needed to confirm this model, Prober says that the preliminary results may offer insights about human sleep as well.

"Zebrafish are vertebrates and their brain is structurally similar to ours. All of the markers that we and others have tested are expressed in the same regions of the zebrafish brain as in the mammalian brain," he says. "Zebrafish sleep and human sleep are likely different in some ways, but all of our drug and genetic data indicate that the same factors—working through the same mechanisms—have similar effects on sleep in zebrafish and mammals. "

Prober's work with the circadian regulation of sleep follows in the conceptual—and physical—footsteps of late Caltech geneticist Seymour Benzer, who founded genetic studies of the circadian clock. In experiments in fruit flies, Benzer and his graduate student, the late Ronald Konopka (PhD '72), discovered the first circadian-rhythm mutants. Benzer passed away in 2007, and when Prober came to Caltech in 2009, he was offered Benzer's former office and lab space. "Seymour Benzer's work in fruit flies launched the beginning of our understanding of the molecular circadian clock," Prober says, "so it's really special to be in this space, and it's gratifying that we're taking the next step based on his work."

The results of Prober's study are published in the journal Neuron in an article titled, "Melatonin is required for the circadian regulation of sleep." Other Caltech coauthors on the paper are graduate student Avni Gandhi and postdoctoral scholars Eric Mosser and Grigorios Oikonomou. This work was funded by grants from the National Institutes of Health, the Mallinckrodt Foundation, the Rita Allen Foundation, the Brain and Behavior Research Foundation as well as a Della Martin Postdoctoral Fellowship to Mosser.

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Feeling Sleepy? Might be the Melatonin
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Fighting a Worm with Its Own Genome

Tiny parasitic hookworms infect nearly half a billion people worldwide—almost exclusively in developing countries—causing health problems ranging from gastrointestinal issues to cognitive impairment and stunted growth in children. By sequencing and analyzing the genome of one particular hookworm species, Caltech researchers have uncovered new information that could aid the fight against these parasites.  

The results of their work were published online in the March 2 issue of the journal Nature Genetics.

"Hookworms infect a huge percentage of the human population. Getting clean water and sanitation to the most affected regions would help to ameliorate hookworms and a number of other parasites, but since these are big, complicated challenges that are difficult to address, we need to also be working on drugs to treat them," says study lead Paul Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and a Howard Hughes Medical Institute investigator.

Medicines have been developed to treat hookworm infections, but the parasites have begun to develop resistance to these drugs. As part of the search for effective new drugs, Sternberg and his colleagues investigated the genome of a hookworm species known as Ancylostoma ceylanicum. Other hookworm species cause more disease among humans, but A. ceylanicum piqued the interest of the researchers because it also infects some species of rodents that are commonly used for research. This means that the researchers can easily study the parasite's entire infection process inside the laboratory.

The team began by sequencing all 313 million nucleotides of the A. ceylanicum genome using the next-generation sequencing capabilities of the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech. In next-generation sequencing, a large amount of DNA—such as a genome—is first reproduced as many very short sequences. Then, computer programs match up common sequences in the short strands to piece them into much longer strands.

"Assembling the short sequences correctly can be a relatively difficult analysis to carry out, but we have experience sequencing worm genomes in this way, so we are quite successful," says Igor Antoshechkin, director of the Jacobs Laboratory. 

Their sequencing results revealed that although the A. ceylanicum genome is only about 10 percent of the size of the human genome, it actually encodes at least 30 percent more genes—about 30,000 in total, compared to approximately 20,000-23,000 in the human genome. However, of these 30,000 genes, the essential genes that are turned on specifically when the parasite is wreaking havoc on its host are the most relevant to the development of potential drugs to fight the worm.

Sternberg and his colleagues wanted to learn more about those active genes, so they looked not to DNA but to RNA—the genetic material that is generated (or transcribed) from the DNA template of active genes and from which proteins are made. Specifically, they examined the RNA generated in an A. ceylanicum worm during infection. Using this RNA, the team found more than 900 genes that are turned on only when the worm infects its host—including 90 genes that belong to a never-before-characterized family of proteins called activation-associated secreted protein related genes, or ASPRs.

"If you go back and look at other parasitic worms, you notice that they have these ASPRs as well," Sternberg says. "So basically we found this new family of proteins that are unique to parasitic worms, and they are related to this early infection process." Since the worm secretes these ASPR proteins early in the infection, the researchers think that these proteins might block the host's initial immune response—preventing the host's blood from clotting and ensuring a free-flowing food source for the blood-sucking parasite.

If ASPRs are necessary for this parasite to invade the host, then a drug that targets and destroys the proteins could one day be used to fight the parasite. Unfortunately, however, it is probably not that simple, Sternberg says.

"If we have 90 of these ASPRs, it might be that a drug would get rid of just a few of them and stop the infection, but maybe you'd have to get rid of all 90 of them for it to work. And that's a problem," he says. "It's going to take a lot more careful study to understand the functions of these ASPRs so we can target the ones that are key regulatory molecules."

Drugs that target ASPRs might one day be used to treat these parasitic infections, but these proteins also hold the potential for anti-A. ceylanicum vaccines—which would prevent these parasites from infecting a host in the first place, Sternberg adds. For example, if a person were injected with an ASPR protein vaccine before travelling to an infection-prone region, their immune system might be more prepared to successfully fend off an infection.

"A parasitic infection is a balance between the parasites trying to suppress the immune system and the host trying to attack the parasite," says Sternberg. "And we hope that by analyzing the genome, we can uncover clues that might help us alter that balance in favor of the host."

These findings were published in a paper titled, "The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families." In addition to Sternberg and Antoshechkin, other coauthors include Erich M. Schwarz of Cornell University; and Yan Hu, Melanie Miller, and Raffi V. Aroian from UC San Diego. Sternberg's work was funded by the National Institutes of Health and the Howard Hughes Medical Institute.

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Genetically Engineered Antibodies Show Enhanced HIV-Fighting Abilities

Capitalizing on a new insight into HIV's strategy for evading antibodies—proteins produced by the immune system to identify and wipe out invading objects such as viruses—Caltech researchers have developed antibody-based molecules that are more than 100 times better than our bodies' own defenses at binding to and neutralizing HIV, when tested in vitro. The work suggests a novel approach that could be used to engineer more effective HIV-fighting drugs.

"Based on the work that we have done, we now think we know how to make a really potent therapeutic that would not only work at relatively low concentrations but would also force the virus to mutate along pathways that would make it less fit and therefore more susceptible to elimination," says Pamela Bjorkman, the Max Delbrück Professor of Biology and an investigator with the Howard Hughes Medical Institute. "If you were able to give this to someone who already had HIV, you might even be able to clear the infection."

The researchers describe the work in the January 29 issue of Cell. Rachel Galimidi, a graduate student in Bjorkman's lab at Caltech, is lead author on the paper.

The researchers hypothesized that one of the reasons the immune system is less effective against HIV than other viruses involves the small number and low density of spikes on HIV's surface. These spikes, each one a cluster of three protein subunits, stick up from the surface of the virus and are the targets of antibodies that neutralize HIV. While most viruses are covered with hundreds of these spikes, HIV has only 10 to 20, making the average distance between the spikes quite long.

That distance is important with respect to the mechanism that naturally occurring antibodies use to capture their viral targets. Antibodies are Y-shaped proteins that evolved to grab onto their targets with both "arms." However, if the spikes are few and far between—as is the case with HIV—it is likely that an antibody will bind with only one arm, making its connection to the virus weaker (and easier for a mutation of the spike to render the antibody ineffective).

To test their hypothesis, Bjorkman's group genetically engineered antibody-based molecules that can bind with both arms to a single spike. They started with the virus-binding parts, or Fabs, of broadly neutralizing antibodies—proteins produced naturally by a small percentage of HIV-positive individuals that are able to fight multiple strains of HIV until the virus mutates. When given in combination, these antibodies are quite effective. Rather than making Y-shaped antibodies, the Caltech group simply connected two Fabs—often from different antibodies, to mimic combination therapies—with different lengths of spacers composed of DNA.

Why DNA? In order to engineer antibodies that could latch onto a spike twice, they needed to know which Fabs to use and how long to make the connection between them so that both could readily bind to a single spike. Previously, various members of Bjorkman's group had tried to make educated guesses based on what is known of the viral spike structure, but the large number of possible variations in terms of which Fabs to use and how far apart they should be, made the problem intractable.

In the new work, Bjorkman and Galimidi struck upon the idea of using DNA as a "molecular ruler." It is well known that each base pair in double-stranded DNA is separated by 3.4 angstroms. Therefore, by incorporating varying lengths of DNA between two Fabs, they could systematically test for the best neutralizer and then derive the distance between the Fabs from the length of the DNA. They also tested different combinations of Fabs from various antibodies—sometimes incorporating two different Fabs, sometimes using two of the same.

"Most of these didn't work at all," says Bjorkman, which was reassuring because it suggested that any improvements the researchers saw were not just created by an artifact, such as the addition of DNA.

But some of the fabricated molecules worked very well. The researchers found that the molecules that combined Fabs from two different antibodies performed the best, showing an improvement of 10 to 1,000 times in their ability to neutralize HIV, as compared to naturally occurring antibodies. Depending on the Fabs used, the optimal length for the DNA linker was between 40 and 62 base pairs (corresponding to 13 and 21 nanometers, respectively).

Taking this finding to the next level in the most successful of these new molecules, the researchers replaced the piece of DNA with a protein linker of roughly the same length composed of 12 copies of a protein called tetratricopeptide repeat. The end product was an all-protein antibody-based reagent designed to bind with both Fabs to a single HIV spike.

"That one also worked, showing more than 30-fold average increased potency compared with the parental antibodies," says Bjorkman. "That is proof of principle that this can be done using protein-based reagents."

The greater potency suggests that a reagent made of these antibody-based molecules could work at lower concentrations, making a potential therapeutic less expensive and decreasing the risk of adverse reactions in patients.

"I think that our work sheds light on the potential therapeutic strategies that biotech companies should be using—and that we will be using—in order to make a better antibody reagent to combat HIV," says Galimidi. "A lot of companies discount antibody reagents because of the virus's ability to evade antibody pressure, focusing instead on small molecules as drug therapies. Our new reagents illustrate a way to get around that."

The Caltech team is currently working to produce larger quantities of the new reagents so that they can test them in humanized mice—specialized mice carrying human immune cells that, unlike most mice, are sensitive to HIV.

Along with Galimidi and Bjorkman, additional Caltech authors on the paper, "Intra-Spike Crosslinking Overcomes Antibody Evasion by HIV-1," include Maria Politzer, a lab assistant; and Anthony West, a senior research specialist. Joshua Klein, a former Caltech graduate student (PhD '09), and Shiyu Bai, a former technician in the Bjorkman lab, also contributed to the work; they are currently at Google and Case Western Reserve University School of Medicine, respectively. Michael Seaman of Beth Israel Deaconess Medical Center and Michel Nussenzweig of the Rockefeller University in New York are also coauthors. The work was supported by the National Institutes of Health through a Director's Pioneer Award and a grant from the HIV Vaccine Research and Design Program, as well as grants from the Collaboration for AIDS Vaccine Discovery and the Bill and Melinda Gates Foundation. Nussenzweig is also an investigator with the Howard Hughes Medical Institute.

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Why Do We Feel Thirst? An Interview with Yuki Oka

To fight dehydration on a hot summer day, you instinctively crave the relief provided by a tall glass of water. But how does your brain sense the need for water, generate the sensation of thirst, and then ultimately turn that signal into a behavioral trigger that leads you to drink water? That's what Yuki Oka, a new assistant professor of biology at Caltech, wants to find out.

Oka's research focuses on the study of how the brain and body work together to maintain a healthy ratio of salt to water as part of a delicate form of biological balance called homeostasis.

Recently, Oka came to Caltech from Columbia University. We spoke with him about his work, his interests outside of the lab, and why he's excited to be joining the faculty at Caltech.

 

Can you tell us a bit more about your research?

The goal of my research is to understand the mechanisms by which the brain and body cooperate to maintain our internal environment's stability, which is called homeostasis. I'm especially focusing on fluid homeostasis, the fundamental mechanism that regulates the balance of water and salt. When water or salt are depleted in the body, the brain generates a signal that causes either a thirst or a salt craving. And that craving then drives animals to either drink water or eat something salty.

I'd like to know how our brain generates such a specific motivation simply by sensing internal state, and then how that motivation—which is really just neural activity in the brain—goes on to control the behavior.

 

Why did you choose to study thirst?

After finishing my Ph.D. in Japan, I came to Columbia University where I worked on salt sensing mechanisms in the mammalian taste system. We found that the peripheral taste system has a key function for salt homeostasis in the body by regulating our salt intake behavior. But of course, the peripheral sensor does not work by itself.  It requires a controller, the brain, which uses information from the sensor. So I decided to move on to explore the function of the brain; the real driver of our behaviors.

I was fascinated by thirst because the behavior it generates is very robust and stereotyped across various species. If an animal feels thirst, the behavioral output is simply to drink water. On the other hand, if the brain triggers salt appetite, then the animal specifically looks for salt—nothing else. These direct causal relations make it an ideal system to study the link between the neural circuit and the behavior.

 

You recently published a paper on this work in the journal Nature. Could you tell us about those findings?

In the paper, we linked specific neural populations in the brain to water drinking behavior. Previous work from other labs suggested that thirst may stem from a part of the brain called the hypothalamus, so we wanted to identify which groups of neurons in the hypothalamus control thirst. Using a technique called optogenetics that can manipulate neural activities with light, we found two distinct populations of neurons that control thirst in two opposite directions. When we activated one of those two populations, it evoked an intense drinking behavior even in fully water-satiated animals. In contrast, activation of a second population drastically suppressed drinking, even in highly water-deprived thirsty animals.  In other words, we could artificially create or erase the desire for drinking water.

Our findings suggest that there is an innate brain circuit that can turn an animal's water-drinking behavior on and off, and that this circuit likely functions as a center for thirst control in the mammalian brain. This work was performed with support from Howard Hughes Medical Institute and National Institutes of Health [for Charles S. Zuker at Columbia University, Oka's former advisor].

 

You use a mouse model to study thirst, but does this work have applications for humans?

There are many fluid homeostasis-associated conditions; one example is dehydration. We cannot specifically say a direct application for humans since our studies are focused on basic research. But if the same mechanisms and circuits exist in mice and humans, our studies will provide important insights into human physiologies and conditions.

 

Where did you grow up—and what started your initial interest in science?

I grew up in Japan, close to Tokyo, but not really in the center of the city. It was a nice combination between the big city and nature. There was a big park close to my house and when I was a child, I went there every day and observed plants and animals. That's pretty much how I spent my childhood. My parents are not scientists—neither of them, actually. It was just my innate interest in nature that made me want to be a scientist.

 

What drew you to Caltech?

I'm really excited about the environment here and the great climate. That's actually not trivial; I think the climate really does affect the people. For example, if you compare Southern California to New York, it's just a totally different character. I came here for a visit last January, and although it was my first time at Caltech I kind of felt a bond. I hadn't even received an offer yet, but I just intuitively thought, "This is probably the place for me."

I'm also looking forward to talking to my colleagues here who use fMRI for human behavioral research. One great advantage about using human subjects in behavioral studies is that they can report back to you about how they feel. There are certainly advantages of using an animal model, like mice. But they cannot report back. We just observe their behavior and say, "They are drinking water, so they must be thirsty." But that is totally different than someone telling you, "I feel thirsty." I believe that combining advantages of animal and human studies should allow us to address important questions about brain functions.

 

Do you have any hobbies?

I play basketball in my spare time, but my major hobby is collecting fossils. I have some trilobites and, actually, I have a complete set of bones from a type of herbivorous dinosaur. It is being shipped from New York right now and I may put it in my new office.

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How Do You Make a Greasy Protein?

Watson Lecture Preview

Every cell is encapsulated and protected by a thin membrane made of greasy molecules called lipids. Assemblies of equally greasy protein molecules span the membrane, forming passageways that control the flow of signaling molecules that, in turn, direct the cell's activities. Because of these proteins' key role in cell-to-cell communication, they have become a prime target for drug design. Professor of Biochemistry Bil Clemons is among those working out the structures of these proteins and, more fundamentally, the biological processes behind them. Clemons will discuss how cells assemble these proteins, and how they deliver them to the membrane, at 8 p.m. on Wednesday, January 7, in Caltech's Beckman Auditorium. Admission is free.

 

Q: What do you do?

A: I am nominally a structural biologist, but I'm really a crystallographer. We purify a protein in solution and then try to crystallize it, which is really, really hard. When we succeed, we make X-ray diffraction patterns of the crystals and work backwards from those patterns to calculate the precise position of every atom. This allows us to make a blueprint for the molecule, and the blueprint helps us understand how the molecule does what it does. That's my group's real interest—figuring out the biological mechanisms that underlie how a protein works. We want to understand, on a molecular level, the processes by which these proteins are targeted and inserted into the membrane.

Proteins are long chains of amino acids that assume very specific three-dimensional shapes, or conformations. The proteins we work on contain hundreds of amino acids and thousands of individual atoms. These proteins interact with other molecules as they do their jobs. When they do, their conformations change, so a large part of our work is trying to understand all these different interactions and motions.

A crystal contains millions of copies of the same molecule held in exactly one conformation, so in that sense, a crystal structure is just one snapshot of a series of biological motions. Eventually we'd like to make movies of all the conformational changes that occur during these interactions—or at least render the important frames. It's almost like producing a cheap cartoon, where the lead animator draws a few key cels, and the rest is filled in later.

 

Q: What do you get from a crystal structure?

A: We get the first glimpse of how something works. Every crystal structure provides a huge amount of information. The beauty of structural biology is that we get to be the first people to peek under the hood of a protein and draw a three-dimensional map of what we see. Science is vast, and most people work in very narrow fields, doing mechanistic studies and drug discovery and all sorts of things. Structural biologists create the platform for everyone else's studies.

 

Q: How did you get into this line of work?

A: Well, I'd like to say it was a series of happy accidents. I've always been passionate about science. In my heart, I think I was born a scientist. I always wanted to know how everything worked, and biochemistry fascinated me. There was so much complexity—so many ways to ask questions.

At Virginia Tech, I was lucky enough to have an undergraduate adviser, Walt Niehaus, who encouraged me to do research in his group. There was really no looking back after that. I just thought, "Wow. This is really fun. I like doing this." Meanwhile, I was paying my way through school. My senior year I was the student manager of one of the food-service facilities. I was working nearly 40 hours a week managing 40 employees plus spending another 20 hours in the lab and 20 hours in school. I wasn't able to look past that to what my future might be, but Walt pushed me to apply for grad school. It was eye-opening the first time he suggested I could do this for a living.

Walt's research was in basic biochemistry. There weren't any structural biologists at Virginia Tech at the time, but the Howard Hughes Medical Institute sent us a booklet with stereo pictures of protein structures. I thought, "You've got to be kidding me. We can look at these things in 3-D?" It blew my mind. So I went to grad school at the University of Utah to be a crystallographer, and I earned my PhD working on the molecular machinery responsible for making proteins. Then I did my postdoctoral work at Harvard Med, trying to understand the complex process of getting greasy membrane proteins into cell membranes. We solved the structure of an important piece of the puzzle there, and now that I'm at Caltech, which has major strengths in X-ray crystallography, we're filling in the details of the bigger picture.

 

 

Named for the late Caltech professor Earnest C. Watson, who founded the series in 1922, the Watson Lectures present Caltech and JPL researchers describing their work to the public. Many past Watson Lectures are available online at Caltech's iTunes U site.

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Douglas Smith
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Improving The View Through Tissues and Organs

On Saturday, October 18, hundreds of undergraduate students will share the results of their projects during SURF Seminar Day. The event, which is open to the public, is an opportunity for students to discuss and explain their research to individuals with a wide-range of expertise and interests.

This summer, several undergraduate students at Caltech had the opportunity to help optimize a promising technique that can make tissues and organs—even entire organisms—transparent for study. As part of the Summer Undergraduate Research Fellowship (SURF) program, these students worked in the lab of Assistant Professor of Biology Viviana Gradinaru, where researchers are developing such so-called clearing techniques that make it possible to peer straight through normally opaque tissues rather than seeing them only as thinly sectioned slices that have been pieced back together.

Gradinaru's group recently published a paper in the journal Cell describing a new approach to tissue clearing. The method they have created builds on a technique called CLARITY that Gradinaru helped develop while she was a research associate at Stanford. CLARITY allowed researchers to, for the first time, create a transparent whole-brain specimen that could then be imaged with its structural and genetic information intact.

CLARITY was specifically developed for studying the brain. But the new approach developed in Gradinaru's lab, which the team has dubbed PARS (perfusion-assisted agent release in situ), can also clear other organs, such as the kidney, as well as tissue samples, such as tumor biopsies. It can even be applied to entire organisms.

Like CLARITY, PARS involves removing the light-scattering lipids in the tissue to make samples transparent without losing the structural integrity that lipids typically provide. First the sample is infused with acrylamide monomers that are then polymerized into a hydrogel that provides structural support. Next, this tissue–hydrogel hybrid is immersed in a detergent that removes the lipids. Then the sample can be stained, often with antibodies that specifically mark cells of interest, and then immersed in RIMS (refractive index matching solution) for imaging using various optical techniques such as confocal or lightsheet microscopy.

Over the summer, Sam Wie, a junior biology major at Caltech, spent 10 weeks in the Gradinaru lab working to find a polymer that would perform better than acrylamide, which has been used in the CLARITY hydrogel. "One of the limitations of CLARITY is that when you put the hydrogel tissue into the detergent, the higher solute concentration in the tissue causes liquid to rush into the cell. That causes the sample to swell, which could potentially damage the structure of the tissue," Wie explains. "So I tried different polymers to try to limit that swelling."

Wie was able to identify a polymer that produces, over a similar amount of time, about one-sixth of the swelling in the tissue.

"The SURF experience has been very rewarding," Wie says. "I've learned a lot of new techniques, and it's really exciting to be part of, and to try to improve, CLARITY, a method that will probably change the way that we image tissues from now on."

At another bench in Gradinaru's lab, sophomore bioengineering major Andy Kim spent the summer focusing on a different aspect of the PARS technique. While antibodies have been the most common markers used to tag cells of interest within cleared tissues, they are too large for some studies—for example, those that aim to image deeper parts of the brain, requiring them to cross the blood–brain barrier. Kim's project involved identifying smaller proteins, such as nanobodies, which target and bind to specific parts of proteins in tissues.

"While PARS is a huge improvement over CLARITY, using antibodies to stain is very expensive," Kim says. "However, some of these nanobodies can be produced easily, so if we can get them to work, it would not only help image the interior of the brain, it would also be a lot less costly."

During his SURF, Kim worked with others in the lab to identify about 30 of these smaller candidate binding proteins and tested them on PARS-cleared samples.

While Wie and Kim worked on improving the PARS technique itself, Donghun Ryu, a third SURFer in Gradinaru's lab, investigated different methods for imaging the cleared samples. Ryu is a senior electrical engineering and computer science major at the Gwangju Institute of Science and Technology (GIST) in the Republic of Korea.

Last summer Ryu completed a SURF as part of the Caltech–GIST Summer Undergraduate Research Exchange Program in the lab of Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering at Caltech. While completing that project, Ryu became interested in optogenetics, the use of light to control genes. Since optogenetics is one of Gradinaru's specialties, Yang suggested that he try a SURF in Gradinaru's lab.

This summer, Ryu was able to work with both Yang and Gradinaru, investigating a technique called Talbot microscopy to see whether it would be better for imaging thick, cleared tissues than more common techniques. Ryu was able to work on the optical system in Yang's lab while testing the samples cleared in Gradinaru's lab.

"It was a wonderful experience," Ryu says. "It was special to have the opportunity to work for two labs this summer. I remember one day when I had a meeting with both Professor Yang and Professor Gradinaru; it was really amazing to get to meet with two Caltech professors."

Gradinaru says that the SURF projects provided a learning opportunity not only for the participating students but also for her lab. "For example," she says, "Ryu strengthened the collaboration that we have with the Yang group for the BRAIN Initiative. And my lab members benefited from the chance to serve as mentors—to see what works and what can be improved when transferring scientific knowledge. These are very important skills in addition to the experimental know-how that they master."  

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