Delivering Genes Across the Blood-Brain Barrier

Caltech biologists have modified a harmless virus in such a way that it can successfully enter the adult mouse brain through the bloodstream and deliver genes to cells of the nervous system. The virus could help researchers map the intricacies of the brain and holds promise for the delivery of novel therapeutics to address diseases such as Alzheimer's and Huntington's. In addition, the screening approach the researchers developed to identify the virus could be used to make additional vectors capable of targeting cells in other organs.

"By figuring out a way to get genes across the blood-brain barrier, we are able to deliver them throughout the adult brain with high efficiency," says Ben Deverman, a senior research scientist at Caltech and lead author of a paper describing the work in the February 1 online publication of the journal Nature Biotechnology.

The blood-brain barrier allows the body to keep pathogens and potentially harmful chemicals circulating in the blood from entering the brain and spinal cord. The semi-permeable blockade, composed of tightly packed cells, is crucial for maintaining a controlled environment to allow the central nervous system to function properly. However, the barrier also makes it nearly impossible for many drugs and other molecules to be delivered to the brain via the bloodstream.

To sneak genes past the blood-brain barrier, the Caltech researchers used a new variant of a small, harmless virus called an adeno-associated virus (AAV). Over the past two decades, researchers have used various AAVs as vehicles to transport specific genes into the nuclei of cells; once there, the genes can be expressed, or translated, from DNA into proteins. In some applications, the AAVs carry functional copies of genes to replace mutated forms present in individuals with genetic diseases. In other applications, they are used to deliver genes that provide instructions for generating molecules such as antibodies or fluorescent proteins that help researchers study, identify, and track certain cells.

Largely because of the blood-brain barrier problem, scientists have had only limited success delivering AAVs and their genetic cargo to the central nervous system. In general, they have relied on surgical injections, which deliver high concentrations of the virus at the injection site but little to the outlying areas. Such injections are also quite invasive. "One has to drill a hole through skull, then pierce tissue with a needle to the injection site," explains Viviana Gradinaru (BS '05), assistant professor of biology and biological engineering at Caltech and senior author on the paper. "The deeper the injection, the higher the risk of hemorrhage. With systemic injection, using the bloodstream, none of that damage happens, and the delivery is more uniform."

In addition, Gradinaru notes, "many disorders are not tightly localized. Neurodegenerative disorders like Huntington's disease affect very large brain areas. Also, many complex behaviors are mediated by distributed interacting networks. Our ability to target those networks is key in terms of our efforts to understand what those pathways are doing and how to improve them when they are not working well."

In 2009, a group led by Brian Kaspar of Ohio State University published a paper, also in Nature Biotechnology, showing that an AAV strain called AAV9 injected into the bloodstream could make its way into the brain—but it was only efficient when used in neonatal, or infant, mice.

"The big challenge was how do we achieve the same efficiency in an adult," says Gradinaru.

Although one might like to design an AAV that is up to the task, the number of variables that dictate the behavior of any given virus, as well as the intricacies of the brain and its barrier, make that extremely challenging. Instead, the researchers developed a high-throughput selection assay, CREATE (Cre REcombinase-based AAV Targeted Evolution), that allowed them to test millions of viruses in vivo simultaneously and to identify those that were best at entering the brain and delivering genes to a specific class of brain cells known as astrocytes.

They started with the AAV9 virus and modified a gene fragment that codes for a small loop on the surface of the capsid—the protein shell of the virus that envelops all of the virus' genetic material. Using a common amplification technique, known as polymerase chain reaction (PCR), they created millions of viral variants. Each variant carried within it the genetic instructions to produce more capsids like itself.

Then they used their novel selection process to determine which variants most effectively delivered genes to astrocytes in the brain. Importantly, the new process relies on strategically positioning the gene encoding the capsid variants on the DNA strand between two short sequences of DNA, known as lox sites. These sites are recognized by an enzyme called Cre recombinase, which binds to them and inverts the genetic sequence between them. By injecting the modified viruses into transgenic mice that only express Cre recombinase in astrocytes, the researchers knew that any sequences flagged by the lox site inversion had successfully transferred their genetic cargo to the target cell type—here, astrocytes.

After one week, the researchers isolated DNA from brain and spinal cord tissue, and amplified the flagged sequences, thereby recovering only the variants that had entered astrocytes.

Next, they took those sequences and inserted them back into the modified viral genome to create a new library that could be injected into the same type of transgenic mice. After only two such rounds of injection and amplification, a handful of variants emerged as those that were best at crossing the blood-brain barrier and entering astrocytes.

"We went from millions of viruses to a handful of testable, potentially useful hits that we could go through systematically and see which ones emerged with desirable properties," says Gradinaru.

Through this selection process, the researchers identified a variant dubbed AAV-PHP.B as a top performer. They gave the virus its acronym in honor of the late Caltech biologist Paul H. Patterson because Deverman began this work in Patterson's group. "Paul had a commitment to understanding brain disorders, and he saw the value in pushing tool development," says Gradinaru, who also worked in Patterson's lab as an undergraduate student.

To test AAV-PHP.B, the researchers used it to deliver a gene that codes for a protein that glows green, making it easy to visualize which cells were expressing it. They injected the AAV-PHP.B or AAV9 (as a control) into different adult mice and after three weeks used the amount of green fluorescence to assess the efficacy with which the viruses entered the brain, the spinal cord, and the retina.

"We could see that AAV-PHP.B was expressed throughout the adult central nervous system with high efficiency in most cell types," says Gradinaru. Indeed, compared to AAV9, AAV-PHP.B delivers genes to the brain and spinal cord at least 40 times more efficiently.  

"What provides most of AAV-PHP.B's benefit is its increased ability to get through the vasculature into the brain," says Deverman. "Once there, many AAVs, including AAV9 are quite good at delivering genes to neurons and glia."

Gradinaru notes that since AAV-PHP.B is delivered through the bloodstream, it reaches other parts of the body. "Although in this study we were focused on the brain, we were also able to use whole-body tissue clearing to look at its biodistribution throughout the body," she says.

Whole-body tissue clearing by PARS CLARITY, a technique developed previously in the Gradinaru lab to make normally opaque mammalian tissues transparent, allows organs to be examined without the laborious task of making thin slide-mounted sections. Thus, tissue clearing allows researchers to more quickly screen the viral vectors for those that best target the cells and organs of interest.

"In this case, the priority was to express the gene in the brain, but we can see by using whole-body clearing that you can actually have expression in many other organs and even in the peripheral nerves," explains Gradinaru. "By making tissues transparent and looking through them, we can obtain more information about these viruses and identify targets that we might overlook otherwise."

The biologists conducted follow-up studies up to a year after the initial injections and found that the protein continued to be expressed efficiently. Such long-term expression is important for gene therapy studies in humans. 

In collaboration with colleagues from Stanford University, Deverman and Gradinaru also showed that AAV-PHP.B is better than AAV9 at delivering genes to human neurons and glia.

The researchers hope to begin testing AAV-PHP.B's ability to deliver potentially therapeutic genes in disease models. They are also working to further evolve the virus to make even better performing variants and to produce variants that target certain cell types with more specificity.

Deverman says that the CREATE system could indeed be applied to develop AAVs capable of delivering genes specifically to many different cell types. "There are hundreds of different Cre transgenic lines available," he says. "Researchers have put Cre recombinase under the control of gene regulatory elements so that it is only made in certain cell types. That means that regardless of whether your objective is to target liver cells or a particular type of neuron, you can almost always find a mouse that has Cre recombinase expressed in those cells."

"The CREATE system gave us a good hit early on, but we are excited about the future potential of using this approach to generate viruses that have very good cell-type specificity in different organisms, especially the less genetically tractable ones," says Gradinaru. "This is just the first step. We can take these tools and concepts in many exciting directions to further enhance this work, and we—with the Beckman Institute and collaborators—are ready to pursue those possibilities." 

The Beckman Institute at Caltech recently opened a resource center called CLOVER (CLARITY, Optogenetics, and Vector Engineering Research Center) to support such research efforts involving tissue clearing and imaging, optogenetic studies, and custom gene-delivery vehicle development. Deverman is the center's director, and Gradinaru is the principal investigator.

Additional Caltech authors on the paper, "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain," are Sripriya Ravindra Kumar, Ken Y. Chan, Abhik Banerjee, Wei-Li Wu, and Bin Yang, as well as former Caltech students Piers L. Pravdo and Bryan P. Simpson. Nina Huber and Sergiu P. Pasca of Stanford University School of Medicine are also coauthors. The work was supported by funding from the Hereditary Disease Foundation and the Caltech-City of Hope Biomedical Initiative, a National Institutes of Health (NIH) Director's New Innovator Award, the NIH's National Institute of Aging and National Institute of Mental Health, the Beckman Institute, and the Gordon and Betty Moore Foundation.

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Kimm Fesenmaier
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Delivering Genes Across the Blood-Brain Barrier
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Caltech biologists have developed a vector capable of noninvasive delivery of genetic cargo throughout the adult central nervous system.

Rosens Recharge Support for Bioengineering

Caltech board chair emeritus and longtime Compaq chairman Benjamin M. (Ben) Rosen (BS '54) and his wife, Donna, have made a bequest commitment to advance scientific exploration at the intersection of biology and engineering. It is anticipated that the couple's latest gift may double the endowment for the Donna and Benjamin M. Rosen Bioengineering Center.

Established in 2008 with $18 million from the Benjamin M. Rosen Family Foundation of New York, the Rosen Center has become a hub for research and educational initiatives that bring together applied physics, chemical engineering, synthetic biology, computer science, and more.

"Just as we had the digital revolution in the last century, we are having a biological sciences revolution in this century," Ben Rosen says. "And Caltech is the place to be."

Read more on the Caltech giving site.

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Rosens Recharge Support for Bioengineering
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Caltech board chair emeritus Ben Rosen (BS ’54) and his wife Donna have made a commitment to scientific exploration at the intersection of biology and engineering.

A Healthy Start

Science and medicine, it would seem, have always gone hand in hand. But for centuries, they were actually two very disparate fields. Identifying a need for "investigators who are well trained in both basic science and clinical research," the National Institutes of Health (NIH) created the Medical Scientist Training Program (MSTP) in 1964 to help streamline completion of dual medical and doctoral degrees. The purpose of developing this highly competitive MD/PhD program was to support "the training of students with outstanding credentials and potential who are motivated to undertake careers in biomedical research and academic medicine."

Recognizing Caltech's strength in the biological and chemical sciences, UCLA—which first established an MSTP in 1983—formed an affiliation with the Institute in 1997 to offer an average of two students the opportunity to perform graduate research at the partner school through the MSTP; PhD thesis work is done at Caltech for UCLA medical students, and when completed they return to UCLA to finish their MD studies.

The vast majority of alumni who have completed their postgraduate training are actively involved in biomedical research as physician-scientists at outstanding research institutions across the country. Although the MSTP represented the first formal affiliation between UCLA and Caltech, the success of the combined UCLA-Caltech MSTP spearheaded and served as a model for several other joint efforts that benefit from the complementary strengths of the two institutions, including the Specialized Training and Advanced Research (STAR) fellowship program for physician-scientists, and the Institute for Molecular Medicine.

A joint program with the University of Southern California soon followed. In 1998, the Kenneth T. and Eileen L. Norris Foundation awarded Caltech funding to support a joint MD/PhD program with the Keck School of Medicine of USC.

The grant established the Norris Foundation MD/PhD Scholars Fund, which supports Caltech PhD candidates from Keck. Administered by Caltech in cooperation with USC, the program accepts two students each year. As with the UCLA program, students spend their first two years in medical school, taking preclinical science courses, with summers spent at Caltech gaining exposure to the academic research environment. They then come to Caltech, spending three to five years on their PhDs before returning to their medical school for the final two clinical years.

The late Caltech biologist Paul Patterson, who passed away in 2014, was instrumental in developing the joint degree program. He believed that Caltech graduate students should also have an opportunity to explore their work in a clinical setting.

"Paul showed creativity both in curriculum development, in student mentoring, and in bringing the Caltech faculty together to support a program, which was in collaboration with another major institution," says Richard Bergman, director of the Cedars-Sinai Diabetes and Obesity Research Institute, who helped Patterson form the initial collaboration with USC. "His contributions in this regard educated several generations of students who, today, continue to make important contributions to medical science. This was a great legacy of Professor Patterson."

Additional funding for students in the MD/PhD programs has come from a provost-directed endowed fund called the W. R. Hearst Endowed Scholarship for MD/PhD Students; from the Lee-Ramo Life Sciences Fund; and through lab support for medical research from the W. M. Keck Foundation Fund for Discovery in Basic Medical Research. The Division of Biology and Biological Engineering also provides support to students and scholars who are headed for careers in medicine through an endowed fund from the Walter and Sylvia Treadway Foundation.

Since the start of the two MD/PhD programs, 64 students have been accepted to work toward dual degrees, and 40 have received PhDs from Caltech.

This story was reprinted from the Winter 2015 E&S magazine. See the full issue online.

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A Healthy Start
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Explore the origins of Caltech's joint MD/PhD programs, which help students develop expertise in both basic science and clinical research.

Identification Tags Define Neural Circuits

The human brain is composed of complex circuits of neurons, cells that are specialized to transmit information via electrochemical signals. Like the circuits in a computer, these neuronal circuits must be connected in particular ways to function properly. But with billions of neurons in a single human brain, how does a neuron make the right connections with the right cells?

Biologists have long searched for some kind of cellular "identification tags" that label which cells should form connections. Now, researchers from the laboratory of Caltech professor of biology Kai Zinn have identified molecules that act like identification tags on neurons in the fruit fly Drosophila. They discovered that proteins from two different molecular subfamilies, called Dpr and DIP proteins, bind together selectively. This binding can cause neurons that express Dpr proteins to form connections with neurons that express the corresponding DIP protein, playing an important role in directing the development of the neuromuscular and visual systems in growing Drosophila.

A paper detailing the findings is published in the December 17 issue of the journal Cell.

In 2013, a collaboration between Christopher Garcia's structural biology group at Stanford and the Zinn group at Caltech mapped the interactions between all 200 different Drosophila cell surface proteins. By separating the proteins from the cell and observing their interactions in a test tube, the group determined which proteins bind together. The group developed a complex model of interacting proteins they called the interactome. This work showed that a 21-member subfamily of "immunoglobulin superfamily" proteins, the Dprs, selectively bind to a 9-member subfamily called DIPs.

"Certain members of the Dprs and the DIPs match up and bind together—kind of like a lock and key—in a test tube," says Zinn. "We wanted to know if they would bind in vivo, in the Drosophila brain, and if that binding would then determine where synapses were formed."

A synapse is a junction where the wire-like axon of one neuron meets the branched dendrites of another. Information, in the form of chemical signals called neurotransmitters, is passed between neurons across these synapses. "We wanted to know if these interacting proteins on the surface of neuronal cells affected the way that the cells themselves interacted," says Robert Carrillo, a postdoctoral scholar in the Zinn group and co-first author on the new paper. "We showed that neural cells that expressed matching proteins often formed synapses with each other, and we theorized that the interaction between these molecules was driving the formation of synapses."

To test this theory, the Zinn group used the well-studied Drosophila visual system to determine the effects of these proteins on development. Neurons in the fly's eye send axons into layered structures in the visual part of the brain, which is known as the optic lobe. One of these structures, the medulla, is divided into ten layers, and each optic lobe neuron forms synapses within a specific subset of these layers. By removing certain DIP and Dpr proteins in the fly pupa, the researchers caused the axons to "overshoot" their target layers. Additionally, they observed developmental defects in the fly's neuromuscular system when removing the same proteins. Another paper in the same issue of Cell, from Larry Zipursky's group at UCLA, also found that expression of Dprs and DIPs correlates with the patterns of synaptic connectivity in the brain.

This finding helps to validate a theory proposed in the 1950s by the late Caltech professor and Nobel Laureate Roger Sperry. Experimenting mostly with fish and frog brains, Sperry discovered that he could manipulate or cut axons between neurons, and the cells would still re-form the right connections.

"Sperry hypothesized that individual neurons must carry some kind of identification tags, whose recognition is used to create the synaptic circuits of the brain," says Kaushiki Menon, a senior postdoctoral scholar in the Zinn group and a co-first author on the paper. "Our group has shown that the Drosophila Dpr and DIP proteins fit the definition of Sperry's proposed cellular identification tags."

Such tinkering with the brain's circuitry is possible because flies, unlike humans, have brains that are predominantly "hard-wired." "In mammals, the brain has a basic initial scaffold laid down by genetics, and then over time there is a lot of complicated experience-dependent rearrangement. Essentially, the brain can rewire itself through experience," Zinn says. "Fly brains can't do that."

While their findings are not immediately generalizable to mammals, Zinn and his group hope that they can provide a starting point to probe the structure of the human brain. "We hope that there might be protein networks that function similarly in humans, and these could be relevant to an understanding of how the scaffold of the human brain that exists at birth is assembled through genetics."

The paper is titled "Control of synaptic connectivity by a network of Drosophila IgSF cell surface proteins." In addition to Carrillo and Menon, structural biologist Engin Özkan at the University of Chicago is a  co-first author. The work was funded by the National Institutes of Health and the Howard Hughes Medical Institute.

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Lori Dajose
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Identification Tags Define Neural Circuits
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Biologists have identified a network of proteins that guides neural synapse formation in Drosophila brains.

Unlocking the Chemistry of Life

In just the span of an average lifetime, science has made leaps and bounds in our understanding of the human genome and its role in heredity and health—from the first insights about DNA structure in the 1950s to the rapid, inexpensive sequencing technologies of today. However, the 20,000 genes of the human genome are more than DNA; they also encode proteins to carry out the countless functions that are key to our existence. And we know much less about how this collection of proteins supports the essential functions of life.

In order to understand the role each of these proteins plays in human health—and what goes wrong when disease occurs—biologists need to figure out what these proteins are and how they function. Several decades ago, biologists realized that to answer these questions on the scale of the thousands of proteins in the human body, they would have to leave the comfort of their own discipline to get some help from a standard analytical-chemistry technique: mass spectrometry. Since 2006, Caltech's Proteome Exploration Laboratory (PEL) has been building on this approach to bridge the gap between biology and chemistry, in the process unlocking important insights about how the human body works.

Scientists can easily sequence an entire genome in just a day or two, but sequencing a proteome—all of the proteins encoded by a genome—is a much greater challenge says Ray Deshaies, protein biologist and founder of the PEL. "One challenge is the amount of protein. If you want to sequence a person's DNA from a few of their cheek cells, you first amplify—or make copies of—the DNA so that you'll have a lot of it to analyze. However, there is no such thing as protein amplification," Deshaies says. "The number of protein molecules in the cells that you have is the number that you have, so you must use a very sensitive technique to identify those very few molecules."

The best means available for doing this today is called shotgun mass spectrometry, Deshaies says. In general, mass spectrometry allows researchers to identify the amount and types of molecules that are present in a biological sample by separating and analyzing the molecules as gas ions, based on mass and charge; shotgun mass spectrometry—a combination of several techniques—applies this separation process specifically to digested, broken-down proteins, allowing researchers to identify the types and amounts of proteins that are present in a heterogeneous mixture.

"Up until this technique was invented, people had to take a mixture of proteins, run a current through a polyacrylamide gel to separate the proteins by size, stain the proteins, and then physically cut the stained bands out of the gel to have each individual protein species sequenced," says Deshaies. "But mass spectrometry technology has gotten so good that we can now cast a broader net by sequencing everything, then use data analysis to figure out what specific information is of interest after the dust settles down."

For more about the PEL, visit E&S+.

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Unlocking the Chemistry of Life
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Caltech has an advantage in the quest to decipher details of the human proteome—the proteins encoded by the human genome.

Popping Microbubbles Help Focus Light Inside the Body

A new technique developed at Caltech that uses gas-filled microbubbles for focusing light inside tissue could one day provide doctors with a minimally invasive way of destroying tumors with lasers, and lead to improved diagnostic medical imaging.

The primary challenge with focusing light inside the body is that biological tissue is optically opaque. Unlike transparent glass, the cells and proteins that make up tissue scatter and absorb light. "Our tissues behave very much like dense fog as far as light is concerned," says Changhuei Yang, professor of electrical engineering, bioengineering, and medical engineering. "Just like we cannot focus a car's headlight through fog, scientists have always had difficulty focusing light through tissues."

To get around this problem, Yang and his team turned to microbubbles, commonly used in medicine to enhance contrast in ultrasound imaging.

The gas-filled microbubbles are encapsulated by thin protein shells and have an acoustic refractive index—a property that affects how sound waves propagate through a medium—different from that of living tissue. As a result, they respond differently to sound waves. "You can use ultrasound to make microbubbles rapidly contract and expand, and this vibration helps distinguish them from surrounding tissue because it causes them to reflect sound waves more effectively than biological tissue," says Haowen Ruan, a postdoctoral scholar in Yang's lab.

In addition, the optical refractive index of microbubbles is not the same as that of biological tissue. The optical refractive index is a measure of how much light rays bend when transitioning from one medium (a liquid, for example) to another (a gas).

Yang, Ruan, and graduate student Mooseok Jang developed a novel technique called time-reversed ultrasound microbubble encoded (TRUME) optical focusing that utilizes the mismatch between the acoustic and optical refractive indexes of microbubbles and tissue to focus light inside the body. First, microbubbles injected into tissue are ruptured with ultrasound waves. By measuring the difference in light transmission before and after such an event, the Caltech researchers can modify the wavefront of a laser beam so that it is focuses on the original locations of the microbubbles. The result, Yang explains, "is as if you're searching for someone in a dark field, and suddenly the person lets off a flare. For a brief moment, the person is illuminated and you can home in on their location."

In a new study, published online November 24, 2015, in the journal Nature Communications, the team showed that their TRUME technique could be used as an effective "guidestar" to focus laser beams on specific locations in a biological tissue. A single, well-placed microbubble was enough to successfully focus the laser; multiple popping bubbles located within the general vicinity of a target functioned as a map for the light.

"Each popping event serves as a road map for the twisting light trajectories through the tissue," Yang says. "We can use that road map to shape light in such a way that it will converge where the bubbles burst."

If TRUME is shown to work effectively inside living tissue—without, for example, any negative effects from the bursting microbubbles—it could enable a range of research and medical applications. For example, by combining the microbubbles with an antibody probe engineered to seek out biomarkers associated with cancer, doctors could target and then destroy tumors deep inside the body or detect malignant growths much sooner.

"Ultrasound and X-ray techniques can only detect cancer after it forms a mass," Yang says. "But with optical focusing, you could catch cancerous cells while they are undergoing biochemical changes but before they undergo morphological changes."

The technique could take the place of other of diagnostic screening methods. For instance, it could be used to measure the concentrations of a protein called bilirubin in infants to determine their risk for jaundice. "Currently, this procedure requires a blood draw, but with TRUME, we could shine a light into an infant's body and look for the unique absorption signature of the bilirubin molecule," Ruan says.

In combination with existing techniques that allow scientists to activate individual neurons in lab animals using light, TRUME could help neuroscientists better understand how the brain works. "Currently, neuroscientists are confined to superficial layers of the brain," Yang says. "But our method of optical focusing could allow for a minimally invasive way of probing deeper regions of the brain."

The paper is entitled "Optical focusing inside scattering media with time-reversed ultrasound microbubble encoded (TRUME) light." Support for the research was provided by the National Institutes of Health, the National Institutes of Health BRAIN Initiative, and a GIST-Caltech Collaborative Research Proposal.

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Microbubbles Help Focus Light Inside the Body
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A new technique could one day provide doctors with a minimally invasive way of destroying tumors with lasers, and lead to improved diagnostic medical imaging.

Viral Videos (and Bacterial Ones, Too)

Grant Jensen is a high-powered movie producer. You won't see his name on any of this fall's Hollywood blockbusters, but in the field of cell biology, he has revolutionized the view that researchers, and even the curious public, get of the insides of cells. He does this through the innovative use of a digital camera and specialized electron microscope, which together enable a field called cryo-electron microscopy, or cryo-EM.

Now, he's taking what he's learned over the past 13 years using cryo-EM and sharing it with the world through a series of online videos that serve as visual textbooks to teach to the world the skills and knowledge needed for cryo-EM studies.

Read the full story on the E&S website

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Viral Videos (and Bacterial Ones, Too)
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Grant Jensen has revolutionized the view that researchers, and even the curious public, get of the insides of cells.

Neurons Encoding Hand Shapes Identified in Human Brain

Neural prosthetic devices, which include small electrode arrays implanted in the brain, can allow paralyzed patients to control the movement of a robotic limb, whether that limb is attached to the individual or not. In May 2015, researchers at Caltech, USC, and Rancho Los Amigos National Rehabilitation Center reported the first successful clinical trial of such an implant in a part of the brain that translates intention—the goal to be accomplished through a movement (for example, "I want to reach to the water bottle for a drink")—into the smooth and fluid motions of a robotic limb. Now, the researchers, led by Richard Andersen, the James G. Boswell Professor of Neuroscience, report that individual neurons in that brain region, known as the posterior parietal cortex (PPC), encode entire hand shapes which can be used for grasping—as when shaking someone's hand—and hand shapes not directly related to grasping, such as the gestures people make when speaking.

Most neuroprostheses are implanted in the motor cortex, the part of the brain controlling limb motion. But the movement of these robotic arms are jerky, probably due to the complicated mechanics for controlling muscle movement. Having eliminated that problem by implanting the device in the PPC, the brain region that encodes the intent, led Andersen and colleagues to further investigate the role specific neurons play in this part of the brain.

The research appears in the November 18 issue of the Journal of Neuroscience.

"The human hand has the ability to do numerous complex operations beyond just grasping," says Christian Klaes, a postdoctoral fellow at Caltech and first author of the paper. "We gesture when we speak, we manipulate objects, we use sign language to communicate with the hearing impaired. Tetraplegic patients rate hand and arm function to be of the highest importance to have better control over their environment. So our ultimate goal is to improve the range of neuroprostheses using control signals from the PPC.

"The more precisely we can identify individual neurons involved with hand movements, the better the capability these robotic devices will provide. Ultimately, we hope to mimic in a robotic hand the same freedom of movement of the human hand."

In the study, the researchers used the rock-paper-scissors game and a variation, rock-paper-scissors-lizard-Spock. The game, says Andersen, is "perfect" for this kind of research. "The addition of a lizard, depicted as a cartoon image of a lizard, and Spock—a picture of Leonard Nimoy in character—was to increase the repertoire of possible hand shapes available to our tetraplegic participant, Erik G. Sorto, whose limbs are completely paralyzed. We assigned a pinch gesture for the lizard and a spherical shape for Mr. Spock."

The game was played in two phases, first rock-paper-scissors and then the expanded game with the lizard and Spock. In the task, Sorto was briefly shown an object on a screen that corresponded to one of the hand shapes—for example, a picture of a rock or Mr. Spock. The image was followed by a blank screen, and then text appeared instructing Sorto to imagine making the corresponding hand shape with his right hand—a fist for the rock, an open hand for paper, a scissors gesture for scissors, a pinch for the lizard, and a spherical shape (loosely analogous to the Vulcan salute) for Spock—and to say which visual image he had seen, as the neuroprosthetic device recorded the activity of neurons in the PPC.

The researchers were able to identify single neurons in the PPC that fired when Sorto was presented with an image of an object to be grasped—a rock, say—and identified a nearly completely separate class of neurons that responded when Sorto engaged in motor imagery (the mental planning and imagined execution of a movement without the subject actually trying to move the limb).

"We found two mostly separate populations of neurons in the PPC that show either visual responses or motor-imagery responses during the task, the former when Erik identified a cue and the latter when he imagined performing a corresponding hand shape," says Andersen.

The researchers discovered that individual neurons in the PPC also responded to hand shapes that did not directly correspond to a grasp-related visual stimulus. The paper shape can be related to the initial opening of the hand to grasp a paper, and the rock closing the hand to grasp a rock—and in fact, these imagined hand shapes were used by Sorto to imagine opening a robotic hand by imagining paper and closing the robotic hand around an object by imagining rock. However, scissors, lizard, and Spock call for imagining hand gestures that are more abstract and iconic than those needed to grasp the visual objects, and suggests, says Andersen, that this area of the brain may also be involved in more general hand gestures, such as ones we use when talking, or for sign language.

The results of the trial were published in a paper titled, "Hand Shape Representations in the Human Posterior Parietal Cortex." In addition to Andersen and Klaes, other authors on the study are Spencer Kellis, Tyson Aflalo, and Kelsie Pejsa from Caltech; Brian Lee, Christi Heck, and Charles Liu from USC; and Kathleen Shanfield, Stephanie Hayes-Jackson, and Mindy Aisen from Rancho Los Amigos National Rehabilitation Center.

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The neurons, identified through brain studies using the game rock-paper- scissors-lizard-Spock, may lead to improved prosthetic devices.

Choosing the T-Cell Profession: Higher Education for Stem Cells

Watson Lecture Preview

Your body is continuously making new blood cells from a reservoir of "starter" cells called stem cells. Blood cells come in many types, including the highly versatile T cells that play a number of key roles in the immune system. All stem cells are alike, and all the T cells that come from them start out alike before choosing specific careers in response to signals from their environment.

On Wednesday, November 18 at 8 p.m. in Caltech's Beckman Auditorium, Ellen Rothenberg, Caltech's Albert Billings Ruddock Professor of Biology, will lead us along the paths that T cells follow and show how her lab has mapped their journeys. Admission is free.

What do you do?

I'm interested in how cells choose their identities through reading out information stored in the genome, which is the entire collection of DNA that makes a creature what it is, and how a cell that begins with one identity can spawn descendants with very different, very durable new identities.

We study T cells, a large family of white blood cells that form a major part of your immune system. T cells have an extremely long and varied life. They come from so-called stem cells, which have the ability to become many, many different kinds of cells. We want to learn how a "blank slate" of a stem cell develops to achieve a rock-solid identity as a T cell—especially because a T cell has an irreversibly defined "T-cell-ness" at its core, yet it remains very dynamic in using genomic information to decide what kind of T cell it will be.

Generating T cells is a three-step process. First, a stem cell develops into a T cell. Second, the T cell circulates around the body, waiting to see how it will first be used by the body to fight an actual infection. And then third, once it has evolved a specialization, it will continue to go around the body for months, years, or even decades in humans, spawning descendants that are also specialized with the same specific type of cellular function the original T cell had when it was activated—as helper T cells, or killer T cells, or whatever other type of T cell was needed. And they may pick a subspecialty—for example, for every infectious agent you encounter, you develop a specific memory cell to recognize that particular bug so that if it comes around again you are ready for it.

Once made, the decisions are locked in. All the T cell's progeny will generally stay in "the family business." However, it sometimes happens that once a T cell has chosen its profession, a particularly strong environmental signal can drive it to change into a different type of T cell. But even so, it will never, ever go back to being a stem cell. My lab is trying to figure out the molecular control mechanisms that allow the former stem cell to achieve a new rock-solid identity as a T cell, yet maintain a level of flexibility within that T-cell-ness.

Why is this important?

I study biology for the same reason that astronomers study the universe. I believe that there are deep biological principles to be learned from T cells, whose import goes way beyond curing a particular disease. I'm ecstatic when things we do are picked up by clinicians, who do make a profession of helping people, but I do basic science.

There are two main branches to the developmental biology of multicellular organisms. The first goes from the fertilized egg through the embryo, and that's the process that makes your body in the first place. It follows well-known rules worked out by people like my late colleague Eric Davidson [Caltech's Norman Chandler Professor of Cell Biology].

I study a second form of development that begins when an embryo sets aside a bunch of cells and programs them to become stem cells. Stem cells do not differentiate further right away; they just make more copies of themselves. Then, whenever you need to make new blood cells or repair a tissue later in life, those cells are called into action. For example, red blood cells only last about three or four months, so the blood circulating in your body today is coming from stem cells, and those stem cells were "set aside" when you were a fetus. This means there's an additional set of rules, going well beyond embryonic development, for making new blood cells in the right balance and at the right time.

The new cells do have some wear and tear from the consequences of your adventures throughout your life, but to a first approximation they're the same. They're getting primed to do the same job. They have to set up all the molecular circuitry needed to retain their identity and maintain a clear one-directional flow from stem-ness to differentiation. The process has to be as accurate at our advanced ages as it was when we were fetuses. That's the genius of stem-cell-based developmental biology. In my view, the collection of stem-cell development mechanisms ranks right up there with the more established mechanisms of embryonic development.

How did you get into this line of work?

I've always been interested in science. The question when I was young was whether I wanted to be a physicist or a biologist, but then I fell completely in love with biochemistry when I was in high school. When I went off to Harvard I didn't know specifically what I was interested in, but I loved what was known about the genome. I thought it would be fantastic to understand how the genome works at a molecular, mechanistic level.

I had the great good fortune to have microbiologist Boris Magasanik as my undergraduate tutor and mentor. He was the head of MIT's biology department, but he had a relationship with Harvard and he liked teaching undergrads. Boris was an extraordinary intellectual. He was studying metabolic pathways in bacteria at the systems-biology level way before it was normal. He was drawing prototype diagrams of gene-regulatory networks back in the early '70s.

A lot of technology had to be invented before we could explain gene regulation on the molecular level, but when I became a graduate student in [Nobel Laureate] David Baltimore's lab at MIT in 1972, he was already doing incredible work on viral genomes. [Baltimore came to Caltech in 1997 and is currently the Robert Andrews Millikan Professor of Biology.] We were pushing the frontiers of knowledge outward on a daily basis, and it was exceptionally exciting.

However, the development of multicelled organisms was still extremely hard to understand back then. It seemed all anecdotal, as if every organism did things in a fundamentally different way. But by the late '70s, Eric Davidson here at Caltech was making it possible to make sense out of developmental systems. His views integrated Boris Magasanik's systems-level view with David Baltimore's molecular-level finesse, and his work was revealing general mechanisms of development in multicellular organisms. I owe a great deal to the conceptual and mechanistic perspectives that I have gotten from these three people.

Also, Caltech's smallness has been fantastic. Most of the people I know who work with T cells are in immunology departments, and most immunologists do the same kinds of things, more or less. The joy for me at Caltech has been doing things that nobody else is doing. Often when my colleagues here solve their problems, I can use those approaches to break new ground in my field. It's been extraordinarily fun, and a tremendous advantage. Science as it should be done.

Long ago at MIT, my labmates and I were studying a retrovirus that caused early T-cell leukemia in mice. Lots of retroviruses cause cancer by putting a gene responsible for normal cell growth into the host cell and then turning the gene on under the wrong conditions. But our retrovirus didn't cause cancer in other cell types, so we wondered why it affected early T cells. I realized that the T-cell development process itself must be an especially sensitive target. The retrovirus nudged the future T cells toward being cancerous, possibly by accident, and then a little push farther down the line would send them over the edge.

That's when I became interested in T-cell development and this question of what controlled the switchover between growth and differentiation. We've found in the last 10 years or so that there are actually two bursts of proliferation during T-cell development. My lab has focused on the first one, which we now know is the transition between stem-cell-ness and T-cell-ness, when the cell commits to becoming a T cell. And it turns out that if a stem-cell regulatory gene stays on during the process, you get an abnormal persistence of stem-cell-like growth and sometimes leukemia. It's ironic that it's taken me, gosh, 40 years to get back to that, but it has been an incredibly satisfying journey.

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T Cells Get Schooled
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Choosing the T-Cell Profession: Higher Education for Stem Cells
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The road to becoming a T cell is fraught with choices, false starts, and dead ends, where a regulatory tug-of-war brings cells close to the border of leukemia.

Yuki Oka Awarded Mallinckrodt Grant

Yuki Oka, an assistant professor of biology, has been awarded a grant from the Edward Mallinckrodt, Jr. Foundation, given to "support early stage investigators engaged in biomedical research that has the potential to significantly advance the understanding, diagnosis, or treatment of disease," according to the foundation website. The grant will provide $60,000 per year for three years.

"I'm thrilled by being selected for the 2015 Mallinckrodt Grant," says Oka, whose lab uses thirst and water-drinking behavior as a simple model system to study how the brain monitors internal water balance and generates signals that drive appetitive behaviors. The long-term goal of the work is to understand how the brain integrates information about the internal body state and external sensory information to maintain homeostasis (a state of internal equilibrium). The research, he notes, will provide a framework for studying the mechanisms that govern innate behaviors such as eating and drinking. Currently, an estimated 30 million people in the U.S. suffer from appetite disorders including polydipsia and bulimia, characterized by excessive water and food intake, respectively. Identifying neural circuits underlying appetite may offer insights into safe treatments for associated disorders, he says.

Oka received his PhD from the University of Tokyo and was a postdoctoral researcher at UC San Diego and Columbia University before joining the Caltech faculty in 2014. He was named a Searle Scholar in April 2015.

Past Mallinckrodt grantees from Caltech include Sarkis Mazmanian, the Luis B. and Nelly Soux Professor of Microbiology; David Prober, assistant professor of biology; Mitchell Guttman, assistant professor of biology; and Viviana Gradinaru, assistant professor of biology and biological engineering.

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Yuki Oka, an assistant professor of biology, has been awarded a grant from the Edward Mallinckrodt, Jr. Foundation.

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