Simpler technique yields antibodies to a range of infectious agents

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Credit: Graphic by Jason Drees for the Biodesign Institute.

Researchers hope to develop vaccines, therapeutics and new diagnostic tests for a broad range of diseases. To accomplish this, they will need to gain a much better understanding of a critical class of biological components. Known as surface membrane proteins, these vital ingredients in the disease process form a structurally and functionally diverse assemblage of enormous complexity.

In a new study, Debra Hansen, a research professor at Arizona State University’s Biodesign Institute, explores an innovative means of investigating membrane proteins produced by a pair of highly pathogenic organisms. The research team showed that DNA-based genetic immunization, using a device known as a gene gun, could successfully express membrane proteins in mice and induce the animals to produce a range of critical antibodies to bacterial and viral targets.

“We learned that our new process of antibody production is incredibly efficient. If the membrane protein is naturally immunogenic, we easily generated high levels of antibodies using the genes alone,” Hansen says. “Rather than laboriously purifying the membrane protein and attempting to maintain the proper protein structure within detergents prior to immunization, we let the immunized host do the work for us.”

The new study also describes a method for expressing and purifying membrane proteins in a test tube and examining their binding activities with specific antibodies in blood extracted from gene immunized mice.

Conventionally, producing an immune response to a foreign protein requires purification of the protein, which is then injected into an animal. The process is cumbersome, challenging and time consuming. In the current study, an immune response is instead produced by directly introducing a gene encoding the protein of interest.

Two of the membrane proteins produced in the study were also successfully introduced into membranes of the bacterium E. coli, through the process of recombinant DNA. Use of the specific antibodies present in blood from gene-immunized mice demonstrated for the first time that both membrane proteins could be recombinantly expressed in a live organism, correctly fold into proper 3-D structures and migrate to the membranes within E. coli. These results now facilitate the structural determination of these two critical virulence proteins.

The research, which appears in the current issue of the Nature Publishing Group journal Scientific Reports, promises to deliver new insights into the structure and function of membrane proteins of critical importance for medicine.

On the surface

Membrane proteins are implicated in innumerable functions in living organisms including cell signaling and communication, energy conversion and utilization, molecular transport and catalysis. Due to their involvement in a range of diseases, they have recently become primary targets for a new range of therapeutics. Indeed, more than 50 percent of all therapeutic targets are membrane proteins. The number is expected to rise as more is learned about membrane protein structure and function.

Despite their important role as the molecular interface in host/pathogen interactions as well as drug/cell relationships, membrane proteins account for less than 1 percent of the 100,000 unique protein structures presently catalogued. This is largely due to the serious challenges involved in producing, purifying, and determining the structures of membrane proteins. Hansen and her colleagues outline new strategies to produce antibodies–specialized proteins produced naturally by the immune system in response to pathogens or other threatening biological agents.

In the current study, they describe the use of a gene gun to introduce DNA information into a mouse. The handheld device uses a burst of gas to propel gold particles impregnated with circular DNAs known as plasmids into the skin in mice. The gold particles are known as micronanoplexes. The gene gun technology was pioneered and developed by Stephen A. Johnston, co-director of the Biodesign Center for Innovations in Medicine.

The genetic material introduced via the gene gun is taken up by mouse dendritic cells and translated into membrane protein in dermal tissues and lymph nodes. The mouse immune system responds by producing specific antibodies capable of binding to the membrane proteins. While the basic technique of genetic immunization has been in use for some time, the study marks the first description of the broad applicability of this approach to membrane proteins, as well as the first application of DNA-gold micronanoplexes to stimulate antibody production.

Bacterial and viral menaces examined

Results showed that genetic immunization successfully produced antibodies specific to 12 out of 17 membrane proteins from two Biosafety Level 3 pathogens: Francisella tularensis and African swine fever virus (ASFV). F. tularensis causes the disease tularemia. It is a widely studied infectious pathogen notorious for its ability to invade numerous cell types and cleverly evade the immune system. It is one of the most pathogenic bacteria on earth, capable of causing a fatal infection with as few as 10 cells. African swine fever virus is carried by arthropods. Infection in pigs causes a lethal and untreatable hemorrhagic disease that has devastated swine populations in areas of Africa and Eastern Europe.

Investigations of endogenous disease proteins produced by these organisms are difficult, requiring specialized safety facilities and protocols due to the potential danger they pose to researchers. The new method described permits the production of these pathogens’ membrane proteins and associated antibodies through DNA-based approaches, permitting the safe handling of biological material without risk of infection.

Once antibodies to specific membrane proteins have been produced in the mouse, the group sought to characterize the resulting mouse blood or sera. To do this, a new system known as in vitro translation in the presence of hydrophobic magnetic beads (IVT-HMB) was developed. Here, a small quality of membrane protein is produced in a test tube, simultaneously extracted using hydrophobic beads, then screened against the sera extracted from gene-immunized mice. Detection of a resulting signal in two types of diagnostic tests or assays (ELISA and Western blot) established the presence in the mouse sera of antibodies specific to each membrane protein. The IVT-HMB method represents a powerful streamlining of the production of membrane proteins, precluding the arduous process of isolation and purification traditionally required.

Stepping-stone to protein structures

Using X-rays to image tiny crystals of proteins is a powerful method to determine detailed protein structure, but the technique faces many challenges, including the difficulty of producing and purifying proteins that assemble properly. The current research marks a starting point for further structural characterization of membrane proteins, using such techniques as cryo-EM and X-ray crystallography.

Petra Fromme, a co-author of the new study and director of the Biodesign Institute’s Center for Applied Structural Discovery, highlights the power of the new research: “The range of antibodies produced through techniques like genetic immunization opens the door to high resolution molecular images of important membrane proteins,” she says. “The resulting antibodies assist structural determination in a variety of important ways, identifying properly assembled proteins, helping to induce proteins to assemble with other proteins into well ordered crystals and stabilizing or trapping proteins in active states that can be imaged using X-rays.”

In the next phase of research, the group plans to produce monoclonal antibodies using the same immunization process. These are essential as co-crystallization binding factors or ligands, used for the structural determination of membrane proteins via X-ray crystallography. The authors further note that the monoclonal antibodies produced through genetic immunization techniques offer attractive candidates for future therapeutics against a broad range of diseases.

This study was a combined effort of faculty in the Biodesign Institute, including Debra Hansen from the Center for Innovations in Medicine (CIM), Center for Applied Structural Discovery (CASD) and the School of Molecular Sciences (SMS), Kathryn Sykes from CIM and Petra Fromme from CASD and SMS, along with their teams of researchers and students, including: research scientists Mark Robida, Andrey Loskutov and Tien Olson, researchers Felicia Craciunescu, John-Charles Rodenberry and Hetal Patel and graduate student Xiao Wang from CIM and postdoc Katerina Dörner from SMS.

All of the necessary clones (plasmid DNAs) for applying this approach are available through the DNASU Plasmid Repository, which is housed in Biodesign’s Center for Personalized Diagnostics.

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This work was funded by the NIH (NIGMS) under the PSI:Biology program, as part of the MPID (Membrane Proteins in Infectious Diseases) U54 grant, directed by Petra Fromme.

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It’s all in our heads

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A typical political scientist is not likely to develop a research plan that employs data from national archives, survey experiments, public health data, and an fMRI study in a single dissertation. But then, Marika Landau-Wells is not your typical political scientist.

Rooted squarely at the “intersection of cognition and conflict,” Landau-Wells, a PhD student in the Security Studies Program, is using psychology and neuroscience to better understand political behavior — specifically, why we respond to perceived threats the way we do. Her interdisciplinary approach opens up a variety of avenues for gathering different types of data.

“My hope is that the theory and language and framework I’m building will help people understand why they disagree about policies made in response to perceived threats,” she says. These “threats,” she explains, can range from nuclear weapons to influxes of immigrants.

“A huge part of conflict — in the blood-and-battlefield sense, but also in the policymaking sense — comes down to not being very good at imagining why the other person thinks what they do,” she adds. “Until they do, the two sides will continue to talk past each other. This can mean the continuation of a real war or of policy deadlock.

Landau-Wells envisions a career in academia, ideally with the chance to do intervention evaluations for organizations like the World Bank that bring “a cognitive-science-informed point of view” to political problems.

The path from Harvard University undergrad to MIT doctoral candidate involved a number of enlightening side-trips along the way, including performing corporate strategy and acquisitions for the Walt Disney Company in her native Los Angeles, and consulting in a range of industries for Bain and Company in London.

“I spent a lot of time in the private sector watching how people make decisions — not life-or-death ones but certainly very expensive ones,” she says. “It seemed to me that the findings from psychology, often just basic heuristics and biases, went a long way toward explaining the decisions I saw. Rational choice and pure economic modeling didn’t.”

Between her stints at Disney and Bain, Landau-Wells earned an MS in global politics from the London School of Economics. “I was very interested in civil wars,” she says, “and in questioning rationalist arguments. I hadn’t found those arguments compelling in the contexts where they should be most applicable — in business — and I found them even less compelling in contexts like warfare, where we know intuitively there’s a lot more at stake than money. People are willing to die for all kinds of things. Limiting [war] to a rational choice framework oversimplifies the problem.”

She decided to educate herself on the cognitive side of things. “I read up on psychology and neuroscience to learn what insights those fields might have that would help me understand the political science problems I was interested in, and found collaborators willing to work with me.” Since coming to MIT, she has become an active member of the Neuroscience and Social Conflict Initiative, a collaboration with Beyond Conflict, a Boston-based non-governmental organization dedicated to finding innovative strategies for conflict resolution and reconciliation.

In her dissertation, which focuses on how people perceive threats — threats posed by others’ identities or ideology — Landau-Wells continues to examine political behavior through the lens of neuroscience and psychology. “It’s worth understanding why we see the responses to perceived threats that we do,” she says. “I think threat-and-response logic drives a lot of political behavior. My favorite example at the moment is why so many people think that a border wall is a good idea in the U.S. I believe it’s something that can be explained, and not just by resorting to the simplistic explanation that the people who think these things are not as smart as the people who disagree with them.”

She would like to take her research to Europe, where immigration is an acute problem. “I hope that by better explaining the sources of these preferences,” she says, “we can actually advance the debates on these types of issues.”

Another hope is that her work will highlight why regarding certain groups as “social contaminants” is problematic, and how we can stop. “Humans are hierarchical; that’s not going to go away,” says Landau-Wells. “But how we treat people we perceive as being at the bottom is very much a social agreement. Talking about people as ‘vermin’ implicitly justifies policies that are brutally exclusionary. If we can become more self-aware about how we use contaminant heuristics, then maybe we can avoid the really negative consequences.”

Landau-Wells’s “cautious optimism” stems from a resistance to accept things as they are — and from a creative turn of mind. She recently took two semesters of creative writing at MIT, and her short story, “Remote Operations,” won a prize sponsored by the Atlantic Council last year. The notion of “worldbuilding” that is central to fantasy fiction — constructing an imaginary world, with its own geography, laws of physics, and history — resonated with her: It was a good way to think about what is really fixed and what is a working assumption in terms of political behavior. She uses worldbuilding in her teaching, especially around public policy.

“[Students] come in thinking so much of the world works like gravity,” she says. “I show them how little of the world works that way and how much influence they can have. It’s not to disabuse their ideals. It’s to show them the world doesn’t work optimally or automatically, but there’s plenty of room for agency.”

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Pigs’ genetic code altered in bid to tackle deadly virus

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Credit: Norrie Russell, The Roslin Institute, University of Edinburgh

Researchers have made an advance in the fight against a deadly virus that affects pigs.

The team used advanced genetic techniques to produce pigs that are potentially resilient to African Swine Fever — a highly contagious disease that kills up to two-thirds of infected animals.

The new pigs carry a version of a gene that is usually found in warthogs and bush pigs, which researchers believe may stop them from becoming ill from the infection.

African Swine Fever is spread by ticks. When standard farmed pigs are infected, they quickly become ill and die, but warthogs and bush pigs show no disease symptoms when infected.

The research is focused on one of the pig genes associated with African Swine Fever Virus infection called RELA. The gene causes the immune system to overreact with devastating effects.

Warthogs and bush pigs carry a different version of the RELA gene from that found in farmed pigs. Scientists believe that this variant — known as an allele — may dampen their immune response and explain why they are more resilient to African Swine Fever.

Researchers at the University of Edinburgh’s Roslin Institute used a gene-editing technique to modify individual letters of the pigs’ genetic code. By changing just five letters in their RELA gene, they converted it to the allele that is found in the warthog.

The work builds on previous research from the team, which used similar techniques to produce pigs with a single letter of their genetic code altered. These animals produce a shorter version of RELA.

This latest study marks the first time researchers have successfully swapped alleles in an animal’s genetic code using gene editing.

All of these changes to the pig’s genetic code could have occurred spontaneously in nature.

Scientists will now conduct controlled trials to test whether the genetic changes have improved the pigs’ resilience to the disease.

African Swine Fever is endemic in Sub-Saharan Africa and some areas of Russia. The disease has never been found in the UK, although recent outbreaks in Eastern Europe have raised concerns amongst farming groups that it could spread.

The study — published in the journal Scientific Reports — involved collaboration between scientists at The Roslin Institute and Sangamo Biosciences Inc. It was funded by Genus plc and the Biotechnology and Biological Sciences Research Council (BBSRC). The Roslin Institute receives strategic support from the BBSRC.

Professor Bruce Whitelaw, Head of Developmental Biology at the University of Edinburgh’s Roslin Institute, said: “Our goal is to improve the welfare of farmed pigs around the world, making them healthier and more productive for farmers.”

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MIT senior takes on double major in brain and cognitive sciences plus theater arts

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Abra Shen didn’t expect to graduate from MIT with a theater degree, but she couldn’t resist adding theater arts to her major in brain and cognitive sciences when the opportunity presented itself. In fact, while the theater program at MIT has existed for a number of years, she will be MIT’s first official theater arts major.

During the past four years, Shen has pursued both science and theater in equal parts, and whether she is helping cancer patients transition to life after treatment or directing a musical, she does not hesitate to throw herself completely into whatever she is doing.

Exploring neuroscience and medicine

Shen, who grew up in Toronto, Ontario, was in middle school when she came across a book called Phantoms in the Brain: Probing the Mysteries of the Human Mind that jump-started her interest in neuroscience. In particular, she was fascinated by neuroscientist V. S. Ramachandran’s descriptions of how he cared for patients with neurological diseases.

“I thought wow, science is really interesting, neuroscience is really great, and medicine has this creative component to it,” she says.

Shen followed her interest in neuroscience to MIT, where she spent her first two years conducting Alzheimer’s research with Li-Huei Tsai, the Picower Professor of Neuroscience. The Tsai lab uses mouse models to explore the memory areas of the brain, and in one study, her team employed a technique known as optogenetics to inject a light-sensitive virus into neurons in different brain regions of mice including the hippocampus and prefrontal cortex. Once the virus was injected, the neurons could be activated or deactivated by turning a light on or off, resulting in either an improved memory or severe memory loss.

The experience allowed Shen to appreciate the many opportunities MIT undergraduates have to participate in cutting-edge research and technology.

“I think that's a huge benefit of going to a place like MIT,” Shen says. “Professors have great connections and great resources, and are involved at the forefront of what's going on.”

Shen also spent two summers at the Princess Margaret Cancer Centre in Toronto. There, she worked with patients in the Survivorship Centre, which helps cancer patients transition from active treatment to follow-up care. Patients often struggle with this transition, particularly because many of them lack information about what to expect in the coming months.

“We worked on a project to understand the specific needs of testicular and endometrial cancer survivors: what sort of information they were looking for, what information they weren't getting, and what they needed as support to be able to transition into follow-up care and return to their regular lives,” Shen explains.

Shen’s team developed personalized documents called Survivorship Care Plans that patients fill out with their oncologists or nurses so they have an accessible, organized record of all the information they might need.

While Shen appreciates the broad scope of laboratory research, she enjoyed working in a clinical setting where she could directly improve the lives of her patients and have contact with them every day.

“I think it adds a personal touch to what you're doing, and it's a lot more encouraging and motivating when you're able to help the people that you're working with,” she explains.

Discovering a new passion

While at MIT, Shen has also developed a deep commitment to theater arts. Shen was first exposed to MIT’s theater program when, as a high school senior visiting for Campus Preview Weekend, she saw a musical performed by the theater group Next Act.

“I thought it was a great program and that the show was beautiful,” she recalls. “I fell in love and have been involved ever since.”

Shen joined Next Act as a freshman, but it was a directing class during her sophomore year that really changed her perspective on theater. The class, a three-hour workshop, was her first theater class at MIT, and it forced her to challenge herself in new ways.

“That's a period of time where I really grew as an artist, as a person,” she explains. “I became more open, more confident about sharing my own ideas, because I realized, in theater, there's not really a bad idea. Everything can be spun to be seen as really interesting, or really creative, or really different.”

The class motivated Shen to join Dramashop, another theater group on campus, and to pursue theater as her concentration for MIT’s Humanities, Arts, and Social Sciences (HASS) requirement. Her concentration expanded into a minor, which eventually became a full-blown theater arts major.

For Shen, the decision was an obvious one. “I realized that I just wanted to take theater classes forever,” she says. “Theater has been one of the most influential and important experiences that I've had at MIT.”

Experiencing the world beyond MIT

Shen has also had the opportunity to experience different cultures during her time at MIT, through the MIT Science and Technology Initiatives (MISTI) program. As a junior, she spent three weeks over winter break living with a host family in Germany and teaching biology, physics, and chemistry to German high school students. The following summer Shen, who is fluent in French, worked at a cancer center in France, where she shadowed oncologists and conducted research.

“Just being immersed in this totally different environment where people think differently, communicate differently, and the culture and the customs are very different … it helps you learn about yourself and what is important to you,” Shen says.

After graduating this May, Shen will pursue a degree in medicine, and she hopes to continue working with cancer patients. Shen sees a lot of overlap between medicine and theater, both of which she views as extremely personal fields that focus on understanding people. She also thinks that the communication skills she has developed through theater will help her interact more successfully with her patients and colleagues.

“I think that the approach that you should take to theater and medicine should be similar in that you are there engaging in that activity because it's meaningful to you and because you are emotionally attached to what you're working on,” she explains.

Shen’s ultimate goal is to merge her two interests by exploring how theater therapy can be used to help patients.

“I would love to study how being involved in theater through vocal expression and physical gestures at the same time can affect your self-confidence, your well-being, your motivation, and desire to get out again,” she says.

Right now, however, Shen is focused on the task at hand — directing “The Little Mermaid,” Next Act’s spring musical. She is currently reading through the script and music to prepare for auditions, while grappling with the artistic challenges of staging a play that takes place largely underwater. She is not sure exactly how the play will turn out, but she is excited to see what happens.

“In theater if you're doing what has been done before, that's boring,” she explains. “You always want to be trying new things.”

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Pinpointing loneliness in the brain

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Humans, like all social animals, have a fundamental need for contact with others. This deeply ingrained instinct helps us to survive; it’s much easier to find food, shelter, and other necessities with a group than alone. Deprived of human contact, most people become lonely and emotionally distressed.

In a study appearing in the Feb. 11 issue of Cell, MIT neuroscientists have identified a brain region that represents these feelings of loneliness. This cluster of cells, located near the back of the brain in an area called the dorsal raphe nucleus (DRN), is necessary for generating the increased sociability that normally occurs after a period of social isolation, the researchers found in a study of mice.

“To our knowledge, this is the first time anyone has pinned down a loneliness-like state to a cellular substrate. Now we have a starting point for really starting to study this,” says Kay Tye, the Whitehead Career Development Assistant Professor of Brain and Cognitive Sciences, a member of MIT’s Picower Institute for Learning and Memory, and one of the senior authors of the study.

While much research has been done on how the brain seeks out and responds to rewarding social interactions, very little is known about how isolation and loneliness also motivate social behavior.

“There are many studies from human psychology describing how we have this need for social connection, which is particularly strong in people who feel lonely. But our understanding of the neural mechanisms underlying that state is pretty slim at the moment. It certainly seems like a useful, adaptive response, but we don’t really know how that’s brought about,” says Gillian Matthews, a postdoc at the Picower Institute and the paper’s lead author.

Only the lonely

Matthews first identified the loneliness neurons somewhat serendipitously, while studying a completely different topic. As a PhD student at Imperial College London, she was investigating how drugs affect the brain, particularly dopamine neurons. She originally planned to study how drug abuse influences the DRN, a brain region that had not been studied very much.

As part of the experiment, each mouse was isolated for 24 hours, and Matthews noticed that in the control mice, which had not received any drugs, there was a strengthening of connections in the DRN following the isolation period.

Further studies, both at Imperial College London and then in Tye’s lab at MIT, revealed that these neurons were responding to the state of isolation. When animals are housed together, DRN neurons are not very active. However, during a period of isolation, these neurons become sensitized to social contact and when the animals are reunited with other mice, DRN activity surges. At the same time, the mice become much more sociable than animals that had not been isolated.

When the researchers suppressed DRN neurons using optogenetics, a technique that allows them to control brain activity with light, they found that isolated mice did not show the same rebound in sociability when they were re-introduced to other mice.

“That suggested these neurons are important for the isolation-induced rebound in sociability,” Tye says. “When people are isolated for a long time and then they’re reunited with other people, they’re very excited, there’s a surge of social interaction. We think that this adaptive and evolutionarily conserved trait is what we are modeling in mice, and these neurons could play a role in that increased motivation to socialize.”

Social dominance

The researchers also found that animals with a higher rank in the social hierarchy were more responsive to changes in DRN activity, suggesting that they may be more susceptible to feelings of loneliness following isolation.

“The social experience of every animal is not the same in a group,” Tye says. “If you’re the dominant mouse, maybe you love your social environment. And if you’re the subordinate mouse, and you’re being beat up every day, maybe it’s not so fun. Maybe you feel socially excluded already.”

The findings represent “an amazing cornerstone for future studies of loneliness,” says Alcino Silva, a professor of neurobiology, psychiatry, and psychology at the David Geffen School of Medicine at UCLA who was not involved in the research.

“There is something poetic and fascinating about the idea that modern neuroscience tools have allowed us to reach to the very depths of the human soul, and that in this search we have discovered that even the most human of emotions, loneliness, is shared in some recognizable form with even one of our distant mammalian relatives — the mouse,” Silva says.

The researchers are now studying whether these neurons actually detect loneliness or are responsible for driving the response to loneliness, and whether they might be part of a larger brain network that responds to social isolation. Another area to be explored is whether differences in these neurons can explain why some people prefer more social contact than others, and whether those differences are innate or formed by experience.

“There’s probably some part that could very well be determined by innate brain features, but I think probably an equal, if not greater, contribution would be from the environment in which individuals have developed,” Tye says. “These are completely open questions. We can only speculate about it at this point.”

Mark Ungless, a senior lecturer at Imperial College London, is also a senior author of the study. MIT graduate students Edward Nieh and Caitlin Vander Weele are also lead authors.

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Neuroscientists reverse autism symptoms

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Autism has diverse genetic causes, most of which are still unknown. About 1 percent of people with autism are missing a gene called Shank3, which is critical for brain development. Without this gene, individuals develop typical autism symptoms including repetitive behavior and avoidance of social interactions.

In a study of mice, MIT researchers have now shown that they can reverse some of those behavioral symptoms by turning the gene back on later in life, allowing the brain to properly rewire itself.

“This suggests that even in the adult brain we have profound plasticity to some degree,” says Guoping Feng, an MIT professor of brain and cognitive sciences. “There is more and more evidence showing that some of the defects are indeed reversible, giving hope that we can develop treatment for autistic patients in the future.”

Feng, who is the James W. and Patricia Poitras Professor of Neuroscience and a member of MIT’s McGovern Institute for Brain Research and the Stanley Center for Psychiatric Research at the Broad Institute, is the senior author of the study, which appears in the Feb. 17 issue of Nature. The paper’s lead authors are former MIT graduate student Yuan Mei and former Broad Institute visiting graduate student Patricia Monteiro, now at the University of Coimbra in Portugal.

Boosting communication

The Shank3 protein is found in synapses — the connections that allow neurons to communicate with each other. As a scaffold protein, Shank3 helps to organize the hundreds of other proteins that are necessary to coordinate a neuron’s response to incoming signals.

Studying rare cases of defective Shank3 can help scientists gain insight into the neurobiological mechanisms of autism. Missing or defective Shank3 leads to synaptic disruptions that can produce autism-like symptoms in mice, including compulsive behavior, avoidance of social interaction, and anxiety, Feng has previously found. He has also shown that some synapses in these mice, especially in a part of the brain called the striatum, have a greatly reduced density of dendritic spines — small buds on neurons’ surfaces that help with the transmission of synaptic signals.

In the new study, Feng and colleagues genetically engineered mice so that their Shank3 gene was turned off during embryonic development but could be turned back on by adding tamoxifen to the mice’s diet.

When the researchers turned on Shank3 in young adult mice (two to four and a half months after birth), they were able to eliminate the mice’s repetitive behavior and their tendency to avoid social interaction. At the cellular level, the team found that the density of dendritic spines dramatically increased in the striatum of treated mice, demonstrating the structural plasticity in the adult brain.

However, the mice’s anxiety and some motor coordination symptoms did not disappear. Feng suspects that these behaviors probably rely on circuits that were irreversibly formed during early development.

When the researchers turned on Shank3 earlier in life, only 20 days after birth, the mice’s anxiety and motor coordination did improve. The researchers are now working on defining the critical periods for the formation of these circuits, which could help them determine the best time to try to intervene.

“Some circuits are more plastic than others,” Feng says. “Once we understand which circuits control each behavior and understand what exactly changed at the structural level, we can study what leads to these permanent defects, and how we can prevent them from happening.”

Gordon Fishell, a professor of neuroscience at New York University School of Medicine, praises the study’s “elegant approach” and says it represents a major advance in understanding the circuitry and cellular physiology that underlie autism. “The combination of behavior, circuits, physiology, and genetics is state-of-the art,” says Fishell, who was not involved in the research. "Moreover, Dr. Feng's demonstration that restoration of Shank3 function reverses autism symptoms in adult mice suggests that gene therapy may ultimately prove an effective therapy for this disease."

Early intervention

For the small population of people with Shank3 mutations, the findings suggest that new genome-editing techniques could in theory be used to repair the defective Shank3 gene and improve these individuals’ symptoms, even later in life. These techniques are not yet ready for use in humans, however.

Feng believes that scientists may also be able to develop more general approaches that would apply to a larger population. For example, if the researchers can identify defective circuits that are specific for certain behavioral abnormalities in some autism patients, and figure out how to modulate those circuits’ activity, that could also help other people who may have defects in the same circuits even though the problem arose from a different genetic mutation.

“That’s why it’s important in the future to identify what subtype of neurons are defective and what genes are expressed in these neurons, so we can use them as a target without affecting the whole brain,” Feng says.

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Tracing a cellular family tree

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By combining sophisticated RNA sequencing technology with a new device that isolates single cells and their progeny, MIT researchers can now trace detailed family histories for several generations of cells descended from one “ancestor.”

This technique, which can track changes in gene expression as cells differentiate, could be particularly useful for studying how stem cells or immune cells mature. It could also shed light on how cancer develops.

“Existing methods allow for snapshot measurements of single-cell gene expression, which can provide very in-depth information. What this new approach offers is the ability to track cells over multiple generations and put this information in the context of a cell’s lineal history,” says Robert Kimmerling, a graduate student in biological engineering and the lead author of a paper describing the technique in the Jan. 6 issue of Nature Communications.

The paper’s senior authors are Scott Manalis, the Andrew and Erna Viterbi Professor of Biological Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research, and Alex Shalek, the Hermann L.F. von Helmholtz Career Development Assistant Professor of Health Sciences and Technology, an assistant professor of chemistry, and a member of MIT’s Institute for Medical Engineering and Science.

See how MIT researchers engineered a microfluidic device that traces detailed family histories for several generations of cells descended from one "ancestor."

Video: Melanie Gonick/MIT (additional video courtesy of the Manalis Lab)

Capturing cell lineage

The new method incorporates a recently developed technology called single-cell RNA-seq, which sequences the messenger RNA from a single cell. These RNAs, known collectively as the transcriptome, reveal which genes are being actively transcribed (that is, copied into messenger RNA instructions for building proteins) inside a cell at a given point in time. This helps scientists understand, for example, what makes a skin cell so different from a heart cell even though the cells share the same DNA.

“Scientists have well established methods for resolving diverse subsets of a population, but one thing that’s not very well worked out is how this diversity is generated. That’s the key question we were targeting: how a single founding cell gives rise to very diverse progeny,” Kimmerling says.

To try to answer that question, the researchers designed a microfluidic device that traps first an individual cell and then all of its descendants. The device has several connected channels, each of which has a trap that can capture a single cell. After the initial cell divides, its daughter cells flow further along the device and get trapped in the next channel. The researchers showed that they can capture up to five generations of cells this way and keep track of their relationships.

To get the cells off the chip, the researchers temporarily reverse the direction of the fluid flowing across the chip, allowing them to remove the cells one at a time to perform single-cell RNA-seq.

In this study, the researchers captured and sequenced immune cells called T cells. These cells are on constant alert in the body, and when they encounter a cell infected with a virus or bacterium, they leap into action, creating two distinct populations — effector T cells, which seek and destroy infected cells, and memory T cells, which retain a memory of the encounter and circulate in the body indefinitely in case of a subsequent encounter.

“A single founding cell can give rise to both effector and memory cell subtypes, but how that diversity is generated isn’t very clear,” Kimmerling says.

The researchers analyzed RNA from recently activated T cells and two subsequent generations. When comparing genes with functions related to T cell activation and differentiation, they found that “sister” cells produced from the same division event are much more similar in their gene expression profiles than two unrelated cells. They also found that “cousin” cells, which have the same “grandmother,” are more similar than unrelated cells, which suggests unique, family-specific transcriptional profiles for single T cells.

The researchers hope that future studies with this device could help to resolve the long-standing debate over how T cells differentiate into effector cells and memory cells. One theory is that the distinction occurs as early as the first T cell division following activation, while a competing theory suggests that the distinction happens later on, as a result of changes in the cells’ microenvironment. To address this question, the researchers believe they would need to analyze the development of T cells taken from a mouse that had been exposed to a foreign pathogen, which would provide a useful model of T cell activation in response to infection.

The new device could also be used to link RNA transcriptome information with other cell traits, the researchers say.

“It opens up possibilities that have never been there before,” Manalis says. “We can further annotate single-cell transcriptome data by applying this strategy to our existing devices for measuring mass, growth rate, density, or deformability.”

“I think this is really beautiful work,” says Dean Felsher, a professor of medicine and pathology at Stanford University School of Medicine. “It builds on what Scott has been doing for a while, which is creating a whole new way of interrogating single-cell measurements. Now he can follow the progeny over multiple generations, which is really hard to do.”

Cellular “age”

In this study, the researchers also discovered that they could use their new technique to learn which genes are expressed at certain points during the cell division cycle. Because they trap each cell and have a record of when it last divided, they can directly link the “age” of each cell to its transcriptome.

They identified a set of about 300 genes that correspond most with time since division (a proxy for cell cycle progression), and found that most of those genes were involved in cell division. Therefore, by measuring the levels of those 300 genes in similar cells, scientists should be able to estimate the ages of those cells. The researchers also found that a leukemia cell line, which proliferates continuously, has a different set of genes that appear to be driving cell division.

“In the future, this approach may be able to provide insight into unique transcriptional regulators of cell cycle progression in various cancer models,” Kimmerling says.

The post Tracing a cellular family tree appeared first on Scienmag.

Targeting cancer from many angles

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BIOENGINEER.ORG http://bioengineer.org/targeting-cancer-from-many-angles/

Since the discovery of the first cancer-causing genes in the 1960s, scientists have uncovered at least 600 genes that contribute to tumor development. Tyler Jacks, the David H. Koch Professor of Biology and director of MIT’s Koch Institute for Integrative Cancer Research, has spent much of his career trying to unravel the roles of some of these genes, in hopes of designing better cancer treatments.

“The challenge is to figure out the contribution of all of those different genes. If you can figure out what’s driving a cancer, you can potentially develop a drug that will help to inhibit it,” Jacks said during yesterday’s James R. Killian Jr. Faculty Achievement Award Lecture.

Jacks received this year’s Killian Award not only for his work in cancer genetics but also for his leadership of MIT’s cancer research community.

“Professor Jacks is described by colleagues as a bold and visionary leader,” reads the award citation. “His nominators say that it takes a village of passionate and dedicated people to invent solutions for the many cancers that affect our society, and this is exactly what he has created in Building 76.”

Chosen to direct MIT’s Center for Cancer Research in 2001, Jacks oversaw the evolution of that center into the Koch Institute in 2007, with the vision of bringing together MIT’s scientists and engineers to pursue innovative approaches to diagnosing, treating, and preventing the disease.

“You don’t find cancer research institutions like this anywhere else,” said Jacks, who thanked others whom he described as critical to the formation of the new institute, including associate directors Jacqueline Lees and Dane Wittrup, former President Susan Hockfield, President L. Rafael Reif, executive director Anne Deconinck, assistant director Cindy Quense, and David H. Koch.

Under Jacks’ leadership, the Koch Institute has also launched new collaborations with local hospitals to help translate new cancer biology knowledge into patient treatments, and 42 companies have been created by Koch Institute faculty members or with intellectual property developed at the institute.

“We want to deliver technologies that can benefit patients,” said Jacks, who pointed out that cancer kills more people each year than HIV, malaria, and tuberculosis combined.

Modeling cancer

Jacks’ roots at MIT run deep. His father was a professor at the MIT Sloan School of Management from 1959 to 1980, and he often accompanied his father to campus.

“Kendall Square in the 1960s was a very different place,” Jacks recalled. “There was literally one place to eat, the F&T Diner. I still remember the taste of the pastrami sandwiches.”

As an undergraduate at Harvard University, Jacks heard a lecture from Robert Weinberg, an MIT biology professor who had discovered the first oncogene (a gene that drives cancer progression), known as H-ras, and the first tumor suppressor gene, known as Rb. Jacks decided that he wanted to study cancer, and after earning his PhD at the University of California at San Francisco he joined Weinberg’s lab as a postdoc at MIT’s Whitehead Institute for Biomedical Research.

There, he began work that he would continue in his own lab at MIT — genetically engineering mice to develop tumors, which allowed researchers to track the progression of the disease and to test new ways to detect and treat it. The strains of mice his lab has developed are now used in labs around the world to study cancer.

Much of Jacks’ research has focused on lung cancer, which in 2012 accounted for 160,000 deaths in the United States. He developed a mouse model for the most common form of lung cancer, adenocarcinoma, by manipulating the oncogene Kras, which is present in 30 percent of such tumors, and the tumor suppressor gene p53, which is missing in 50 percent of adenocarcinomas.

Tracking the progression of this type of cancer has led to the discovery of other genes involved in the process, which Jacks hopes may lead to new targeted drugs, just as the discovery of the HER2 breast cancer oncogene led to the drug Herceptin, which is very effective for the patients who have an overactive form of HER2.

New directions

Several years ago, as cancer genome sequencing studies turned up more and more genes involved in tumor progression, Jacks says he and his students began to feel a bit overwhelmed at the sheer number of genes they were facing: The typical lung tumor has about 175 mutated genes, and using traditional genetic engineering techniques, it takes two to three years to develop a strain of mice that express a particular cancerous mutation.

“How could we look at all of those genes and their potential contributions to cancer?” Jacks said. “We didn’t know quite what to do.”

Just in time, scientists reported the development of a new genome-editing technique known as CRISPR. This system, originally discovered in bacteria, allows researchers to create gene-editing complexes that can precisely target genes and delete or replace them.

Jacks and colleagues immediately began using the new technology and showed that it could be used to create cancerous mutations in mice much faster than previously possible. In just seven months, they created hundreds of different tumors bearing multiple distinct mutations — a feat that previously would have taken years and cost hundreds of thousands of dollars.

That project illustrates one of the key philosophies that Jacks has tried to instill at the Koch Institute — to embrace alternative approaches to solving problems. Asked after his talk what advice he would give students and postdocs just beginning their scientific careers, Jacks reinforced that message.

“If you just focus on what you know, you’re blinding yourself to new opportunities,” he said.

The post Targeting cancer from many angles appeared first on Scienmag.

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Closer to detecting when and why blood clots form

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BIOENGINEER.ORG http://bioengineer.org/closer-to-detecting-when-and-why-blood-clots-form/

Scientists at the Wyss Institute have created a better assay for testing blood’s clotting tendency, which could prove to be a lifesaver for patients with abnormal blood coagulation and platelet function.

As reported in today’s Nature Communications, this bioinspired advance by the Wyss Institute for Biologically Inspired Engineering at Harvard University takes a biophysical approach by subjecting blood to what it would experience inside a patient’s vascular network. It can be used with blood samples or potentially be integrated into patients’ blood-flow lines, offering clinicians the foresight they need to prevent life-threatening blood clotting or internal hemorrhaging.

Led by Donald Ingber, founding director of the Wyss, the team has developed a novel microfluidic device in which blood flows through a lifelike network of small “vessels.” It is here that it’s subjected to true-to-life shear stresses and force gradients of the human vascular network. Using automated pressure sensors and a proprietary algorithm developed by the Wyss team, data acquired from the device is analyzed in real time, precisely predicting when a certain blood sample will obstruct the blood vessel network.

The device’s hollow channels mimic the pathology of the narrowing of small blood vessels, which occurs in patients as a side effect of medical conditions or treatments and can often cause a shift in the fluid mechanics of blood flow, possibly leading to life-threatening blood clots or internal bleeds.

Known clinically as hemostasis, the body’s ability to stop bleeding is critical for survival. For a patient with a blood-clotting disorder or medical condition, it is essential that care providers have the ability to quickly monitor the patient’s ability to maintain healthy hemostasis while preventing clotting.

“By combining our fabricated microfluidic device that mimics blood-flow dynamics of small arterioles with our novel data-analysis software, we can rapidly quantitate hemostasis in real time and predict if blood clots will develop in an individual or blood sample,” said Ingber, who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School, a member of the Pathology, Surgery and Vascular Biology Programs at Boston Children’s Hospital, and professor of bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

The real-time monitoring ability of the device could also assess patients’ coagulation status almost continuously, in stark contrast to today’s standard of once- or twice-daily testing procedures, thereby reducing the likelihood of toxic side effects resulting from anticoagulation therapies. The team also demonstrated that the device could detect abnormal platelet function in patients with a rare bleeding disorder that is not easily identified using conventional assays.

“The physics of what’s happening inside our bodies is a major contributor to the reasons why blood clots form or why clotting fails during surgeries, traumas, or extracorporeal medical procedures,” said Abhishek Jain, a postdoctoral fellow at the Wyss Institute and the Division of Hemostasis and Thrombosis at Beth Israel Deaconess Medical Center and Harvard Medical School, the lead author on new study. “By mimicking the physics of blood clotting in our device more precisely, we hope this technology can one day be used to save lives.”

In a large animal experiment already conducted, the team perfused blood directly from a living vessel into a microfluidic device to measure clinical clotting parameters over time. From this research they recorded precise predictions for clotting times for blood samples, which were far more accurate and faster than currently used clinical assays.

According to the Wyss Institute, its hemostasis-monitoring device has been developed with translation and versatility in mind, using inexpensive in-line pressure sensors to measure clot formation. As a result, the device does not require additional instrumentation and can be integrated directly into the blood lines of extracorporeal devices.

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The above post is reprinted from materials provided by Harvard News

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