Dietary Fiber and Microbes Change the Gel That Lines Our Gut

In the ongoing hustle and bustle of our intestines, where bacteria and food regularly intermingle, there is another substance that, to the surprise of researchers, has been found to rapidly change: the gel that lines the gut. A new Caltech study is the first to show how the structure of this gut gel, or mucus, can change in the presence of certain substances, such as bacteria and polymers—a class of long-chained molecules that includes dietary fiber.

The work, to be published online the week of June 13 in the Proceedings of the National Academy of Sciences, could lead to the development of new drugs or diets for intestinal conditions such as irritable bowel disease.

Our intestinal tracts are lined with a mucus gel that acts as a protective barrier between the insides of our bodies and the outside world. The gel lets in nutrients and largely blocks out bacteria, preventing infections. It also regulates how some drugs are delivered elsewhere in our bodies.

Researchers had previously studied how the gel can be damaged, for instance when bacteria feed on the gut's lining. The Caltech study is the first to look at the structure of the gel and how it morphs in the presence of other substances naturally found in the gut.

Performing their experiments in mice, the team tested the effects of polymers, which include dietary fiber as well as therapeutics such as medicines for constipation. The researchers fed some mice a diet rich in polymers and others (the controls) a polymer-free diet. Using a technique called confocal reflectance microscopy they measured the thickness of the gut gel and the degree to which the gel was compressed as a result of the consumed polymers. Mice given a high-polymer diet, they found, had a more compressed gel layer.

"The gel is like a sponge with holes that let material through," says the paper's lead author, Sujit Datta, a postdoctoral scholar in the laboratory of Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering. "We are seeing that polymers, including dietary fiber, can compress the gel, potentially making the holes smaller, and we think that this might offer protective benefits," Datta adds.

In addition, the researchers applied different kinds of polymers—including dietary fibers like pectin, found in apples—directly to the gel lining to test its response. All of the polymers tested compressed the gel layer.

"It's too early to draw any conclusions, but it may be that eating an apple a day will affect the shape of the lining in your gut," says Asher Preska Steinberg, a Caltech graduate student and coauthor of the study.

The researchers also found that dietary fiber and gut bacteria—which are part of a community of microorganisms collectively known as gut microbiota—can work together to influence how the gut gel changes shape. They performed the same polymer/fiber experiments in germ-free mice, which are mice carefully raised to not have any bacteria in their gut. The results showed that the polymers compressed the gut gels of these germ-free mice to a greater degree. This implies that species of bacteria in our gut that are known to break down polymers can weaken the compressing effect.

"We previously thought of the gel as a static structure, so it was unexpected to find an interplay between diet and gut microbiota that rapidly and dynamically changes the biological structures that protect a host," says Ismagilov.

Both dietary fiber and certain gut microbes have been linked to good health. Fiber has been shown to lower cholesterol and regulate blood sugar levels—factors in heart disease and diabetes, respectively. Meanwhile, some bacteria, including the good "probiotics," can help treat digestive disorders and may even play a beneficial role in mental health. For instance, a separate Caltech-led study found that probiotics can alleviate autism-like behaviors in mice—a finding that could potentially lead to new therapies for the disorder in humans.

The entire collection of bacteria in our gut can include 1,000 different species or more and weigh a total of three pounds. Exactly how these microscopic organisms influence our health, for good and bad, is an area of active research with many unanswered questions. The White House recently announced the National Microbiome Initiative, with federal funding worth $121 million, to investigate the mysteries of microbes not only living in our bodies but all over the planet. In addition, more than 100 nonfederal agencies have pledged money and support toward researching microbial communities.

"Our study gives biologists and scientists studying diseases of the gut something else to think about," says Datta. "Now they can take the structure of the gut mucus, and how it responds to its environment, into account."

This research was funded by the Defense Advanced Research Projects Agency (DARPA) and National Science Foundation.

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The study is the first to look at the structure of the gut's mucus gel lining and how it morphs in the presence of other substances naturally found in the gut.

Natural Quasicrystals May Be the Result of Collisions Between Objects in the Asteroid Belt

Naturally formed quasicrystals—solids with orderly atomic arrangements with symmetries impossible for conventional crystals—are among the rarest structures on Earth. Only two have ever been found.

A team led by Paul Asimow (MS '93, PhD '97), professor of geology and geochemistry at Caltech, may have uncovered one of the reasons for that scarcity, demonstrating in laboratory experiments that quasicrystals could arise from collisions between rocky bodies in the asteroid belt with unusual chemical compositions.

A paper on their findings was published on June 13 in the advance online edition of the Proceedings of the National Academy of Sciences.

At an atomic level, crystals are both ordered and periodic, meaning that they have a well-defined geometric structure composed of atomic clusters that repeat like building blocks with equal spacings along the repeat directions. Over one hundred years ago, it was shown the crystal can only exhibit one of four types of rotational symmetry: two-fold, three-fold, four-fold, or six-fold.

The number refers to how many times an object will look exactly the same within a full 360-degree rotation about an axis. For example, an object with two-fold symmetry appears the same twice, or every 180 degrees; an object with three-fold symmetry appears the same three times, or every 120 degrees; and an object with four-fold symmetry appears the same four times, or every 90 degrees.

Prior to 1984, it was believed that it would be impossible for a solid to grow with any other type of symmetry, and no examples of materials with other symmetries had ever been discovered in nature or grown in a lab. In that year, however, Princeton physicist Paul Steinhardt (BS '74) and his student Dov Levine (now at the Technion – Israel Institute of Technology) theorized a set of conditions under which other types of symmetry could potentially exist and Dan Shechtman of the Israel Institute of Technology and collaborators published a paper announcing the synthesis in the laboratory of a material with a five-fold rotational symmetry.

The atomic arrangements of these materials were ordered so that, like crystals, X-rays and electrons passing through them form a pattern of sharp spots. However, whereas the spots obtained from a crystal are equally spaced and only form patterns with symmetries from the restricted list, the spots obtained from a quasicrystal form a fractal snowflake pattern that include forbidden symmetries, such as five-fold. Steinhardt and Levine dubbed them "quasiperiodic crystals" or "quasicrystals" for short.

Over the next few decades, researchers figured out how to manufacture more than 100 different varieties of quasicrystals by melting and homogenizing certain elements and then cooling them at very specific rates in the lab. Still, though, no naturally existing quasicrystals were known. Indeed, researchers suspected their formation would be impossible. That is because most lab-grown quasicrystals were metastable, meaning that the same combination of elements could arrange themselves into a crystalline structure using less energy.

Everything changed in the late 2000s, when Steinhardt and colleague Luca Bindi from the Museum of Natural History at the University of Florence (currently in the Faculty of the Department of Earth Sciences of the same University) found a tiny grain of an aluminum, copper, and iron mineral that exhibited five-fold symmetry. The grain came from a small sample of the Khatyrka meteorite, an extraterrestrial object known only from a few pieces found in Russia's Koryak Mountains. Steinhardt and his collaborators found a second natural quasicrystal from the same meteorite in 2015, confirming that the natural existence of quasicrystals was possible, just very rare.

A microscopic analysis of the meteorite indicated that it had undergone a major shock at some point in its lifetime before crashing to Earth – likely from a collision with another rocky body in space. Such collisions are common in the asteroid belt and release high amounts of energy.

Asimow and colleagues hypothesized that the energy released by the shock could have caused the quasicrystal's formation by triggering a rapid cycle of compression, heating, decompression, and cooling.

To test the hypothesis, Asimow simulated the collision between two asteroids in his lab. He took thin slices of minerals found in the Khatyrka meteorite and sandwiched them together in a sample case that resembles a steel hockey puck. He then screwed the "puck" to the muzzle of a four-meter-long, 20-mm-bore single-stage propellant gun, and blasted it with a projectile at nearly one kilometer per second, about equal to the speed of the fastest rifle-fired bullets.

It is important to note that those minerals included a sample of a metallic copper-aluminum alloy, which has only been found in nature in the Khatyrka meteorite.

After the sample was shocked with the propellant gun, it was sawed open, polished, and examined. The impact smashed the sandwiched elements together and, in several spots, created microscopic quasicrystals, according to X-ray and electron diffraction studies at Caltech, Princeton, and Florence.

Armed with this experimental evidence, Asimow says he is confident that shocks are the source of naturally formed quasicrystals. "We know that the Khatyrka meteorite was shocked. And now we know that when you shock the starting materials that were available in that meteorite, you get a quasicrystal."

Sarah Stewart (PhD '02)—a planetary collision expert from the University of California, Davis, and reviewer of the PNAS paper—admits she was surprised by the findings. "If you had called me before the study and asked if this would work I would have said 'no way.' The astounding thing is that they did it so easily," she says. "Nature is crazy."

Asimow acknowledges that the experiments leave many questions unanswered. For example, it is unclear at what point the quasicrystal formed during the shock's pressure and temperature cycle. A bigger mystery, Asimow says, is the origin of the copper-aluminum alloy in the meteorite, which has never been seen elsewhere in nature.

Next, Asimow plans to shock various combinations of minerals to see what key ingredients are necessary for natural quasicrystal formation.

These results are published in a paper titled "Shock synthesis of quasicrystals with implications for their origin in asteroid collisions." In addition to Asimow, Steinhardt, and Bindi, other coauthors on the paper are Chi Ma, director of analytical facilities in the Geological and Planetary Sciences division at Caltech; Lincoln Hollister (PhD '66) and Chaney Lin from Princeton University; and Oliver Tschauner from the University of Nevada, Las Vegas. Their work was supported by the National Science Foundation (NSF), the University of Florence, and the NSF-Materials Research Science & Engineering Centers Program through New York University and the Princeton Center for Complex Materials.

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A Feeling Touch

Using funding from the BRAIN Initiative, Caltech biologists are developing neuroprosthetics to bring tactile sensations to the users of robotic arms.

Caltech biologist Richard Andersen is working to incorporate a sense of touch into the neural prosthetics he has been helping develop for years—devices implanted in the brain that allow a paralyzed patient to manipulate a robotic arm.

Andersen and colleagues first reported success of their original implant in early 2015. The team, led by Andersen, placed their prosthesis in the posterior parietal cortex, an area that controls the intent to move rather than controlling movement directly as previous experiments had done. This allowed Erik Sorto, a 35-year-old man who has been paralyzed from the neck down for more than 10 years, to use a robotic arm placed next to his body to perform a fluid hand-shaking gesture, play rock-paper-scissors, and even grasp a bottle of beer and bring it to his mouth for a sip—something he had long dreamed of doing.

This research on how to make a robotic arm move resulted in a 2015 National Science Foundation grant to Andersen from President Obama's Brain Research through Advancing Innovative Neurotechnology—or BRAIN—Initiative, as well as seed money from the California Blueprint for Research to Advance Innovations in Neuroscience (Cal-BRAIN) program, the California complement to the federal initiative, which gave out its first-ever monetary awards last year to a group of researchers that included Andersen.

Andersen is now using those Cal-BRAIN funds—designed to bring together interdisciplinary teams of scientists and engineers from diverse fields for fundamental brain research—to take his team's work to the next level. His hope is to enable people using robotic arms to literally regain their sense of touch—their ability to feel an object in their "hands."

For more on Andersen's work, read A Feeling Touch on E&S+

 

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When Beneficial Bacteria Knock But No One is Home

The community of beneficial bacteria that live in our intestines, known as the gut microbiome, are important for the development and function of the immune system. There has been growing evidence that certain probiotics—therapies that introduce beneficial bacteria into the gut—may help alleviate some of the symptoms of intestinal disorders such as Crohn's disease. By studying the interplay between genetic risk factors for Crohn's and the bacteria that populate the gut, researchers at Caltech have discovered a new potential cause for this disorder in some patients—information that may lead to advances in probiotic therapies and personalized medicine.

The results were published online in the May 5 edition of the journal Science.

Previously, scientists had found that patients with Crohn's disease often exhibit alterations in both their genome and their gut microbiome—the diverse collection of bacteria that reside in the intestine. More than 200 genes have been implicated as having a role in the susceptibility to Crohn's. For years, researchers in the field have believed that these are genes that normally function by sensing pathogenic bacteria and deploying an immune response to kill the unwanted microbes; when these genes are defective, the pathogenic bacteria survive, multiply in the gut, and lead to disease.

"While we believe that all of that is true, in this study we were curious to see if some of the genes that are important in sensing pathogenic bacteria may also be important in sensing beneficial bacteria to promote immune health," says the study's first author, Hiutung Chu, a postdoctoral scholar in biology and biological engineering at Caltech. "Typically, the signals from these beneficial commensal microbes promote anti-inflammatory responses that dampen inflammation in the gut. However, mutations in genes that sense and respond to pathogenic bacteria would also impair the response to the beneficial ones. So it's kind of a new spin on the existing dogma."

To figure this out, Chu and her colleagues in the laboratory of Sarkis Mazmanian, the Luis B. and Nelly Soux Professor of Microbiology and a Heritage Principal Investigator, designed several experiments to study how genetic mutations might interrupt the immune-enhancing effects of a known beneficial bacterium, Bacteroides fragilis. The researchers tested their new theory by using B. fragilis to treat mice that had nonfunctional versions of two genes known to play a role in Crohn's disease risk, called ATG16L1 and NOD2.

The researchers found that if just one of these two genes was absent, the mice were unable to develop disease-protective immune cells called regulatory T cells in response to B. fragilis—and that even after treatment with B. fragilis, symptoms in an ATG16L1-deficient mouse model of intestinal disease remained unchanged.

Chu and Mazmanian then obtained blood samples from both healthy patients and patients with Crohn's disease at the Cedars-Sinai Medical Center in Los Angeles. "We could see that certain patients' immune cells responded to Bacteroides fragilis, while immune cells from other patients didn't respond at all," Chu says. "Because the cells from Cedars had already been genotyped, we were able to match up our results with the patients' genotypes: immune cells from individuals with the protective version of ATG16L1 responded to the treatment, but cells from patients who had the mutated version of the gene showed no anti-inflammatory response to B. fragilis."

Mazmanian says the results suggest that the faulty versions of these genes may cause Crohn's disease in two different ways: by being unable to assist in destroying pathogenic bacteria and by preventing the beneficial immune signals usually elicited by "good" bacteria. "What Hiutung has shown is that there are specific bacteria in the human microbiome that appear to utilize the pathways that are encoded by these genes—genes normally involved in killing bacteria—to send beneficial signals to the host," he says.

This work reveals the important relationship between the genome and the microbiome—and it may also one day be used to improve the use of probiotics in clinical trials, Mazmanian says. "For example, our previous work has suggested using B. fragilis as a probiotic treatment for certain disorders. What this new study suggests is that there are certain populations that wouldn't benefit from this treatment because they have this genetic predisposition," he notes. "Right now, clinical trials don't do a good job of identifying which patients might respond best to treatment, but our experiments in mouse models suggest that, conceptually, you could design clinical trials that are more effective."

The research described in the paper, "Gene-Microbiota Interactions Contribute to the Pathogenesis of Inflammatory Bowel Disease," was funded by the National Institutes of Health, the Cedars-Sinai F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, the Lupus Research Institute, the European Union, the Crohn's and Colitis Foundation of America, the Leona M. and Harry B. Helmsley Charitable Trust, and the Heritage Medical Research Institute.

In addition to Chu and Mazmanian, other Caltech coauthors include former graduate students Arya Khosravi (PhD '14) and Yue Shen (PhD '12); research technician assistants Indah Kusumawardhani and Alice Kwon; and Wei-Li Wu, a postdoctoral scholar in biology and biological engineering. Coauthors from other institutions include: Anilton Vasconcelos and Peter Ernst from UC San Diego; Larissa Cunha and Douglas Green from St. Jude Children's Research Hospital in Memphis; Anne Mayer, Amal Kambal, and Herbert Virgin from the Washington University School of Medicine in St. Louis; Stephan Targan and Dermot McGovern from Cedars-Sinai Medical Center; and Ramnik Xavier from Harvard Medical School.

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Michael Watkins Named Next JPL Director

Michael M. Watkins, the Clare Cockrell Williams Centennial Chair in Aerospace Engineering and Director of the Center for Space Research at The University of Texas at Austin, has been appointed director of the Jet Propulsion Laboratory and vice president at Caltech, the Institute announced today. 

Watkins will formally assume his position on July 1, 2016. He succeeds Charles Elachi, who will retire as of June 30, 2016, and move to the Caltech faculty.   

Watkins is an internationally recognized scientist and engineer. Prior to assuming his current position at The University of Texas in 2015, he worked at JPL for 22 years, where he held leadership roles on some of NASA's highest-profile missions. Watkins served as mission manager and mission system manager for the Mars Science Laboratory Curiosity Rover; led review or development teams for several missions including the Cassini, Mars Odyssey, and Deep Impact probes; and was the project scientist leading science development for the GRAIL moon-mapping satellites, the GRACE Earth science mission, and the GRACE Follow-On mission, scheduled for launch in 2017. He last served at JPL as manager of the Science Division, and chief scientist for the Engineering and Science Directorate.

"Michael's record of successful mission leadership and impressive management skills quickly distinguished him as a leading candidate for this position," says Caltech president Thomas F. Rosenbaum, the Sonja and William Davidow Presidential Chair and professor of physics. "As JPL director, Michael will build upon the laboratory's outstanding achievements in planetary exploration and earth science, strengthening the connections between Caltech's campuses and partnering with NASA to deliver highly complex and nuanced missions."

"I've known Mike Watkins for more than twenty years now," Elachi says. "Mike has played important and varying roles in a number of important JPL areas. His intimate knowledge of the lab and staff, combined with his highly diversified set of skills and knowledge base in science and engineering, will serve JPL very well in the years to come."

A committee composed of Caltech trustees, faculty, senior administrative leaders, and a member of the JPL executive council conducted an extensive search and recommended Watkins to Caltech's president.

Watkins holds a Bachelor's degree, Master's degree, and PhD in aerospace engineering from The University of Texas at Austin. He has published broadly in both engineering and science, contributed more than 100 conference presentations, and has served on the boards of numerous international scientific and engineering societies.

"JPL has such a talented and deeply committed staff," says Watkins. "It is a privilege to have this opportunity to lead the laboratory to even greater discoveries. I look forward to working with my colleagues on campus and across NASA to forge new directions in space exploration and earth science."

 

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Michael M. Watkins has been appointed director of the Jet Propulsion Laboratory and vice president at Caltech, the Institute announced today.

The Global History of Space Exploration

When talking about the history of space exploration, people are often quick to reference the Soviet and U.S. victories of the 1950s and 1960s, such as Sputnik and the Apollo program. However, these memorable advances are only a slice of a broader global history that includes significant contributions from dozens of nations, including many within the developing world. To science historian Asif Siddiqi—who specializes in the history of space exploration—these lesser-known stories of the global space race are just as interesting.

"For a long time, historians have said, 'Science is global!' but their claims were largely theoretical," Siddiqi says. "I'm interested in empirical examples of the global circulation of scientific knowledge and expertise, and one way I wanted to track this was through examples of leftover infrastructure from space exploration."

A professor of history at Fordham University, Siddiqi is this year's Eleanor Searle Visiting Professor of History at Caltech and The Huntington Library. The Searle visiting professorship is offered every year to a historian who wishes to conduct research in the Huntington's collections and teach courses in the Division of the Humanities and Social Sciences at Caltech.

"This professorship is a very wonderful opportunity for people in the middle of their careers to take a year off to start some new projects," he says.

Siddiqi has used this time, which continues until the end of the academic year, to focus his work on the history and impact of India's space program. "I'm interested in how developing nations allocated resources for very high-technology projects, despite their apparent social and economic problems. I wanted to look at India because it is a country with some obvious societal inequalities, but at the same time, they also prioritized this very modern technology. So I'm looking at the Indian space program through that lens," he says.

Siddiqi believes that one reason the Indian government initially became interested in space exploration was an urge to modernize after becoming independent from Great Britain in 1947. "Things like space exploration and nuclear energy were markers of the modern world, and they didn't want to have to spend 200 years trying to play catch up with the Western countries," he says. "That urge to 'leapfrog' over the West, often framed in terms of the modernization theory of the 1960s, gets manifested in a kind of fixation on certain things, and space was the most cutting-edge thing at the time."

The Indian government was also motivated by the introduction of technologies, developed by NASA, that the American government gave to other countries—such as India—in an effort to gain allies during the Cold War, Siddiqi says. The countries could keep these millions of dollars of scientific equipment and materials at no cost, allowing them to build an infrastructure for space exploration, with one unspoken but implicit caveat: they had to agree that, politically speaking, they would side with the U.S. against the Soviet Union.

Siddiqi also wanted to investigate how India's commitment to space exploration had an impact on those people not involved with science or politics. For instance, the Searle professorship allowed Siddiqi to travel to a small fishing village on the southern tip of India, to see how the space race impacted the local community.

"It turns out that this village's location intersects with a particular cosmic ray phenomenon that only happens around the magnetic equator," he says. The phenomenon, called the equatorial electrojet, results when solar winds cause an intensification of the Earth's magnetic field in a small patch directly above equatorial regions, including the Indian Ocean.

Because of this phenomenon, top scientists from around the world wanted to build an observatory in the village to study it. The Indian government got behind the plan, insisting that the international scientists leave behind all of their expensive equipment when they left, infrastructure which was later used for their space program. However, making room for the observatory also meant that the entire local fishing village would have to be packed up and relocated.

"One of my goals in this project was to recover the history of the Catholic fishing community that lived there for centuries, but had to be moved," Siddiqi says.

He was also interested in what it was like for the international community of scientists that was created on the former site of the fishing village. "It was 1963—the height of the Cold War—and there were scientists from the U.K., America, Russia, Germany, Japan, and all over the world working in this remote village," he notes, pointing out how unusual such a collaboration was during the Cold War.

The observatory only lasted a few decades, as expanding capabilities of satellites eventually made the ground-based technologies obsolete for these types of studies. But it had a lasting impact.

"The cosmic phenomenon above this Indian fishing village was a total accident of geography, and that's what was interesting about that story," Siddiqi says. "However, there are many more stories like that across the global landscape. Our narrative of the space race is mostly about astronauts and the moon, and maybe a little bit of deep-space exploration, such as Mars. But in my work, I hope to shed some light on some of these other types of contributions on the earth that are largely forgotten."

Siddiqi recently presented a summary of this work in a lecture at The Huntington Library titled, "A Different Space: NASA in the Postcolonial World."

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Mapping Neurons to Improve the Treatment of Parkinson's

Because billions of neurons are packed into our brain, the neuronal circuits that are responsible for controlling our behaviors are by necessity highly intermingled. This tangled web makes it complicated for scientists to determine exactly which circuits do what. Now, using two laboratory techniques pioneered in part at Caltech, Caltech researchers have mapped out the pathways of a set of neurons responsible for the kinds of motor impairments—such as difficulty walking—found in patients with Parkinson's disease.

The work—from the laboratory of Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering and a Heritage Principal Investigator—was published on April 20 in the journal Neuron.

In patients with Parkinson's disease, gait disorders and difficulty with balance are often caused by the degeneration of a specific type of neuron—called cholinergic neurons—in a region of the brainstem called the pedunculopontine nucleus (PPN). Damage to this same population of neurons in the PPN is also linked to reward-based behaviors and disorders, such as addiction.

Previously, researchers had not been able to untangle the neural circuitry originating in the PPN to understand how both addictions and Parkinson's motor impairments are modulated within the same population of cells. Furthermore, this uncertainty created a barrier to treating those motor symptoms. After all, deep brain stimulation—in which a device is inserted into the brain to deliver electrical pulses to a targeted region—can be used to correct walking and balance difficulties in these patients, but without knowing exactly which part of the PPN to target, the procedure can lead to mixed results.

"The circuits responsible for controlling our behaviors are not nicely lined up, where this side does locomotion and this side does reward," Gradinaru says, and this disordered arrangement arises from the way neurons are structured. Much as a tree extends into the ground with long roots, neurons are made up of a cell body and a long string-like axon that can diverge and project elsewhere into different areas of the brain. Because of this shape, the researchers realized they could follow the neuron's "roots" to an area of the brain less crowded than the PPN. This would allow them to more easily look at the two very different behaviors and how they are implemented.

Cheng Xiao, a senior research scientist at Caltech and first author on the study, began by mapping the projections of the cholinergic neurons in the PPN of a rat using a technique developed by the Gradinaru lab called Passive CLARITY Technique, or PACT. In this technique, a solution of chemicals is applied to the brain; the chemicals dissolve the lipids in the tissue and render that region of the brain optically transparent—see-through, in other words—and able to take up fluorescent markers that can label different types of neurons. The researchers could then follow the path of the PPN neurons of interest, marked by a fluorescent protein, by simply looking through the rest of the brain.

Using this method, Gradinaru and Xiao were able to trace the axons of the PPN neurons as they extended into two regions of the midbrain: the ventral substantia nigra, a landmark area for Parkinson's disease that had been previously associated with locomotion; and the ventral tegmental area, a region of the brain that had been previously associated with reward.

Next, the researchers used an electrical recording technique to keep track of the signals sent by PPN neurons—confirming that these neurons do, in fact, communicate with their associated downstream structures in the midbrain. Then, the scientists went on to determine how this specific population of neurons affects behavior. To do this, they used a technique that Gradinaru helped develop called optogenetics, which allows researchers to manipulate neural activities—in this case, by either exciting or inhibiting the PPN neural projections in the midbrain—using different colors of light.

Using the optogenetic approach in rats, the researchers found that exciting the neuronal projections in the ventral substantia nigra would stimulate the animal to walk around its environment; by contrast, they could stop the animal's movement by inhibiting these same projections. Furthermore, they found that they could stimulate reward-seeking behavior by exciting the neuronal projections in the ventral tegmental area, but could cause aversive behavior by inhibiting these projections.

"Our results show that the cholinergic neurons from the PPN indeed have a role in controlling both behaviors," Gradinaru says. "Although the neurons are very densely packed and intermingled, these pathways are, to some extent, dedicated to very specialized behaviors." Determining which pathways are associated with which behaviors might also improve future treatments, she adds.

"In the past it's been difficult to target treatment to the PPN because the specific neurons associated with different behaviors are intermingled at the source—the PPN. Our results show that you could target the axonal projections in the substantia nigra for movement disorders and projections in the ventral tegmental area for reward disorders, as addiction is," Gradinaru says. In addition, she notes, these projections in the midbrain are much easier to access surgically than their source in the PPN.

Although this new information could inform clinical treatments for Parkinson's disease, the PPN is only one region of the brain and there are many more important examples of connectivity that need to be explored, Gradinaru says. "These results highlight the need for brain-wide functional and anatomical maps of these long-range neuronal projections; we've shown that tissue clearing and optogenetics are enabling technologies in the creation of these maps."

These results are published in a paper titled, "Cholinergic mesopontine signals govern locomotion and reward through dissociable midbrain pathways." In addition to Gradinaru and Xiao, other Caltech coauthors include Jounhong Ryan Cho, Chunyi Zhou, Jennifer Treweek, Ken Chan, Sheri McKinney, and Bin Yang. The work was supported by the National Institutes of Health, the Heritage Medical Research Institute, the Pew Charitable Trust, the Michael J. Fox Foundation, and the Sloan Foundation; the Beckman Institute supports the Resource Center on CLARITY, Optogenetics, and Vector Engineering (CLOVER) for technology development and broad dissemination.

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Rerouting Cancer

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Midnight Blue: A New System for Color Vision

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Biochemists Solve the Structure of Cell's DNA Gatekeeper

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Solved: Structure of the Cell's DNA Gatekeeper
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The detailed map is the first to determine the structure of a massive protein machine with near-atomic resolution.

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