January/Febuary 2020 The Scientist

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STOPPING AFRICAN SWINE FEVER ALTERNATIVE SPLICING OHIO: GENE THERAPY HUB

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DANGERS OF PEER REVIEW GHOSTWRITING

A LIGHT IN THE DARK UNPACKING THE COMPLEX NEUROBIOLOGY OF SUICIDE

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JANUARY/FEBRUARY 2020

Contents

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THE SCIENTIST

THE-SCIENTIST.COM

VOLUME 34 NUMBER 01

Features

ON THE COVER: © LYNN SCURFIELD

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As a devastating outbreak of African swine fever continues to spread across East Asia, researchers are rushing to develop a vaccine.

Can neurobiology shed light on why people end their own lives?

It’s now clear that cells can carve up transcripts in different ways to generate a variety of proteins from the same gene. Yet many questions remain about alternative splicing and its effects.

Pig Plague

BY KATARINA ZIMMER

The Roots of Suicide

BY CATHERINE OFFORD

Alternative Endings

BY GABRIELLE M. GENTILE, HANNAH J. WIEDNER, EMMA R. HINKLE, AND JIMENA GIUDICE

01/02.2020 | T H E S C IE N T IST

3

70 % 50 %

of surveyed scientists admitted that they could not replicate someone else’s research.1 admitted that they couldn’t replicate their own research.1

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JANUARY/FEBRUARY 2020

Department Contents 14

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FROM THE EDITOR

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44

BIO BUSINESS

Ohio, Gene Factory

On the crest of a new decade, science is poised to change the world . . . again.

The state is emerging as a nascent gene therapy hub.

BY BOB GRANT

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51

Into the Future

BY SHAWNA WILLIAMS

55

READING FRAMES

CRITIC AT LARGE

Slip Sliding Away

Exorcising Peer Review Ghosts

Training young scientists to review submitted manuscripts should be an academic exercise, not a facet of professional scientific publishing.

Navigating a wintry landscape forces the mind and body to come to a constructive equilibrium and reveals the fascinating dialogue between the two elements of a human being.

BY JAMES L. SHERLEY

BY SCOTT GRAFTON

NOTEBOOK

60 FOUNDATIONS

Island Frog Rescue; Jumping Disease; Very Hungry Caterpillars; Autism’s Cuffs

20 MODUS OPERANDI Capturing Elusive Microbes

Using reverse genetics, researchers create antibodies to reel in previously uncultured bacteria.

A Woman of Firsts, Early 20th Century BY EMILY MAKOWSKI IN EVERY ISSUE

9 11 59

CONTRIBUTORS SPEAKING OF SCIENCE THE GUIDE

BY RUTH WILLIAMS JENNIFER PARKER; © ISTOCK.COM, LUISMOLINA; © YADID LEVY PHOTOGRAPHY

CORRECTIONS:

44 THE LITERATURE Viruses mediate bacteria-sponge interactions; aneuploidy and female fertility; diving beetles eat tadpoles

49

The author of the November 2019 article "Poet of the Sea" was erroneously listed as Shawna Williams; in fact, it was Ashley Yeager. The December article "Marking the Way" stated that spinal taps are painful. In fact, the procedure is performed under local anesthesia and is rarely painful. The Scientist regrets the error.

46 PROFILE Switch Master

University of California, Berkeley molecular biologist Barbara Meyer’s work with bacteriophages and nematodes exposed the role of genetic switches in early development. BY DIANA KWON

49 SCIENTIST TO WATCH Oded Rechavi: Epigenetic Expressionist BY EMILY MAKOWSKI

ANSWERPUZZLE

T E A R G A S J A N U S

ON PAGE 11

A P I R C Y E I O UDU BON A V O L DCR E S E X C I ON S Z N T T F K I CHN Y P E A CR E S E A D I A P I E N S

G R E A T D A N E

N E T S L W D EMA M M E S P N N T A C O UMON A C QUO I A A R V T R I B E

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JANUARY/FEBRUARY 2020

Online Contents

THIS MONTH AT THE-SCIENTIST.COM: VIDEO

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Inheriting Memories

Saving Mountain Chickens

Eating Up the Food Chain

Tel Aviv University neuroscientist Oded Rechavi discusses his studies on the inheritance of acquired traits.

Peek inside the effort to save this critically endangered Caribbean frog species.

Watch a predaceous diving beetle dine on a tadpole.

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Contributors

UNC-CHAPEL HILL; COURTESY OF GABRIELLE GENTILE; COURTESY OF SCOTT GRAFTON; COURTESY OF REBECCA ROLLINS

When cell biologist Jimena Giudice was an undergraduate studying chemistry at the University of Buenos Aires, Argentina, one of the few biology-related classes she took was on alternative splicing, a process by which different proteins can be produced from the same gene. While she continued to study chemistry, she remained fascinated by the topic, and when she became a visiting fellow at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, during her doctoral work at the University of Buenos Aires, she got the chance to take a week-long course on splicing. “Instead of taking vacation days, I took those days to do that course,” she says. After receiving her PhD in 2011, Giudice did a postdoc at Baylor College of Medicine in Houston, Texas, until 2016, when she started her own lab at the University of North Carolina at Chapel Hill School of Medicine. Her lab studies how alternative splicing controls the function of proteins necessary for heart and skeletal muscle development. Giudice, collaborated with her graduate students Gabrielle Gentile, Hannah Wiedner, and Emma Hinkle to write an article on the biological importance of alternative splicing, which can be found on page 38. Gentile, who played the key role in organizing the article, received her bachelor’s degree in microbiology from the University of Pittsburgh in 2018. She recalls staying up late one night studying for a genetics exam because she was so intrigued by the concept of alternative splicing. “Looking back, I laugh because that was perhaps a foreshadowing.”

Neuroscientist Scott Grafton has been interested in using math to study the brain for decades. He majored in mathematics and psychobiology as an undergraduate at University of California, Santa Cruz—but what really got him “hooked on doing neuroscience” was a semester at the National Institutes of Health’s clinical hospital, where he worked in a neurochemistry lab and volunteered to take part in several research studies. “Researchers would come to me and ask, ‘Hey, can we inject this protein in you and see what happens?’” Grafton recalls. The clinical experience helped him decide to attend medical school at University of Southern California. After he graduated in 1984, he completed residencies at the University of Washington and the University of California, Los Angeles, and developed brain imaging programs at the University of Southern California, Emory University, and Dartmouth College. In 2006, he joined the faculty at the University of California, Santa Barbara, where he is now director of the university’s imaging center. Grafton wrote his first book, Physical Intelligence: The Science of How the Body and the Mind Guide Each Other Through Life, “to celebrate how much intelligence is required to even do the simplest of things. It’s not just speech and memory and social intelligence,” he says. Even simple tasks take “an enormous amount of computational horsepower.” Read his essay about walking on ice—which Grafton describes as a surprisingly complex task—on page 55.

Researcher and entrepreneur James Sherley knew early on that he wanted to be a scientist. After graduating from Harvard University in 1980 with a degree in biology, Sherley pursued medicine to gain a clinical perspective on cancer research. He received his medical degree, along with a doctorate in molecular biology, from Johns Hopkins University School of Medicine in 1988. Afterward, Sherley did postdoctoral work at Princeton University before taking his first principal investigator position at Fox Chase Cancer Center. Later, he moved to MIT, where his lab focused on stem cell research. After leaving MIT, he became a senior scientist at Boston Biomedical Research Institute until founding Asymmetrex, a stem cell biotechnology company, in 2013. On page 12, Sherley criticizes ghostwriting, the widespread practice of having early-career researchers peer review journal articles under their principal investigator’s name. He explains that the onus is on academia to properly train manuscript reviewers instead of involving them in the process of scientific publication too early in their careers. “The real issue is changing the culture,” says Sherley. He’s facing a similar issue at Asymmetrex, where he and others have developed a method of counting stem cells that could help precisely measure out stem cell treatment doses. It’s a challenge to convince researchers that the technique is possible, he says. “[We’re] really trying to introduce this new technology to a field that has operated for 60 years without any counting of adult tissue stem cells.” 01 /02. 202 0 | T H E S C IE N T IST

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FROM THE EDITOR

Into the Future On the crest of a new decade, science is poised to change the world . . . again. BY BOB GRANT

I

10 T H E SC I EN TIST | the-scientist.com

top of these watershed moments, the 2010s saw the rise and rapid maturation of CRISPR genome editing, which is set to change the landscape of clinical practice and biomedical research for decades to come. As is often the case with science, these breakthroughs do not represent the crossing of finish lines, but rather serve as starting points. The 2020s and decades to come likely hold in store discoveries and applications that will prove even more momentous. As biologists forge ahead, a common theme emerges: as one teases apart more and more of life’s complexity, the chore of gathering, sorting, storing, and analyzing data becomes tougher and tougher. But in this arena, too, science has answered the call. Bioinformatics has made impressive strides in recent years, especially in enhancing researchers’ ability to handle unwieldy datasets using artificial intelligence. That’s not to say that the recent past, and likely the near future, are without problems. Widespread inequality continues to plague societies around the globe. And as the human population grows, our environment is in desperate need of thoughtful, forward-looking, evidence-based research and action. But if one considers how far we’ve come in some arenas, it starts to look like the future has truly arrived. In the space of mere decades, science has transformed several diseases—HIV and some cancers, for example—from death sentences into treatable and survivable illnesses. We may not have consumer-grade flying cars and jetpacks yet, but 2020 is feeling pretty futuristic. g

Editor-in-Chief [email protected]

ANDRZEJ KRAUZE

vividly recall, as a child in the 1980s, spending an inordinate amount of time watching classic Hanna-Barbera cartoons such as The Flintstones, The Yogi Bear Show, and The Jetsons in syndication. That last show, in particular, fed what I assume is a natural fascination that most young people have with the future. I’m sure if I looked hard enough through the ephemera of my childhood, I could find a few grade school notebooks festooned with poorly drawn images of flying cars, robot servants, and personal jetpacks. In those halcyon days of boyhood, one date stuck in my mind as “the future”—2020. That year, difficult to imagine but endlessly entertaining to dream about, was when everything would be different. World peace would be a reality. Technology would solve humanity’s and the planet’s ailments. And yes, cars would fly. Alas, this “future” date has arrived, and though we each walk around with supercomputers in our pockets or purses, and some cars can indeed drive themselves, little of the Jetsonian vision of the future has come to pass. But that doesn’t mean that we’re not living a futuristic existence with regard to the leaps and bounds that science has made, particularly in the past decade. Since 2010, humanity has learned much about our place in the universe. In 2016, astrophysicists provided the first-ever direct evidence of gravitational waves in the fabric of space and time—these caused by two massive black holes colliding more than a billion years ago—a discovery half a century in the making. Then, just last year, astronomers published the first image of a black hole. Mind-bending stuff. The past decade of life science advancement and innovation has been similarly remarkable. In the 2010s, the FDA approved the first-ever gene therapy to hit the market, a CAR T cell immunotherapy for cancer (which some have called the first truly personalized medicine), the first-ever RNAi-based therapy, and, at the very end of the decade, the first vaccine for the deadly Ebola virus that ravaged parts of Africa earlier in the decade. Life scientists working in the 2010s also arrived at the long-awaited $1,000 whole human genome sequence, peered further than ever into the internal landscape of the living cell, and helped rewrite human history with newly discovered species and hybrids added to our ancient family tree and with new models for prehistoric human migrations. On

QUOTES

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Mammal with a prehensile nose trunk Young of pens and cobs Eponym of an environmental society Watery swelling of plant organs Eurasian kinglet with a bright yellow crown 12. Psi phenomenon 13. Upper parts of horticultural grafts 15. Trade name of the antacid ranitidine 18. POTUS who gave NASA a mandate 19. Genus of parasitoid wasps 21. Secretion of epithelial cells in some molluscs 22. Redwood conifer of the Northwest US 24. Latin adjective humans apply to humans 25. Crow, or Fox

Lachrymatory agent (2 wds.) Part of an organ not in one’s body One of the primary veins of a leaf The opposite of 16-Down Big German breed of domestic dog (2 wds.) 6. Item tabled by Mendeleev 7. Denser, darker, deader kin of a marsh 11. Arthropod that never has exactly 100 legs 14. Mushroom named for its top (2 wds.) 16. Shaped like the inside of a bowl 17. Proposition to be proved 18. God depicted with two opposite faces 20. Native New Zealander 23. Chewable stimulant of Arabia

I certainly hope that over the coming decade we see an increasing global effort to put in place appropriate regulations for using genome editing, especially in applications that could have a very profound impact on everyone. —Jennifer Doudna, University of California, Berkeley, biologist and seminal figure in the development of CRISPR genome editing, talking to Fortune about genomics in the 2020s (December 19)

Perhaps an obvious response to these findings is to encourage women to act more like men and be more positive; . . . this ‘fix the women’ approach lacks an understanding of the current evidence base on gender equity. We must fix the systems that support gender disparities. —Reshma Jagsi of the University of Michigan and Julie Silver of Harvard Medical School, in a commentary published alongside a BMJ study reporting that male researchers were far more likely than female researchers to describe their studies using superlatives such as “novel” and “unique” (December 16)

Answer key on page 5 01/02.2020 | T H E S C IE N T IST 1 1

CRITIC AT LARGE

Exorcising Peer Review Ghosts Training young scientists to review submitted manuscripts should be an academic exercise, not a facet of professional scientific publishing.

O

n November 4, 2019, The Scientist ran a revealing Q&A highlighting a recent survey published in eLife. Responses from early career researchers (ECRs) and other scientists drew attention to a widespread, unethical practice to which academic scientists have too long resigned themselves—peer review ghostwriting (8:e48425, 2019). As defined in that paper, peer review ghostwriting occurs when scientists hand over manuscripts that they have agreed to review for journal editors to

12 T H E SC I EN TIST | the-scientist.com

graduate students or postdocs in their research groups. The involvement of the junior scientists is not typically disclosed to the journal, so editors work under the impression that the invited reviewer developed and wrote the resulting manuscript review themselves. Survey results reported in the eLife paper provided the first quantitative evidence for the prevalence of this practice, as well as for the practice the study authors refer to as co-reviewing. In a strict sense, co-reviewing happens when a trainee is involved in developing

and writing the review and their contribution is disclosed to journal editors. Some consider this transparent form of collaborative peer review a valuable part of scientific training, and the eLife study authors even argue that journals should codify co-review. But in my experience, the involvement of co-reviewers is sometimes not disclosed to the journals, just as is the case with ghostwriters. Whether co-reviewers are named or not, this practice, along with the more patently unethical ghostwriting, has no defensible place in the live

© ISTOCK.COM, ANDREYPOPOV

BY JAMES L. SHERLEY

arena of academic publishing. In this professional context, journal publications are major forces in determining academic career success, supporting the livelihoods of researchers, influencing government policy, furthering research funding, advancing scientific and medical progress, and supporting the academic enterprise. Having inexperienced reviewers usher manuscripts through the essential process of peer review is a disservice to submitting authors. Coreviewing, even when trainee reviewers have been named and credited, has the potential to harm the careers of the scientists who submitted the manuscript under review in the event that poorly informed, deficient reviews result in rejections of papers that are crucial to obtaining research funds, academic promotion, professional reputation, salary compensation, and so on. Rather, training in peer review practice and ethics should be at least started, if not completed, before a young scientist has the opportunity to review actual submitted manuscripts and potentially alter that process in a negative way. Both ghostwriting and co-reviewing can also have the effect of denying trainees credit for the work they have contributed. The potential for exploitation of graduate student and postdoc ghostwriters and co-reviewers is certainly a good reason for concern and intervention, but the more significant problem for academic science—one that was not considered by the authors of the eLife paper—is how these practices contribute to the general erosion of academic integrity. Prospective study authors submit their manuscripts for peer review by professional journals with the reasonable expectation and agreement that their submissions will be provided fair, expert, and confidential peer review by another qualified member of their field. Ghostwriting and co-review completely violate the professional ethics of this contract. Ghostwriting is clearly unethical. But arguing that co-reviewing is acceptable because it trains ECRs to be better

manuscript reviewers is a convenient rationalization to excuse a similarly unethical practice. The place to teach journal manuscript review is in open working forums such as departmental journal clubs, and in graduate-level subject courses and special topics seminars. It is interesting that the survey in the eLife paper did not list graduate classes as a response choice for where respondents had obtained training for reviewing manuscripts, though the authors did propose the introduction of compulsory teaching of manuscript reviewing in graduate courses.

Whether co-reviewers are named or not, this practice, along with the more patently unethical ghostwriting, has no defensible place in the live arena of academic publishing.

Most journals provide reviewers with detailed instructions for the desired content and format of manuscript reviews. What they do not do, and should not be expected to do, is teach reviewers how to evaluate and judge the significance of manuscripts, their technical quality, the soundness of their arguments and conclusions, the integrity of their conduct, and their overall scientific value. This expertise should be learned and developed in the course of an academic career by attention to it at every stage of training. And this essential aspect of a scientist’s education needs to be complemented with an emphasis on proper ethical conduct in journal manuscript review.

The eLife paper authors rightly advise that something needs to be done about these aspects of peer review in the interest of improving the quality of academia. They recommend ending the practice of ghostwriting and crafting more-substantive guidelines around co-reviewing. They suggest that journal editors codify mechanisms for disclosing and crediting the contributions of noninvited reviewers, who are often members of the invited reviewer’s lab. But journals already have a process for invited reviewers to decline the invitation and propose alternative reviewers, such as a trainee, that the journal editor can then decide to accept or not. In this way, interested and properly trained ECRs can begin to establish their own credentials in the eyes of journal editors with appropriate instruction and guidance, without compromising the integrity of the journal manuscript review process and of academia as a whole. It is up to institutions of higher learning and their members to remedy the breach of publishing integrity that ghostwriting and co-review cause. Often in academia, ethical conduct is taught but not practiced. A cultural shift toward more-ethical practices will require that all academics work to better align their actions with the well-reasoned ideals of ethical conduct. It is simple; we need to begin teaching ethical manuscript review as a core principle of academic life and responsibility. The eLife paper shows that the scientific community is ready for this change. College and university faculty simply need to start teaching it and following it themselves. g James L. Sherley is the founder and current director of Asymmetrex LLC, a company focused on developing adult tissue stem cell technologies and applying them to clinical drug discovery and cellular medicine. Before starting Asymmetrex, he spent more than 20 years as a principal investigator leading laboratory research programs in cancer center, independent research institute, and research university settings. 01 /02. 202 0 | T H E S C IE N T IST 1 3

NEWS AND ANALYSIS

Island Frog Rescue

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uke Jones spent the early evening of July 12 last year crouched in several inches of dusty ash in a secluded region of Montserrat’s tropical dry forest. After gently unzipping three nylon tents and laying their flaps flat to the ground, the program coordinator for Mountain Chicken Reintroduction and his team waited silently in a moonlit clearing for hordes of giant frogs to leap to freedom. After 30 minutes of waiting, it became clear that the frogs, commonly

14 T H E SC I EN TIST | the-scientist.com

known as mountain chickens (Leptodactylus fallax), were not going to emerge in the triumphant cavalry the researchers had anticipated. It had been 10 years since the amphibians’ ancestors had lived on the Caribbean island; the frogs in the tents were more accustomed to small biosecure homes in Jersey and London Zoos in the UK than to the 25-square-meter, semi-wild enclosure waiting for them outside. When rustling the tents failed to encourage the frogs to hop into their new world, the researchers gently tipped the 14 animals onto the forest floor. Although it wasn’t the striking entrance that the team had antici-

JANUARY/FEBRUARY 2020

SKIN TEST: Mountain chicken frogs are being

monitored for signs of fungal disease on the Caribbean island of Montserrat.

pated, what followed was spectacular, Jones says. As the frogs sat blending into their surroundings, a sudden deluge of warm rain welcomed the mountain chickens home. One frog started calling, and then the next. For the first time in years, the atmosphere rippled with the enchanting sounds of these amphibians, which, weighing up to a kilogram each, were once apex predators on the island. This frog release—one of two such efforts Jones oversaw that summer—

JENNIFER PARKER

Notebook

marked the beginning of an innovative reintroduction program that not only has implications for Montserrat’s mountain chickens but could inform the conservation of amphibians around the planet. By manipulating the frogs’ habitat within the semi-wild enclosure, Jones and his colleagues hope to support the population they’ve released and protect it from disease—and to learn more about amphibian biology and conservation methods along the way. Montserrat hit the headlines in 1995, when the Soufrière Hills volcano began to erupt, wiping out two-thirds of the mountain chicken’s range on the island. Despite the severity of the disaster, the frogs survived and continued to thrive in the island’s northern regions—that is, until the deadly fungal disease chytridiomycosis arrived on Montserrat in 2009, probably via the accidental importation of infected tree frogs. The disease spread rapidly, decimating the island’s mountain chicken population within just one year—the fastest decline of any vertebrate ever recorded.

Any effort which takes that scary step into the wild . . . is a huge step forward. —Jonathan Kolby, Honduras Amphibian Research and Conservation Center

Ben Scheele, a population ecologist at the Australian National University who recently reported the latest figures on the disease (Science, 363:1459–63, 2019), says that chytridiomycosis has contributed to the decline of at least 501 species worldwide. “This estimate is likely conservative as the disease has likely also affected undescribed or poorly known species,” he says. What’s more, chytridiomycosis is widely recognized as “the most important cause of species extinctions” in amphibians,

says Andrew Cunningham, a veterinary pathologist at Zoological Society London (ZSL) who led the team that first identified the chytrid fungus, Batrachochytrium dendrobatidis. (See “A Race Against Extinction,” The Scientist, December 2014.) To date, researchers have been unable to stop the disease. The fungus kills amphibians by degrading the keratin in their skin, making it harder for them to absorb the oxygen and trace minerals they need for good health and, in turn, causing heart failure. Back when the fungus first arrived on Montserrat, 50 disease-free mountain chickens were swiftly gathered up and bred in biosecure facilities at zoos in Europe. In the years that followed, Durrell Wildlife Conservation Trust—in collaboration with Montserrat’s Department of Environment, ZSL, and various European partners—composed the reintroduction program that Jones, along with his supervisor Mike Hudson and the Department of Environment team, has been putting into action over the past year. The frogs’ enclosure contains solarheated ponds, areas in which tree canopy has been removed to allow the sun to heat the ground, and large rocks that the frogs can use as basking sites. Jones explains that the chytrid fungus struggles to survive at high temperatures; he and his colleagues hope that the abundance of warm areas will give the frogs an advantage over the fungus, and perhaps time to develop resilience to the disease. One possible route to resilience could be behavioral adaptation: frogs that learn to make greater use of hot spaces might be able to keep their chytrid load low enough to avoid harm. But Jones and colleagues suspect that there might be other mechanisms—for example, changes to the epigenome or to the skin microbiome—that make the frog’s surface a less favorable environment for the fungus. On a longer timescale, there’s the additional potential for genetic mutations that promote

survival to spread throughout the frog population. To monitor the project’s success, the frogs are subjected to a monthly health check: their sizes and weights are recorded, and the animals are examined for any signs of disease. The frogs are also swabbed, and samples are sent to ZSL’s laboratory in London to determine how the chytrid load of each individual changes over time. The team has yet to publish its results, but Jones says that the return of this charismatic species will offer a “beacon of hope” for the people of Montserrat, who have been struggling to recover from the volcanic eruptions that left the capital city buried in ash some 25 years ago. Cunningham adds that simultaneous work being carried out by other researchers with genetic material from a handful of mountain chickens that survived for years with the fungus in Dominica—the only other island where the frog species is found—could aid conservation plans by identifying DNA sequences that confer resistance to the disease. “That could inform selective breeding or selective releasing on Montserrat,” he says. The work could also help researchers protect amphibians more broadly. “The experimental management implemented in Montserrat is very innovative and exciting,” Scheele says. “Hopefully it is successful and the approach can be used to help the conservation of other species.” But he cautions that “there are no silver bullet approaches. . . . We need to try a range of different things and it’s likely that different actions will work for some species, but not be so effective for others.” Jonathan Kolby, director of the Honduras Amphibian Research and Conservation Center, says that the Montserrat project represents an important step in researchers’ fight against chytridiomycosis. “Any effort which takes that scary step into the wild . . . and does any kind of experimental effort to see how we can have these animals coexist with chytrid is a huge step forward.” —Jennifer Parker 01/02. 2020 | T H E S C IE N T IST 1 5

NOTEBOOK

When Wellcome Sanger Institute geneticist Eugene Gardner set out to look for a specific type of genetic mutation in a massive database of human DNA, he figured it’d be a long shot. Transposons— also known as jumping genes because they can move around the genome— create a new mutation in one of every 15 to 40 human births, but that’s across the entire 3 billion base pairs of nuclear DNA that each cell carries. The sequencing data that Gardner was working with covered less than two percent of that, with only the protein-coding regions, or exons, included. Doing a quick calculation, he determined that, in the bestcase scenario, he could expect to find up to 10 transposon-generated variants linked to a developmental disease. And “we really might get zero,” he says. “This whole thing might be for naught.” But Gardner had recently developed the perfect tool to find the sort of de novo mobile element insertions that come about as a result of transposon movements and are often overlooked in genetic screens and analyses. As a graduate student in Scott Devine’s lab at the University of Maryland, Baltimore’s Institute for Genome Sciences, he had spent many hours making the software for the mobile element locator tool he dubbed MELT. The program was easy to use, so when Gardner moved across the Atlantic for a postdoc in Matthew Hurles’s lab at Sanger near Cambridge and gained access to a database of exomes from 13,000 patients with developmental disorders, he figured running the tool was worth a try. Of those 13,000, Gardner focused on 9,738 people in the Deciphering Developmental Disorders (DDD) study whose parents’ exomes had also been sequenced, making it easier to single out variants present in the child but not in mom and dad. And as it turned out, he did get some hits. MELT picked up 40 potentially transposon-generated 16 T H E SC I EN TIST | the-scientist.com

variants, which Gardner sat down at his computer to review using the raw sequencing data. Nine appeared to be true de novo mobile element insertions. “I remember being in my desk doing the visualization of all the putative de novo variants after I got the first results off the pipeline,” he recalls. “I remember being excited: I think I might have found a diagnostic de novo!” Discussing the literature on the genes affected by such insertions with clinicians and other colleagues, Gardner narrowed the list down to four insertions found in genes that may be causing or contributing to four different patients’ disorders (Nat Commun, 10:4630, 2019). He sent these results off to the physicians who had referred each of the patients to the database, and all the doctors confirmed that the results made sense to them given what had been published on those genes and what they knew about other cases involving patients with mutations in the same sequences. In one case, the physi-

cian had already linked the patient’s disorder to the gene Gardner had identified; in the other three cases, the patients were still undiagnosed. “There is tremendous value for these families that get a diagnosis,” says human geneticist Dan Koboldt, who has collaborated with Hurles in the past and has used MELT in his studies of rare disease at the Steve and Cindy Rasmussen Institute for Genomic Medicine at Nationwide Children’s Hospital in Columbus, Ohio, but who was not involved in Gardner’s recent study. A genetic answer not only can help physicians connect patients to appropriate medical and counseling resources; it puts an end to the diagnostic odyssey that families affected by rare disease often endure. What’s more, the finding of four potentially causative hits out of the nearly 10,000 cases provides first estimate of how commonly such mobile element insertions underlie developmental disorders. “What’s interesting about this study is that it’s taking a very broad

ANDRZEJ KRAUZE

Jumping Disease

approach,” says Ian Adams, a developmental biologist at the University of Edinburgh’s MRC Human Genetics Unit who was not involved in the research. Rather than look for transposon activity in a specific disorder, “it’s casting a much broader net in trying to find what type of diseases this class of mutations could be contributing to.”

There is tremendous value for these families that get a diagnosis.

SARA GREGG

— Dan Koboldt, Steve and Cindy Rasmussen Institute for Genomic Medicine Nationwide Children’s Hospital

This approach is important, agrees Adams’s MRC Human Genetics Unit colleague Jose Garcia-Perez, a transposable elements expert who was also not involved in the new research. In the last few years, two studies have used a tool developed around the same time as MELT to search for de novo mobile elements in people with autism spectrum disorder, but neither identified any that were likely to be responsible for the patients’ symptoms (Am J Hum Genet, 98:667–79, 2016; Science, 360:327–31, 2018). “[Gardner’s] study shows that, no matter what’s [been found] recently, it’s something that should be explored in further detail in the future,” says GarciaPerez. “[The study] actually shows a real connection between . . . transposition with that particular [type of ] disorder.” Koboldt adds: “The reason this is an important study is that it establishes [that these] variants do occur and [that] they can be pathogenic.” Gardner says he hopes that his methods can be used to explore other diseases, from both a research and a clinical perspective. Adams says MELT does appear to be “widely applicable to other datasets.” Such a tool could be a boon to research on transposons, given that their movements are often missed by normal screening tools, Adams adds. “I think [MELT is] something that could be readily built into existing pipelines.” —Jef Akst

Very Hungry Caterpillars Armed with a paring knife, evolutionary biologist Genevieve Kozak often ventures into cornfields in the Northeastern US to cut open plant stalks and look for larvae of the European corn borer moth. The caterpillars burrow inside the plants to overwinter, leaving a telltale hole that signals to researchers such as Kozak which plants to open up. Slicing through the stalks can be a challenge: back in 2011, “the first time I went out there, I cut my thumb,” says Kozak, then a postdoc in Erik Dopman’s lab at Tufts University, and now a professor at the University of Massachusetts–Dartmouth. Once she has the plants cracked open, though, the caterpillars inside aren’t that difficult to gather. Each winter, after going through their last molt before becoming pupae, corn borers (Ostrinia nubilalis) enter a dormant state of activity known as dia-

pause. To survive this vulnerable time, the larvae hide out someplace where they won’t be disturbed. For corn borers, that place is inside the stalks of the plants, which also serve as their primary food source when they wake up in the spring. Although most commercially grown corn in the US is genetically engineered to be resistant to corn borers, the moths are a major problem for organic farmers, who can’t plant engineered crops or use chemical pesticides if they are to meet regulatory standards for organic products set by the United States Department of Agriculture. And over the last few decades, a new challenge has emerged in the battle against these pests. As global temperatures rise and winters become shorter, some populations of corn borer larvae in the US have CHANGING WITH THE TIMES: Mutations in

two genes associated with circadian rhythms may help the corn borer moth adapt to climate change.

01/02. 2020 | T H E S C IE N T IST 1 7

NOTEBOOK

been emerging from diapause almost three weeks earlier than usual—giving them more time to harm corn plants and potentially infest other crops, too. “At some spots, you have [corn borers] that are coming out of diapause earlier,” says Dopman. “So early, in fact, that they can squeeze in a second generation at the end of the year, whereas the ones that wait around in diapause only have a single generation.” Those early-emerging larvae live mostly in the southern US; in the northern states, corn borers still don’t emerge until late May or early June. But there’s no clear geographical boundary between the phenotypes, and in upstate New York and Pennsylvania, where Dopman’s lab has been collecting caterpillars, the researchers found both early- and lateemerging populations. To find out how O. nubilalis might be adjusting its emergence time, the group recently scanned the genomes of larvae in five populations—three earlyemerging and two late-emerging. The team found that larval emergence time was linked to variation in two genes known to be involved in circadian rhythms. One is period, or per, a gene that regulates sleep-wake cycles in Drosophila and has been linked to seasonal timing in many other insect species. The other gene, Pdfr, produces a receptor that binds to a neurotransmitter called pigment-dispersing factor, which in Drosophila helps to regulate the activity of clock neurons in the brain. Dopman and his colleagues speculate that variation in the sequences of these two genes could provide a way for corn borers, and perhaps other insects as well, to adapt to changes in season length (Curr Biol, 29:3501–509, 2019). “This paper is incredibly interesting and important because it sheds light on the molecular basis of differences in seasonal responses,” says Megan Meuti, an entomologist at Ohio State University who was not involved with the study. The fact that the genes are already known to be involved in circadian rhythms suggests “that the differences in the 18 T H E SC I EN TIST | the-scientist.com

sequences of the clock genes are not only affecting seasonal responses, but also daily responses as well.” Daniel Hahn, an evolutionary physiologist at the University of Florida, adds that while previous research has focused on how circadian clock gene polymorphisms are associated with the timing of insects entering dormancy, this new study shows that such variation is also “associated with when animals are going to come out of dormancy. That’s a completely new facet that nobody’s been able to do before.”

If an organism has genes that cause it to enter diapause or break diapause at inappropriate times, then that population can crash. —Erik Dopman, Tufts University

Hahn, who was not involved with the current study but has collaborated with Dopman in the past, is planning to work with the Dopman team to expand on this research. In addition to uncovering more about the basic biology of the corn borer, the scientists hope that their findings could inform genetic engineering approaches to disrupt the annual cycle of these pests, says Dopman. “One possibility that we’re looking into is whether we can create an ecological mismatch,” he says. “If an organism has genes that cause it to enter diapause or break diapause at inappropriate times, then that population can crash.” —Emily Makowski

Autism’s Cuffs About four years ago, pathologist Matthew Anderson was examining slices of postmortem brain tissue from an individual with autism under a microscope when he noticed something extremely odd: T cells swarming around a narrow

space between blood vessels and neural tissue. The cells were somehow getting through the blood-brain barrier, a wall of cells that separates circulating blood from extracellular fluid, neurons, and other cell types in the central nervous system, explains Anderson, who works at Beth Israel Deaconess Medical Center in Boston. “I just have seen so many brains that I know that this is not normal.” He soon identified more T-cell swarms, called lymphocytic cuffs, in a few other postmortem brains of people who had been diagnosed with autism. Not long after that, he started to detect another oddity in the brain tissue—tiny bubbles, or blebs. “I’d never seen them in any other brain tissue that I’ve looked at for many, many different diseases,” he says. Anderson began to wonder whether the neurological features he was observing were specific to autism. To test the idea, he and his colleagues examined postmortem brain tissue samples from 25 people with autism spectrum disorder (ASD) and 30 developmentally normal controls. While the lymphocytic cuffs only sporadically turned up in the brains of neurotypical individuals, the cuffs were abundant in a majority of the brains from individuals who had had ASD. Those same samples also had blebs that appeared in the same spots as the cuffs. Staining the brain tissue revealed that the cuffs were filled with an array of different types of T cells, while the blebs contained fragments of astrocytes, non-neuronal cells that support the physical structure of the brain and help to maintain the bloodbrain barrier. Reading the literature and drawing on his experience as a pathologist, Anderson started to think about blebs and what they do when they show up in tissues beyond the brain. For example, in cancer, “blebs are generated when T cells attack a tumor cell,” he explains. “The tumor cells will spit out surface membrane pieces . . . as a way to protect [themselves] from the attack, but also possibly to deliver signals to other cells around them.” In the brain samples from individuals with ASD, the blebs visually resembled blebs created in response to

M.M. DISTASIO ET AL., ANN NEUROL, 86:885–98, 2019.

tumors. The brain blebs may be formed in response to the infiltration of T cells into the space between blood vessels and neural tissue, Anderson suggests, while the cell fragments they contain could come from the astrocytes that make up the glia limitans—the final wall of defense separating neural tissue from foreign and toxic substances circulating in the blood. Lymphocytic cuffs, meanwhile, are common in diseases such as skeletal muscle polymyositis, a type of chronic muscle inflammation. That disease has many traits of autoimmune disorders, in which the body perceives and attacks parts of itself as foreign, and it’s a disease that Anderson had often seen in biopsies. “I’ve seen many [cuffs] under the microscope,” he says. “So I know what a T-lymphocyte attack of an organ looks like.” The cuffs also show up in response to toxins, or antigens given off by a virus, and cause brain inflammation. The finding of both cuffs and blebs in the postmortem brains of autistic people suggests that the individuals’ T cells were also responding to some antigen—either a molecule considered foreign even though it’s created by the person’s own body, or a viral or bacterial one encountered in utero, Anderson says. Except for rare cases in which an autism-linked genetic mutation can be identified, the cause of ASD is unknown. According to the new data, a majority of the unexplained cases could have arisen as an autoimmune disorder or an inflammatory condition triggered during pregnancy, Anderson and colleagues concluded in a recent paper (Ann Neurol, 86:885–98, 2019). “It’s really a very striking finding,” says Dan Littman, an immunologist at New York University Langone Health who was not involved in the work. The team’s results, he notes, fit well with recent animal research showing a connection between the immune system and autism—specifically that interleukin-17 (IL-17), a signaling molecule produced by T cells to help fend off pathogens, can cause rodents to exhibit behaviors associated with autism. In 2016, Littman and colleagues reported that blocking the

production of IL-17 in pregnant mice prevented their pups from developing an autism-like condition (Science, 351:933– 39). “You could imagine that if cytokineproducing cells in the central nervous system are localizing in particular places, they could be contributing to behavioral changes,” Littman says. The findings also dovetail with what little has been described in the way of neuropathological features of autism in humans, Anderson says. Fifteen years ago, Carlos Pardo-Villamizar of Johns Hopkins University and colleagues studied postmortem brain tissues and cerebrospinal fluid from individuals with autism and found signs of neuroinflammation in the cerebral cortex, white matter, and cerebellum—regions essential for sensory perception and for motor skills such as balance and coordination (Ann Neurol, 57:67–81, 2005). Transcriptional profiling of postmortem brains from individuals with autism revealed elevated levels of messenger RNAs that make inflammatory proteins (Neurobiol Dis, 30:303–11, 2008), and more-recent data support the conclusion that the brains of individuals with autism are typically in an inflammatory state (PNAS, 116:21659–65, 2019). While the Boston team’s discovery of T cell–induced inflammation associated with autism is noteworthy, the astrocyte blebs are particularly intriguing, notes Duke University neuroscientist Staci Bilbo, who did not participate in the new study. The development of the blebs in reaction to the cuffs “points to a role for the blood-brain barrier breaking down,” something rarely studied in autism, she says. Looking further into the interaction between the cuffs and the blebs could reveal not only how, but why T cells are getting into the brain, giving clues to the origins of autism in cases driven by immune dysfunction. Anderson says his team has already started follow-up experiments, running transcriptome profiling of the cuffs and blebs. Infiltration of T cells into the space between blood vessels and neural tissue, and the subsequent generation of blebs,

FRIENDLY FIRE: Swarms of lymphocytes (pur-

ple) in the space between blood vessels and neural tissue are more common in postmortem brain samples from people with autism (below) than in samples from controls (above). (Scale bar: 40μm)

“almost for sure is going to trigger the expression of unique genes and proteins” in the astrocytes and “may dissociate autism even more specifically from other conditions,” he says. His team is also looking at the receptors on the T cells in the cuffs to determine what’s provoking the immune cells to swarm. The researchers are studying all of the genes linked to autism and to autoimmunity as well—an analysis that has begun to reveal “a signature of an autoimmune genetics within the existing autism genetics,” Anderson says. “It’s a multi-pronged approach.” —Ashley Yeager 01/02. 2020 | T H E S C IE N T IST 1 9

MODUS OPERANDI

Capturing Elusive Microbes Using reverse genetics, researchers create antibodies to reel in previously uncultured bacteria. Membrane proteinencoding gene

BY RUTH WILLIAMS

B

acteria and archaea make up most of the living world, but the vast majority of species, including some that are intimately associated with humans, have never been isolated or cultured. Sequencing of DNA from natural microbe populations has allowed the identification of previously unknown taxa and in some cases provided detailed genomic information about the organisms. But having sequence data is “like having the parts list” for a machine, says microbiologist Karsten Zengler of the University of California, San Diego. This alone “does not tell you what this machine will do.” For a better understanding of a microbe’s physiology and functions, researchers need to study living specimens, or at least whole cells. To that end, microbial geneticist Mircea Podar of Oak Ridge National Laboratory and colleagues are examining the sequence data of uncultured microbes to design tools with which to capture the bugs. Focusing on bacteria present in the human mouth, Podar’s team compared available sequence data from uncultured organisms therein with sequences of previously cultured bacteria to identify codes for potential cellsurface proteins with regions unique to the uncultured organisms’ DNA. The researchers then generated peptides from these candidate codes, injected them into rabbits to create antibodies against them, and labeled the antibodies with fluorescent markers. Adding

Bacterial DNA

 6

 4 Antibody

Sequencing

 1

 2

 5

Flow cytometer

Fluorescent tag

Unique membraneprotein epitope

 3

Target Fluorescent antibodies added microbes to sample of mixed microbes isolated

CAPTURE AND CHARACTERIZE: To isolate an uncultured microbe of interest, researchers can

examine available sequence data to find likely cell-surface proteins  1 and select a peptide region as unique to the desired organism as possible  2 . This peptide is then used to generate antibodies in rabbits  3 , and the antibodies are labeled with a fluorescent tag  4 . Next, researchers add the labeled antibodies to the microbial sample  5 and use flow cytometry to isolate those that bind  6. The isolated bacteria can then be sequenced, characterized, and ideally, cultured.

these fluorescent antibodies to microbe samples from human saliva enabled the detection of bacteria with corresponding surface proteins and their isolation via fluorescence-activated cell sorting (FACS). The team isolated two different taxa of previously uncultured oral bacteria—TM7 and SR1—both of which the team was able to cultivate using carefully selected media. In addition to enriching for the target organisms themselves, the antibody-driven technique pulled out specific types of bacteria with which the TM7 and SR1 organisms were physically associated. In fact, the success of subsequent cultivation of TM7 and SR1 may be due in part to the co-isolation of microbes required for their growth.

While in principle the technique could be applied to any bacteria, it will be necessary with each new target strain to carefully design the peptides used for antibody generation, says Podar. The design is a careful balance between having similarities to known surface proteins in other microbes and being specific for the desired organism. So “it’s never going to be a kit,” he says. Rather, “it’s a guided approach that enhances your chance of success.” Zengler, who was not involved in the research, says the technique “will help us to get more organisms in culture and to learn more about organisms that we only know from their genomic fingerprint.” (Nat Biotechnol, 37:1314–21, 2019) g

ADVANTAGES

DISADVANTAGES

CULTURING POTENTIAL

Single-cell sequencing

Individual cells from a mixed population are sorted into droplets for genomic amplification and sequencing.

The approach can be high-throughput and uses the increasingly commonplace techniques of microfluidics and single-cell sequencing.

Yields no morphological or direct physiological information. Difficult to gather data from especially rare organisms. Little or no information on interspecies interactions.

None

Reverse genomics–enabled isolation

Bacterial DNA sequences are used to generate peptides for antibody production. The antibodies are then used to isolate the bacteria of interest from a mixed population.

Target organisms are greatly enriched, facilitating genomic, morphological, and physiological characterization. Associated organisms may also be isolated, enabling insights into relationships.

Choosing peptides for antibody generation requires a tricky balancing act between homology and specificity.

Culturing involves some trial and error, but an abundance of the target organism and isolation of associated microbes improves chances of success.

20 T H E SC I EN TIST | the-scientist.com

© GEORGE RETSECK

AT A GLANCE STUDYING UNCULTURED ISOLATING CELLS BACTERIA

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As a devastating outbreak of African swine fever continues to spread across East Asia, researchers are rushing to develop a vaccine.

BY KATARINA ZIMMER

© ISTOCK.COM, ZAYATSSV

PIG PLAGUE

I

n the fall of 2017, a year before an unfamiliar virus captured the world’s attention with an explosive outbreak in East Asia that left tens of millions of pigs dead, immunologist Waithaka Mwangi and his graduate students were already aware of the culprit and its imminent threat to the swine industry. Behind the glass of a biosafety cabinet at Kansas State University’s Biosecurity Research Institute—one of two sites in the US authorized to con duct research on the deadly pathogen—they carefully extracted a few milliliters of fluid from a test tube containing live African swine fever virus (ASFV) collected from the spleens of infected pigs. In another room down the hall, the researchers administered droplets of the fluid into the nostrils of piglets. In total, more than 60 young pigs were exposed to the virus, and the team waited to see how they’d fare. ASFV is typically harmless to humans, but it can be devastating to domestic pigs (Sus scrofa domesticus), and this particular strain of the virus, known as Georgia 2007 after appearing in the country that year, was typically fatal. Within a week, infected animals would succumb to a lethal hemorrhagic fever, the same type of illness as that caused by Ebola and Marburg viruses in humans. But a few days earlier, Mwangi’s team had given 32 of the piglets a cocktail of proteins that they hoped would help the animals survive the infection. This prototype vaccine consisted of an inactivated adenovirus—a mostly harmless pathogen that is often used as a vector for therapeutics—that had been genetically engineered to express one of two combinations of ASFV proteins. Mwangi knew from his group’s previous studies that the modified adenovirus could trigger the porcine immune system to attack those proteins and generate antibodies against them. Now, he was about to find out if that was enough to fight off an infection with the deadly Georgia 2007 strain. The scientists monitored the animals daily, checking their temperature and general health. To the team’s dismay,

after only a few days, some of the piglets began to huddle together, a sign of feverish chills. The first group of vaccinated pigs developed a fever and deteriorated quickly, even faster than control animals that hadn’t received the adenovirus, and had to be put down. Eight of 10 animals that had received a different protein cocktail also fell ill and were euthanized. A different formulation of this cocktail showed a little promise, with five of nine animals surviving.1 But overall, “it was disappointing that we didn’t get a positive outcome,” Mwangi tells The Scientist. The results foreshadowed worse news to come. Almost exactly a year later, China reported an outbreak of the Georgia 2007 strain in Shenyang, a city in the country’s northeast. From there, it swept

cerned that the virus could slip into their countries via contaminated pork products or animal feed imported from infected countries. Seemingly overnight, finding a vaccine for ASFV—a virus that had long stood at the periphery of the scientific community’s attention—became a global research priority. But as Mwangi’s results suggest, there are still significant challenges to overcome. The most successful vaccine candidates are not yet appropriate to use in agricultural settings, while safer options, such as Mwangi’s cocktail approach, have yet to prove effective. Researchers need to understand more about the virus, its origin, and its interaction with the porcine immune system to complete their mission.

There’s so many pigs in China, it was just a matter of time. —Dirk Pfeiffer, City University of Hong Kong

through the world’s largest congregations of pigs and among countless small farms, killing hundreds of thousands of animals across China. By the end of October 2019, nearly 200 million animals had been culled in a desperate effort to stop the virus, but ASFV continued to spread, popping up in Mongolia, Vietnam, Cambodia, Laos, Myanmar, South Korea, and the Philippines. In October, Mark Shipp, the president of the World Council of Delegates of the World Organization for Animal Health, told reporters that around a quarter of the global pig population could die due to the disease. Unrelated to the East Asian epidemic, new outbreaks have also been reported in Eastern Europe. There, low levels of the virus have been circulating in wild boar and domestic pig populations for more than a decade since it arrived from its native Africa, where it often leaps from wild pigs to domestic animals. The rapid spread of ASFV across Eastern Europe and Asia alarmed officials in Asia, Western Europe, and North America, con-

“To get to the stage of making a vaccine that can be used in the field requires a lot more [research],” says Linda Dixon, a virologist at the UK’s Pirbright Institute, part of the government’s Biotechnology and Biological Sciences Research Council. “I don’t think there are any [candidates] at that stage yet.”

Out of Africa ASFV infection was first documented in the early 20th century in Kenya, then a British colony. People there noted that pigs brought from England quickly succumbed to a “contagious pneumonia,” as veterinarian Robert Montgomery described it in 1921.2 When antibodies against classical swine fever, which also causes feverish chills in pigs, failed to offer protection, scientists concluded that a different pathogen, later christened ASFV, must be responsible. (African swine fever is also not to be confused with swine flu, caused by an unrelated virus of the influenza group that can cause respiratory symptoms in pigs and sometimes in people.) 01 /02. 2020 | T H E S C IE N T IST 2 3

benign, suggesting they’ve coevolved with the virus for a long time, but in domestic pigs, infection unleashes chaos in the animals’ immune systems. Upon infecting macrophages and other white blood cells, many ASFV strains proliferate rapidly and trigger inflammatory reactions while simultaneously releasing proteins that blunt the animals’ immune response. Infection also induces cell death in white blood cells and endothelial cells lining blood vessels. Ultimately, infected pigs

Later research revealed that ASFV likely arose in eastern and southern Africa3 and subsequently spread throughout much of Sub-Saharan Africa. It has diversified into at least 24 different genotypes, each of which can encompass many different strains. In eastern and southern Africa, ticks of the Ornithodoros genus transmit the virus between common warthogs (Phacochoerus africanus) and domestic swine. ASFV infections in African wild species are typically

develop hemorrhagic shock and die. For farmers in many parts of Africa, “it is devastating,” says Mary-Louise Penrith, a veterinary pathologist at the University of Pretoria in South Africa. For most of its evolutionary history, ASFV has been limited to its continent of origin. Before the current outbreak in Asia, the virus was known to have journeyed out of Africa only twice: in 1957, when an ASFV genotype 1 strain infected Portuguese pigs that ate food

ASFV’S DEADLY ESCAPES FROM AFRICA For centuries, African swine fever virus (ASFV) has circulated between ticks and warthogs in Africa as part of a natural lifecycle, occasionally spilling over to domestic pigs. (See graphic on opposite page.) The virus became a global concern when it left the continent and spread to the Iberian Peninsula—twice in the mid-20th century. The second time, it traveled across the Atlantic to the Americas. These outbreaks were successfully quelled through strict eradication programs, but a devastating epidemic now spreading across Asia has intensified global research into understanding ASFV and finding a way to stop it.

1957: ASFV traveled out of Africa for the first time, likely via food waste from airline flights that was fed to pigs near an airport in Lisbon, Portugal. The ensuing outbreak of the disease was quickly eradicated. Portugal

1960: ASFV once again surfaced in Portugal. This time, it also spread to the Caribbean and Brazil. The virus hasn’t been sighted in the Americas since the 1980s and was eradicated from Europe, with the exception of Sardinia, by the 1990s.

Georgia

WHERE IS ASFV ENDEMIC? ASFV is endemic in eastern and southern Africa where it has been circulating among wild warthogs for hundreds of years, and also in West Africa where it is routinely spread among domestic pig populations. Following its emergence in Georgia in 2007, the virus also became endemic throughout Eastern Europe. It’s not clear yet whether wild swine populations in Asia have begun transmitting ASFV to domestic pigs.

2007: ASFV traveled to the country of Georgia, where it caused a major outbreak, killing nearly 90,000 pigs in six months. Over the next few years, it jumped to eastern Europe and western Russia. Millions of pigs and wild boar died from infection or were culled to contain the virus, but it is still causing hundreds of outbreaks as it continues to spread west and south. It was this strain of the virus, genotype 2, that eventually reached China in the spring of 2018. Millions more animals have been culled across East Asia, but the virus continues spreading to this day.

waste from airline flights traveling from Africa; and in 1960, when the same strain revisited the Iberian Peninsula, then crossed the Atlantic to Brazil and several Caribbean island nations. Scientists and public health officials in Spain and Portugal were able to quash the outbreaks through careful surveillance of the disease, culling animals on infected farms, and keeping pigs away from wildlife. By the late 1990s, almost all countries affected by those midcen-

tury outbreaks were virus free, and for the next decade, things were silent. Because of its long-time restriction to Africa, ASFV “probably hasn’t received the amount of attention that it should receive due to the threat it poses,” says Daniel Rock, a virologist at the University of Illinois at Urbana-Champaign. In 2004, the US Department of Homeland Security decided to shutter a research program dedicated to studying the disease, notes Rock, who used to lead the

ASFV’S LIFECYCLE ASFV is transmitted by ticks of the genus Ornithodoros to common warthogs (Phacochoerus spp.) when they feed on the wild animals’ blood. Domestic pigs (Sus scofra domesticus) can catch the virus through tick bites in areas of Africa where warthogs exist, as well as through contact with contaminated food or materials. In Eastern Europe, where the disease is also endemic, pigs can contract ASFV by coming into contact with bodily fluids or carcasses of infected wild boar (also Sus scofra). Since the late 2000s, ASFV is thought to have gained a foothold in Europe, especially the eastern part of the continent where infections often spill over to small-scale pig farms. It’s not yet clear whether ASFV has infected wild boar populations in China or other East Asian countries it has spread to. If it has, the virus will be near-impossible to eradicate there. Warthogs and other wild swine

Soft ticks transmit ASFV through bites.

Contaminated food sources and manmade materials can spread ASFV to domestic pigs and occasionally wild swine.

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Domestic pigs

Domestic pigs can also catch ASFV through contact with Eurasian wild boar or their carcasses.

program—ASFV wasn’t considered a priority due to being widely perceived as “an African thing.” That thinking changed in 2007, when the deadly genotype 2 strain of ASFV now making its way across Asia first surfaced in Georgia, possibly arriving via ships from Africa carrying infected pork products that were then fed to domestic pigs. In as little as five years, it swept through the Caucasus and into Russia—a “game-changing moment” for ASFV and the world, says Rock. That this strain eventually surfaced in China in 2018 was not a surprise to anyone in the field. “There’s so many pigs in China, it was just a matter of time,” veterinary epidemiologist Dirk Pfeiffer of City University of Hong Kong told The Scientist last year. By the summer of 2019, the epidemic had escalated into what Pfeiffer calls “the biggest animal disease outbreak ever.” Some feared that it would further escalate into a worldwide pandemic. In response, the field has seen an influx of funding from the European Union, Bulgaria, and China, with governments funding researchers in the hope that they develop a vaccine quickly. But that’s easier said than done, notes Luis Rodriguez, a virologist at the United States Department of Agriculture’s (USDA) Plum Island Animal Disease Center, which relaunched a longinactive ASFV program the year after the virus’s spread to Europe in 2007. “We’re doing the best to move forward as fast as we can in developing these vaccines, but that is a process that takes time and effort, and there are major challenges.”

Live vaccines As early as 1967, researchers discovered that the traditional approach to making vaccines doesn’t work for ASFV. 4 Pigs injected with killed or inactivated forms of the virus—intended to provoke their B cells into generating virus-targeting antibodies—weren’t protected against virulent forms of the disease. In 2014, a team of German scientists tried the experiment for themselves, and found that while pigs did develop antibodies against ASFV proteins, it wasn’t enough 01 /02. 2020 | T H E S C IE N T IST 2 5

Wildtype virus

to fight off the virulent Georgia 2007 strain. 5 “Antibodies alone are not fully effective,” Dixon says. Over the past few decades, researchers have begun to understand why. Studies suggest that pigs rely heavily on killer T cells—and potentially other immune cells—to fend off ASFV, and stimulation of T cells can only occur if living viruses infect host cells.6 Only then are viral peptides processed and presented via cell surface receptors to T cells. This doesn’t happen with the dead viruses traditionally used in the vaccine experiments. Recognizing this, researchers developed vaccine candidates with live, but weakened, forms of ASFV. They took advantage of the fact that many ASFV strains have mutated over time, becoming less aggressive to domestic pigs and their wild relatives. In 2019, a group of Spanish researchers injected a number of domestic pigs with a weak strain of ASFV genotype 2 that had been isolated from a wild boar captured in Latvia. The vaccine caused mild, transient symptoms involving fever and joint swelling in some animals, but they all survived after being exposed to pigs that carried the virulent genotype 2 strain Georgia 2007.7 As researchers have amassed more knowledge about ASFV’s biology, they’ve adopted a more targeted approach in attenuating viruses by removing specific genes that make it so deadly. As Dixon puts it, the goal is to strategically disarm the virus so the porcine immune system has a chance to develop long-lasting antibodies and to prime T cells to attack the virus. In 2016, for example, her group created an attenuated form of ASFV genotype 1 by knocking out eight genes and interrupting two genes the virus uses to dampen pigs’ interferon type 1 response, a pathway that helps curtail viral replication. All five animals immunized with this gene-deleted virus survived a challenge with a virulent genotype 1 strain.8 Dixon’s team has achieved similar success with other gene deletions9,10 and, with funding from the Biotechnology and Biological Sciences Research Council in the

Genes encoding harmless proteins

DNA

Macrophage

Virulent proteins supress immune system

Virus infects macrophages and produces virulent and harmless ASFV proteins. Nucleus Virulence gene

Overreplication of virus kills cell

UV irradiation

Antibodies Antibodies bind to the virus particle and stop it from infecting macrophages.

Stimulates B cells (not pictured) to produce antibodies but no T cells

Destroyed DNA

Deletion of virulent genes T cell

Virus infects macrophages and produces harmless ASFV proteins that are presented on the immune cell’s surface.

Stimulates B cells to produce antibodies and cytotoxic T cells to recognize and kill virus-infected macrophages

Antigens expressed in viral vector

Viral vector infects antigenpresenting cells such as macrophages and produces harmless ASFV proteins that are presented on the immune cell’s surface.

As above, stimulates B cells and T cells

26 T H E SC I EN TIST | the-scientist.com

A VACCINE HUNT Researchers have tested three main approaches to develop a vaccine candidate for the ASFV strain that is currently killing pigs throughout Asia.

VACCINE STRATEGY #1: INACTIVATED VIRUSES The traditional approach involves killing or inactivating viruses—for instance, through UV irradiation—so that they’re no longer virulent but retain viral antigens that stimulate the production of protective antibodies. EFFICACY: These vaccines stimulate an antibody response in pigs, but they don’t pro-

tect against intact forms of ASFV. Researchers think this is because inactivated viruses don’t activate killer T cells.

SAFETY: Based on limited studies, no side effects have been shown so far. COMMERCIAL PROSPECTS: Researchers have abandoned this approach because of

the shortfalls in efficacy.

VACCINE STRATEGY #2: LIVE VIRUSES Injecting tamer forms of virulent viruses could potentially stimulate antibody production and the all-important T cell responses without killing vaccinated animals. EFFICACY: Both gene-deleted and naturally attenuated forms of ASFV stimulate the immune system to generate antibodies and killer T cells and usually offer protection against virulent genotypes of ASFV. SAFETY: Vaccinated pigs can develop mild to debilitating symptoms, from fever to joint swelling. COMMERCIAL PROSPECTS: Researchers are both testing ASFV strains that have naturally attenuated over time and genetically modifying virulent forms of the virus by removing sequences that code for harmful proteins. Scientists have yet to find a stable cell line capable of generating live vaccine candidates in bulk, but these types of vaccines are expected to be the first to hit the market.

VACCINE STRATEGY #3: SUBUNIT VACCINES

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A third approach involves genetically engineering viral vectors such as adenoviruses to express combinations of ASFV antigens. Inside the body, the vector-encoded antigens are produced in the absence of the pathogen. EFFICACY: Inoculations provoke the production of antibodies and killer T cells, but don’t seem to protect pigs against virulent forms of ASFV. SAFETY: Vaccinated animals typically experience few or no side effects. COMMERCIAL PROSPECTS: Researchers are testing different antigen combinations. Many consider this to be the preferred strategy for developed countries, although it’s expected to reach the market much later than live virus vaccines. Subunit vaccines can be easily synthesized in bioreactors and rapidly generated in bulk.

UK, is working with a New Jersey–based biotechnology company to create a viable vaccine candidate for the Georgia 2007 strain using this approach. Meanwhile, at Plum Island Animal Disease Center, microbiologist Manuel Borca has developed four gene-deleted vaccine candidates that protect animals against the same strain—each carrying one, two, or three deletions.11 A number of other groups, including one in China, are also working on similar genedeletion approaches. Although they’re effective, safety is still a significant concern. Some of Dixon’s immunized pigs developed a slight fever, which most veterinary vaccine companies would consider an unacceptable safety risk, though further gene deletions and modifications have greatly reduced or eliminated this fever while maintaining good efficacy, she says. Part of the challenge in identifying the right genes to remove to strike that balance between safety and efficacy is ASFV’s unusual genomic complexity. Like some other DNA viruses, ASFV has a very large genome—“this thing is about 190 kilobases,” notes Mwangi, making it longer than the RNA genomes of Ebola, HIV, Lassa, Marburg, and rabies viruses put together. Another major barrier in developing live vaccine candidates is that they’re difficult to produce in bulk. ASFV replication requires macrophages, but there aren’t any porcine macrophage cell lines that last for more than a few weeks. Instead, researchers have to continuously harvest fresh macrophages from animal blood or other body tissues. “You will never get a uniform, reproducible [vaccine] product” this way, remarks Yolanda Revilla of the Spanish National Research Council’s Center for Molecular Biology “Severo Ochoa” in Madrid. Finding a cell line that lasts is “one of our most important objectives at the moment.”

Protein cocktails To get around these issues, researchers such as Mwangi are trying another strategy: a subunit vaccine. Viral vectors—such as adenoviruses—are engineered to express cocktails of ASFV antigens. Once the vector 01 /02. 2020 | T H E S C IE N T IST 27

viruses infect porcine cells, the antigens are presented on the cells’ surfaces, triggering B and T cells to target ASFV. Because the viral vectors are genetically modified so that they’re not capable of replicating quickly, there is no risk of uncontrolled spread, Mwangi says. Another advantage of subunit vaccines is that they can be easily grown in bio reactors using several well-established cell lines, Dixon adds. However, the challenge, as Mwangi’s adenovirus study suggests, is finding an antigen combination that induces a protective immune response.

To that end, in 2018 Dixon teamed up with researchers at Arizona State University to design a systematic screen. 12 The scientists selected 47 ASFV proteins with a range of functions and injected pools of as many as 20 of the proteins into pigs. Then, they exposed cytotoxic T cells from the inoculated pigs to each of the proteins in culture. Those proteins to which the lymphocytes reacted most strongly—releasing interferon gamma, a cytokine that prompts the cells to differentiate into long-lasting memory T cells targeting specific antigens—were selected as good

building blocks for a subunit vaccine. From their ranking of the most immunogenic proteins, the team created four different cocktails and engineered vaccinia viruses, a popular vaccine vector, to express one of the cocktails and replicate only a few times. To Dixon’s disappointment, vaccinated pigs that received a lethal dose of ASFV Georgia 2007 died. But the pigs did have reduced concentrations of live ASFV in their blood compared with control animals—a sign that the researchers should be able to refine their approach “to get stronger protective responses,” Dixon says.

PREPARING FOR THE NEXT ASFV OUTBREAK compliance among the millions of people involved in the country’s giant pork supply chain. Meanwhile, some in the US and Canada, concerned that the virus could slip into North America via contaminated pork products or animal feed imported from countries that have the virus, have taken precautions in their own swine industries. In April 2019, for example, the Canadian Pork Council issued new guidelines for importing swine feed ingredients, recommending that feed be held in a sealed container for up to 100 days, depending on storage temperature, before being accessed, to allow time for the virus to perish. In the past, many nations outside Africa, including Spain, were able to eradicate the disease through extensive culling of pigs, notes MaryLouise Penrith, a veterinary pathologist at South Africa’s University of Pretoria who has consulted the FAO on how to manage ASFV. And many African farmers, especially in South Africa, succeed in managing the virus’s spread through relatively straightforward biosecurity measures such as keeping pigs and their feed away from wildlife. Preventing ASFV’s spread “is not rocket science,” Penrith says. But a key to containing the virus is altering human habits—from making sure workers change into clean clothes and footwear once they enter a farm to quarantining sick animals. “All of us are agreeing more and more that what really spreads African swine fever is people. And that’s a hard thing to change.”

© SHUTTERSTOCK, PRESSLAB

Many researchers and vaccine companies are concentrating their efforts on finding a vaccine for ASFV genotype 2, the virus responsible for the ongoing outbreak in Asia. But some recognize the need to find defenses against other African genotypes that create immense economic difficulties for African farmers while threatening to unleash havoc on the rest of the world if they escape to another continent. For instance, Linda Dixon, a virologist at the UK’s Pirbright Institute, is studying a virulent genotype 1 virus widespread in West and central Africa, while immunologist Lucilla Steinaa of the International Livestock Research Institute is focusing on the dominant genotypes in East Africa, genotypes 9 and 10. University of Illinois virologist Daniel Rock is taking a different approach by looking for protective antigens—those that trigger specific, long term responses of the immune system—that different ASFV strains have in common, which may help researchers design vaccines that target multiple forms of the virus (J Gen Virol, 100:259–65, 2019). Engineering a vaccine that protects against multiple strains and genotypes is “the longer-term goal,” he says. But even if such a vaccine existed, it wouldn’t be a panacea, warns City University of Hong Kong epidemiologist Dirk Pfeiffer. A major reason why ASFV has spread so explosively in China is that most pigs there live scattered across thousands of small-scale farms with little to no biosecurity—as a report by the United Nations Food and Agriculture Organization (FAO) pointed out just months before Chinese pigs caught the virus. Pfeiffer expects that officials have little chance of eradicating ASFV for good without improving that biosecurity—making sure feed isn’t contaminated with ASFV, for instance. Moreover, if the virus infects Chinese wild boars and becomes endemic in Asia, officials would have to continuously vaccinate pigs for years to safeguard them from spillover from wild animals. A vaccine would “keep a lid on the spread of the virus, but it doesn’t remove the virus,” Pfeiffer says. Last year, the Chinese government started providing some biosecurity recommendations to farmers, such as using appropriate cleaning and disinfection on farms and prohibiting feeding of food waste to domestic pigs, although Pfeiffer questions how feasible it is to encourage

Lucilla Steinaa, an immunologist at the International Livestock Research Institute in Nairobi, Kenya, has been conducting a similar screen with her team, focusing on ASFV genotypes 9 and 10, which circulate throughout East Africa. Instead of looking at whole proteins, her team is monitoring T cell responses to specific peptides, to identify the precise amino acid sequences that elicit immune responses. Other organizations, such as the US-based Phibro Animal Health Corporation and the Madrid-based vaccine company Algenex, are also developing subunit vaccines. In addition to finding the right proteins, researchers must also consider delivery mechanisms. That vaccinia and adenovirus vectors fail to curtail replication of ASFV virus could be partially due to the fact that the vectors themselves don’t induce a strong enough immune response, Dixon explains. For instance,

deleted live vaccine to be the first to enter the market there, optimistically within two years, says Pfeiffer. These approaches already offer good protection against ASFV, and with China’s $130 billion/year pork industry at stake, researchers there may be willing to compromise a little in terms of vaccine efficacy and safety, Rock says— but not by much. Having an unsafe, replication-competent vaccine virus floating around East Asia would spell disaster, he adds. For other regions that have more time to spare, for example, in the US or Europe, a subunit vaccine would likely be a preferred alternative, José Escribano, founder and chief scientific officer of Algenex, tells The Scientist by email. This type of vaccine would be the only approach considered safe enough for regulatory bodies in developed countries to approve, he says.

To get to the stage of making a vaccine that can be used in the field requires a lot more research. —Linda Dixon, Pirbright Institute of the UK’s Biotechnology and Biological Sciences Research Council

her team has tried boosting an antigenencoding adenovirus vaccine with a vaccinia virus containing the same proteins five weeks later, in an attempt to amplify the pigs’ immune response. But once again, although this reduced the amount of circulating ASFV, it didn’t save the animals from death.13 “There’s quite a number of approaches that folks are pursuing, but so far, no viable or very promising [delivery mechanism] has been demonstrated,” Mwangi notes.

The road ahead The two main approaches—genedeleted live vaccines and subunit vaccines—aren’t mutually exclusive. To address the rapidly spreading outbreak in China, researchers expect a gene-

An effective vaccine for the Georgia 2007 strain would be useful to have on hand for any country, but it won’t be the end of the story, Rock warns. There are numerous known ASFV strains circulating in Africa—many of them just as deadly to domestic swine as Georgia 2007—and likely more yet to be discovered, Rock says, and a vaccine against one would be unlikely to protect pigs against others. (See “Preparing for the Next ASFV Outbreak” on previous page.) With China having an everstronger economic presence in Africa, the chances that other strains will find their way to Asia in the future is high, he adds. “It’s the Georgia [2007] strain today. What’s the strain tomorrow?” g

Katarina Zimmer  is a New York–based freelance journalist. Find her on Twitter @katarinazimmer.

References 1. S. Lokhandwala et al., “Adenovirus-vectored African Swine Fever Virus antigen cocktails are immunogenic but not protective against intranasal challenge with Georgia 2007/1 isolate,” Vet Microbiol, 235:10–20, 2019. 2. R.E. Montgomery, “On a form of swine fever occurring in British East Africa (Kenya colony),” J Comp Pathol, 34:159–91, 1921. 3. M.A. Alkhamis et al., “Phylodynamics and evolutionary epidemiology of African swine fever p72-CVR genes in Eurasia and Africa,” PLOS ONE, 13:0192565, 2018. 4. S.S. Stone, W.R. Hess, “Antibody response to inactivated preparations of African swine fever virus in pigs,” Am J Vet Res, 28:475–81, 1967. 5. S. Blome et al., “Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation,” Vaccine, 32:3879–82, 2014. 6. C.A.L. Oura et al., “In vivo depletion of CD8+ T lymphocytes abrogates protective immunity to African swine fever virus,” J Gen Virol, 86:9, 2005. 7. C. Gallardo et al., “Attenuated and nonhemadsorbing (non-HAD) genotype II African swine fever virus (ASFV) isolated in Europe, Latvia, 2017,” Transbound Emerg Dis, 86:1399–404, 2019. 8. A.L. Reis et al., “Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response,” Vaccine, 34:4698–705, 2016. 9. A.L. Reis et al., “Deletion of the African Swine Fever Virus gene DP148R does not reduce virus replication in culture but reduces virus virulence and induces high levels of protection against challenge,” J Virol, 91:e01428-17, 2017. 10. P.J. Sánchez-Cordón et al., “Evaluation of protection induced by immunization of domestic pigs with deletion mutant African swine fever virus Benin ΔMGF by different doses and routes,” Vaccine, 36:707–15, 2018. 11. V. O’Donnell et al., “Simultaneous deletion of the 9GL and UK genes from the African Swine Fever Virus Georgia 2007 isolate offers increases safety and protection against homologous challenge,” J Virol, 91:e01760-16, 2016. 12. J.K. Jancovich et al., “Immunization of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African Swine Fever Virus immunogenic and protective proteins,” J Virol, 92:e02219-17, 2018. 13. C.L. Netherton et al., “Identification and immunogenicity of African Swine Fever Virus Antigens,” Front Immunol, 10:1318, 2019.

01 /02. 2020 | T H E S C IE N T IST 2 9

The Roots of Suicide Can neurobiology shed light on why people end their own lives?

BY CATHERINE OFFORD

T

he first time Kees van Heeringen met Valerie, the 16-yearold girl had just jumped from a bridge. It was the 1980s and van Heeringen was working as a trainee psychiatrist at the physical rehabilitation unit at Ghent University Hospital in Belgium. As he got to know Valerie, who’d lost both legs in the jump and spent several months at the hospital, he pieced together the events leading up to the moment the teenager tried to end her life, including stressful interactions with people around her and a steady accumulation of depression symptoms. Van Heeringen, who would later describe the experience in his 2018 book The Neuroscience of Suicidal Behavior, says Valerie’s story left a permanent impression on him. “I found it very difficult to understand,” he tells The Scientist. He asked himself why anyone would do “such a horrible thing,” he recalls. “It was the first stimulus for me to start studying suicidal behavior.”

30 T H E SC I EN TIST | the-scientist.com

In 1996, van Heeringen founded the Ghent University Unit for Suicide Research. He’s been its director ever since, helping to drive scientific research into the many questions he and others have about suicide. Many of the answers remain as elusive as they seemed that day in the rehabilitation unit. Suicide rates are currently climbing in the US and many other countries, and suicide is now the second leading cause of death among young people globally, after traffic accidents. The World Health Organization recently estimated that, worldwide, one person ends their own life every 40 seconds. (See Sidebar on page 37.) Suicide is as complicated as it is tragic. Suicidal behaviors come in many varieties, ranging from suicidal thinking, or ideation, to suicide attempt and completion, all of which may be associated with various levels of violence or intent. The behaviors themselves differ in incidence among genders, ethnicities, and other

demographic categories, and almost always occur against a background of depression or some other mood disorder—although only a fraction of people with mood disorders become suicidal. No field of scientific inquiry can single-handedly untangle a phenomenon as complex as suicide. But van Heeringen and many other scientists are hoping to shed light on the problem by digging into the neurobiological processes underlying thoughts about ending one’s own life and attempts to do so. This work is building support for the idea that suicide is tied to specific biochemical changes that can be measured and targeted independently of, and possibly in parallel with, the mental health disorders they often accompany. Findings from this work, researchers hope, could help reveal new treatments, and perhaps even opportunities to identify the people most at risk in time to intervene. “The knowledge we have today is way larger than what we had twenty years

© LYNN SCURFIELD

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Pressure points Valerie’s account shared elements with the stories of many other people who have attempted to end their lives. She showed signs of depression and social stress, and, as van Heeringen later discovered, she had a family history of suicide—a known risk factor for suicidal behaviors, independent of any psychiatric disorders.

IN THE US, MORE THAN 3.5 TIMES AS MANY MEN AS WOMEN DIE BY SUICIDE. Source: American Foundation for Suicide Prevention, 2017 data

Scientists now think about suicide risk in terms of stress-diathesis models, which treat suicide as a product of both so-called precipitating factors such as elevated stress or mood disorders and predisposing factors—the “diathesis”—such as family history, particular genetic variants, or early-life adversity such as abuse or neglect. “Suicide is more than . . . being very depressed,” explains Colum bia University’s John Mann, a psychi atrist and translational neuroscientist who helped develop the conceptual framework with Columbia neurobiolo gist Victoria Arango. 32 T H E SC I EN TIST | the-scientist.com

This framework has helped focus research on biochemical pathways that regulate the brain’s response to stress, and how those pathways could be altered in people who become suicidal. The brain has multiple stress responses, but the best-studied in relation to suicide is the hypothalamic-pituitaryadrenal (HPA) axis, which controls the release of the stress hormone cortisol and is known to be upregulated in clinical depression. Early clues regarding the link between the HPA axis and suicide include findings of higher concentrations of corticotropin-releasing hormone (CRH), which triggers the synthesis of cortisol and other glucocorticoids involved in stress signaling, in postmortem brain samples from people who died by suicide than in samples from people who died by other means. Other research has hinted that people who died by suicide have enlarged adrenal glands—sites of cortisol production. Due to the high incidence of depression and other mood disorders among people who end their own lives, however, studies such as these didn’t attempt to determine whether the observed effects were specific to suicide or to mood disorders more generally. More recently, the case for a central role for the HPA axis in suicide has gained support from work led by Turecki and others revealing that earlylife adversity, one of the strongest risk factors for suicide even when psychiatric disorders are controlled for, can have long-term effects on HPA axis function. In the mid-2000s, Turecki teamed up with McGill University geneticist Moshe Szyf, who had shown that rats neglected by their mothers exhibit altered epigenomes in the hippocampus—a brain region involved in stress, learning, and memory—and dysfunctional HPA responses to stress. 1 In the hippocampi of people who have died by suicide and had a history of childhood abuse, Turecki, Szyf, and their colleagues found evidence of hypermethylation and reduced expression of the

gene coding for NR3C1, a glucocorticoid receptor that helps dampen cortisol signaling, compared with healthy controls or people who died by suicide but hadn’t experienced abuse.2 Research since then has linked suicidal behaviors to methylation abnormalities in other HPA-related genes. One 2018 assessment of nearly 90 people who had attempted suicide identified reduced methylation at the CRH gene in blood samples from some of the study’s subjects—specifically, those who made attempts that were more violent or more likely to result in death.3 And several studies have identified hypermethylation and reduced expression of SKA2, which codes for a protein that interacts with NR3C1, in people who died by suicide compared with healthy controls and with nonsuicidal patients with depression, schizophrenia, or other psychiatric disorders. The relationship between the HPA axis and suicidal behavior is complicated. For example, while some studies imply that the HPA axis overreacts to stress in people who die by suicide, others indicate that people who attempt suicide have lower baseline cortisol levels and/or blunted HPA reactivity to stress compared with controls. “It is a confusing literature,” says Nadine Melhem, a psychiatric genetic epidemiologist at the University of Pittsburgh School of Medicine who found a few years ago that, among around 200 people whose parents had mood disorders, those who attempted suicide had overall lower HPA activity.4 “Almost every [possible] finding has been reported.” Part of this inconsistency likely stems from small study samples and variations in experimental design, Melhem notes. But variability may also come from differences in the drivers of suicidal behavior in different groups of people. Mann’s group reported last year that, of 35 people who attempted suicide, only those who scored highly for impulsive aggression in personality tests had significantly elevated cortisol responses to stress compared with non-

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ago,” says Gustavo Turecki, a psychiatrist at McGill University and the director of the McGill Group for Suicide Studies at the Douglas Research Centre in Montreal. “[We’ve] made tremendous advances . . . in terms of understanding the complexity of the problem, understanding the neurobiology, understanding the causes.”

suicidal controls.5 And one meta-analysis published a few years ago found a positive correlation between cortisol levels and risk of suicide attempt in studies of people under 40 years old, but a negative correlation in studies of older people.6 Until now, “we have not been able in the literature to capture the dynamic nature of these pathways in relation to suicide risk,” says Melhem, adding that she and colleagues are beginning to build longitudinal datasets to address this gap. “It needs a lot more work.”

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Communication issues Mann first became interested in the neurobiology of suicide while studying a rather different aspect of brain chemistry. Throughout the 1980s and ’90s, he and others found deficits in serotonin (5-hydroxytryptamine, or 5-HT) signaling and in the neurotransmitter’s main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in the brains of people who died by suicide, regardless of psychiatric diagnosis, compared with brains of people with or without psychiatric disorders who had died by other means. The findings were key in the realization that there might be biochemical changes specific to suicide, Mann says. Since then, the serotonergic system has become one of several neurotransmitter systems being probed for clues about suicidality. Like the HPA axis, serotonin signaling appears to be modulated by earlylife adversity. For example, methylation of HTR2A, which codes for a serotonin receptor known as 5-HT2A, is altered in children who have suffered early-life adversity—although it’s not yet clear how those methylation changes affect HTR2A expression. 7 A 2016 study of twins in the UK revealed that children who were bullied had hypermethylation at SERT—a gene coding for a protein that helps transport serotonin from the synapse back into the presynaptic neuron—compared with children who weren’t. Bullied children also showed blunted cortisol responses to stress,

hinting at a link between the serotonergic system and HPA functioning.8 How such physiological changes might influence suicidal behavior remains to be seen, but groups such as Mann’s are working to disentangle some of the details. For example, he and his colleagues recently published a more concrete link between serotonin and HPA-axis activity: even when psychiatric diagnoses are controlled for, levels of the serotonin receptor 5-HT1A are correlated with cortisol reactivity to stress.9 The team has also explored levels of serotonin receptors in depressed and nondepressed people exhibiting suicidal behaviors, and found that levels of 5-HT 1A in some regions of the cortex are higher in people who attempt or die by suicide, regardless of psychiatric diagnosis, than in controls.10,11 Somewhat counterintuitively, higher levels of 5-HT 1A could contribute to a deficit in serotonin signaling, Mann explains, because the receptor is part of a neural feedback response that inhibits further serotonin release into synapses. Accordingly, it seems that in people who are suicidal, “the problem is not the capacity to make serotonin but . . . the capacity to use that serotonin,” he says. This role for 5-HT1A could also help explain why selective serotoninreuptake inhibitors (SSRIs) do a better job of dampening suicidal thoughts and behaviors than some other antidepressants, he adds: among other effects, SSRIs reduce the number and responsiveness of 5-HT 1A receptors and may thereby quiet the negative feedback loop that suppresses serotonin signaling. Neurotransmitters besides serotonin, including glutamate, GABA, and dopamine, have also been investigated in the context of suicidal behavior— particularly following recent findings that drugs such as ketamine and esketamine, which interact with the glutamate receptor NMDAR, reduce suicide risk in patients with clinical depression. However, the literature on these neurotransmitters is fairly inconsistent, spurring researchers to continue look-

ing for new mechanisms to explain suicidal behaviors.

Neural irritation A couple of years ago, researchers in Denmark reported a link between suicide and infectious disease. Analyzing three decades’ worth of health records from more than 7 million people, the team found that being hospitalized with an infection was associated with more than a 40 percent greater probability of suicide. Spending more than three months in the hospital was linked to a more than doubled suicide incidence. While acknowledging that such observational data can’t demonstrate causation, the team calculated that the statistical risk associated with hospitalization for infections could account for about 10 percent of the Denmark’s suicides.12

IN THE US, AN AVERAGE OF NEARLY 130 PEOPLE DIE BY SUICIDE EACH DAY. Source: CDC, 2017 data

There are many possible explanations for this finding—one being that treatment of infections with antibiotics or other hospital medications influences mental health. But van Heeringen and others point out that the study ties into another hypothesis about suicidal behavior, one that involves a role for inflammation. Elevated suicide risk has previously been reported in people with autoimmune disorders and traumatic brain injury—conditions that, like infections, typically involve inflammation. Further 01 /02. 2020 | T H E S C IE N T IST 3 3

NEUROBIOLOGICAL PATHWAYS LINKED TO SUICIDE RISK Scientists have identified several key neurobiological pathways with ties to suicidal behaviors. Research in the field addresses only a fraction of the complexity of this serious public health problem, and the literature on the topic is complicated by variation in study design, but the clues point to several interacting moderators of suicide risk. Three of the systems best-studied in relation to suicide are depicted below.

STRESS RESPONSES Many studies have linked suicidal behaviors to dysregulation of the hypothalamic-pituitaryadrenal (HPA) axis and other mediators of the body’s responses to stress.

NOTE: The findings shown in this graphic come from studies with very different approaches to investigating suicide. Some studies control for psychiatric disorders, others don’t; different studies focus on different brain areas; and many of the findings are preliminary.

Cortisol levels appear to be correlated with levels of certain serotonin receptors, and increases in CRH may boost serotonergic activity in some brain areas.

POSSIBLE CONNECTIONS

HYPOTHALAMUS

CRH

Corticotropin-releasing hormone (CRH) has been found in higher concentrations in the brains of people who die by suicide.

Serotonergic neurons influence the release of HPA components such as CRH, and drugs that target serotonin receptors have been shown to affect HPA-axis function.

PITUITARY GLANDS

ADRENOCORTICOTROPIC HORMONE (ACTH)

ADRENAL GLANDS

People who die by suicide, and particularly those who die by violent means, may have enlarged adrenal glands. CORTISOL

Basal cortisol levels have been found to be both higher and lower than normal in people who have attempted suicide. The reactivity of cortisol to stress may also be dysfunctional in people with suicidal behaviors. NR3C1

NR3C1, also known as the glucocorticoid receptor, may be in lower abundance in people who die by suicide, particularly those with a history of childhood abuse.

Cortisol released as part of a stress response can help suppress inflammation.

POSSIBLE CONNECTIONS

The release of certain cytokines can stimulate HPA axis activity.

NEURAL TRANSMISSION Neural communication via serotonin and other neurotransmitters such as glutamate often shows signs of dysregulation in people who die by suicide.

Presynaptic neuron

SEROTONIN

Disruption of serotonin signaling has repeatedly been found in the brains of people who die by suicide. SERT

Levels of the serotonin transporter SERT, which shuttles serotonin back into the presynaptic neuron, may be lower in people who die by suicide. 5-HT1A AND 5-HT2A

Levels of the serotonin receptors 5-HT1A and 5-HT2A may be higher in people who attempt or die by suicide.

Postsynaptic neuron

Inflammation may dysregulate the serotonin system via several pathways.

POSSIBLE CONNECTIONS

Serotonin may be involved in directing immune cells to sites of inflammation.

INFLAMMATION People who die by suicide show signs of increased inflammation in the brain while epidemiological data reveal that some inflammationrelated health conditions are associated with higher suicide risk.

© LISA CLARK

MICROGLIA The brains of people who die by suicide show higher levels of microglia activation.

CYTOKINES Blood levels of inflammatory cytokines, particularly some types of interleukins, have been found at higher levels in people who attempt suicide.

clues come from epidemiological studies of Toxoplasma gondii—a parasite that causes chronic, low-level neuroinflammation in humans. A 2018 study of nearly 300 people in Korea found that 14 percent of people who attempted suicide tested positive for the parasite, compared to just 6 percent of healthy controls—mirroring a correlation found in several US cohorts. 13 Together, the findings paint a compelling picture that neuroinflammation “is part of the story,” says Melhem. While depression is not thought of as an inflammatory disease, signs of neuro inflammation in the brain have been repeatedly documented in people who suffer from depression, and a number of anti-inflammatory drugs show antidepressant effects. Microglia, the central nervous system’s primary immune cells and mediators of inflammation, tend to show increased activation in the brains of people who die by suicide, Melhem adds, and several studies have identified elevated concentrations of inflammatory cytokines such as interleukins IL-2, IL-6, and IL-8 in people with fatal and nonfatal suicidal behaviors. One 2019 analysis of nearly 2,000 Mexican-Americans, for example, found that blood levels of IL-8 were elevated in depressed and nondepressed women who had attempted suicide.14 How exactly neuroinflammation might contribute to suicidal behavior is still unclear, and some recent epidemiological studies have raised doubts about whether the association exists independently from depression. One route that researchers are exploring is neuroinflammation’s interaction with the serotonergic system. In a process thought to be mediated by microglia, neuroinflammation triggers a shift in the metabolism of serotonin’s molecular precursor, tryptophan, away from the production of serotonin and towards other chemical pathways—potentially reducing serotonin signaling and triggering other suicide-related changes in the brain. That’s just one hypothesis, says Melhem, who recently won a grant 01 /02. 2020 | T H E S C IE N T IST 3 5

Predicting suicide One of the defining moments in psychiatrist David Brent’s career happened during his medical residency some 40 years ago. Brent had been assigned to work with young people admitted for intentional drug overdoses at the University of Pittsburgh Medical Center Children’s Hospital. He had to determine who should be referred to a psychiatric ward and who could safely go home. “I found that I really didn’t have a very good way of making that determination,” says Brent, now a professor at Pitt. As he learned more about how other clinicians made such decisions, “I realized I was in good company—that nobody really knew what they were doing.” It’s still a dilemma facing anyone attempting to provide care for people at risk of suicide. Today’s clinicians often rely on patients to report their intentions in order to decide on appropriate interventions. But the approach has limitations. One 2019 meta-analysis of studies on suicidal ideation found that around 60 percent of people who ended their lives had denied having suicidal thoughts when asked by a clinician or doctor in the weeks or months before their death.15 This problem has led some researchers to look for ways to translate findings from neurobiology, however preliminary, into the identification of biomarkers to predict the onset of suicidal behaviors. Given its strong association with suicide, the HPA axis has long been a focus of this work, and there’s some evidence that abnormal cortisol levels—higher or lower than normal— in blood or saliva could hold promise as a biomarker. A few months ago, for example, Melhem, Brent, and colleagues published findings from a longterm study of teenagers that suggested a person’s baseline cortisol levels might 36 T H E SC I EN TIST | the-scientist.com

be used to predict future suicidal thinking, with higher cortisol associated with increased ideation within the next couple years.16 Cortisol tests may help provide predictive power to other measures of suicidality, such as questionnaires about social and academic stress. One recent analysis showed that while survey data were good predictors of who among 220 teenage girls with mental health concerns would be thinking about suicide within the next few months, they were poor predictors of who would attempt suicide during that period. But when the researchers focused only on girls who had shown blunted cortisol responses in lab tests, the questionnaire data predicted suicide attempts much better. 17

NEARLY 800,000 PEOPLE WORLDWIDE DIE BY SUICIDE EVERY YEAR. Source: WHO, 2016 data

Looking beyond stress responses, other groups have attempted to identify biomarkers related to neurotransmission. A few years ago, Mann’s group used positron emission tomographic (PET) imaging to assess levels of 5-HT1A serotonin receptors in the midbrains of 100 patients with major depressive disorder. The scientists found that higher 5-HT1A levels predicted greater suicidal ideation and more-lethal suicidal behavior within the next two years.18 Last summer, a team led by Yale University neuropsychologist Irina Esterlis reported that levels of glutamate receptor mGluR5,

as measured by PET, was linked to current suicidal ideation in patients with post-traumatic stress disorder—though the results didn’t hold for patients with major depressive disorder.19 Opinions differ among researchers about the potential of such biochemical signatures to assess suicide risk. Greg Ordway, a pharmacologist studying depression at East Tennessee State University, says that while biology might identify people predisposed to suicidal behavior, it’s unlikely to produce one or a handful of biomarkers that reliably reveal whether a person is about to end their life. “Suicide is extremely difficult to predict,” he says. “People are always trying to do it—people like me are looking for markers. But in reality, I don’t think we’ll probably ever find that.” Some of the most promising tools for assessing immediate risk might instead come from other areas of neuroscience that measure more-complex, emotional signals in the brain as opposed to biochemical signatures. In 2017, Brent, along with Carnegie Mellon University neuroscientist Marcel Just and other colleagues, used functional MRI to image the brains of 34 people as they contemplated words such as “death,” “trouble,” and “carefree.” Using machine learning algorithms to process the data, the team could distinguish between people who were thinking about suicide, as selfreported during the study, and those who weren’t with 91 percent accuracy. Among those who were, the team identified people who’d already attempted suicide with 94 percent accuracy.20 The researchers recently received $3.8 million from the National Institute of Mental Health to scale up the project and are planning long-term monitoring of people with and without various types of mood disorders. As part of the study, the researchers hope to extend their tool to identify people who might attempt suicide in the future, not just those who are thinking about it at the time of the scan or who have attempted it in the past. Just tells The Scientist that the team also plans to adapt the

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with Mann to investigate the suicideinflammation link. These pathways haven’t “been interrogated enough,” Melhem says. “We’ ll be looking more into that in future work.”

UP TO 90 PERCENT OF PEOPLE WHO DIE BY SUICIDE ARE THOUGHT TO HAVE HAD A DIAGNOSABLE MENTAL HEALTH CONDITION PRIOR TO THEIR DEATH.

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Source: NATIONAL ALLIANCE ON MENTAL ILLNESS, various datasets

technique to a cheaper, more clinicfriendly technology than MRI, such as electroencephalography (EEG). Melhem says she’s hopeful that combining techniques will improve predictive approaches in the coming years. In 2019, she and colleagues published a model that improved on the accuracy and performance of existing models to predict suicide attempts based on factors such as the severity and variability of a person’s depression symptoms over time. 21 Integrating this sort of easyto-collect clinical data with biological information from brain scans or other diagnostic tests should lead to moreaccurate predictions, she says. The search for such tests has important consequences for suicide prevention even beyond their potential to assess risk. “When we introduce biological markers, just like [for] any other area

of medicine, then stigma will be reduced at the level of the patient,” Melhem says. Patients are often surprised to hear that researchers are studying the biology underlying suicide “because they’ve been thinking that this is a behavioral flaw in their character, and they feel guilty about it. That’s part of the stigma that we want to break.” g

References 1. I.C.G. Weaver et al., “Epigenetic programming by maternal behavior,” Nat Neurosci, 7:847–54, 2004. 2. P.O. McGowan et al., “Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse,” Nat Neurosci, 12:342–48, 2009. 3. J. Jokinen et al., “Epigenetic changes in the CRH gene are related to severity of suicide attempt and a general psychiatric risk score in adolescents,” EBioMedicine, 27:123–33, 2018. 4. N.M. Melhem et al., “Blunted HPA axis activity in suicide attempters compared to those at high risk for suicidal behavior,” Neuropsychopharmacology, 41:1447–56, 2016. 5. B. Stanley et al., “Suicidal subtypes, stress responsivity and impulsive aggression,” Psychiatry Res, 280:112486, 2019. 6. D.B. O’Connor et al., “Cortisol levels and suicidal behavior: A meta-analysis,” Psychoneuroendocrinology, 63:370–79, 2016. 7. S.H. Parade et al., “Stress exposure and psychopathology alter methylation of the serotonin receptor 2A (HTR2A) gene in preschoolers,” Dev Psychopathol, 29:1619–26, 2017. 8. I. Ouellet-Morin et al., “Increased serotonin transporter gene (SERT) DNA methylation is associated with bullying victimization and blunted cortisol response to stress in childhood: a longitudinal study of discordant monozygotic twins,” Psychol Med, 43:1813–23, 2012. 9. L.J. Steinberg et al., “Cortisol stress response and in vivo PET imaging of human brain serotonin 1A receptor binding,” Int J Neuropsychopharmacol, 22:329–38, 2019.

10. G.M. Sullivan et al., “Positron emission tomography quantification of serotonin1A receptor binding in suicide attempters with major depressive disorder,” JAMA Psychiatry, 72:169–78, 2015. 11. M.D. Underwood et al., “Serotonin receptors and suicide, major depression, alcohol use disorder and reported early life adversity,” Transl Psychiatry, 8:279, 2018. 12. H. Lund-Sørensen et al., “A nationwide cohort study of the association between hospitalization with infection and risk of death by suicide,” JAMA Psychiatry, 73:912–19, 2016. 13. J. Bak et al., “The association between suicide attempts and Toxoplasma gondii infection,” Clin Psychopharmacol Neurosci, 16:95–102, 2018. 14. E.E.M. Knowles et al., “Family-based analyses reveal novel genetic overlap between cytokine interleukin-8 and risk for suicide attempt,” Brain Behav Immun, 80:292–99, 2019. 15. C.M. McHugh et al., “Association between suicidal ideation and suicide: meta-analyses of odd ratios, sensitivity, specificity and positive predictive value,” BJPsych Open, 5:e18, 2019. 16. A. Shalev et al., “Cortisol response to stress as a predictor for suicidal ideation in youth,” J Affect Disord, 257:10–16, 2019. 17. T.A. Eisenlohr-Moul et al., “HPA axis response and psychosocial stress as interactive predictors of suicidal ideation and behavior in adolescent females: a multilevel diathesisstress framework,” Neuropsychopharmacology, 43:2564–71, 2018. 18. M.A. Oquendo et al., “Positron emission tomographic imaging of the serotonergic system and prediction of risk and lethality of future suicidal behavior,” JAMA Psychiatry, 73:1048–1055, 2016. 19. M.T. Davis et al., “In vivo evidence for dysregulation of mGluR5 as a biomarker of suicidal ideation,” PNAS, 116:11490–95, 2019. 20. M.A. Just et al., “Machine learning of neural representations of suicide and emotion concepts identifies suicidal youth,” Nat Hum Behav, 1:911–19, 2017. 21. N.M. Melhem et al., “Severity and variability of depression symptoms predicting suicide attempt in high-risk individuals,” JAMA Psychiatry, 76:603–13, 2019.

PREVENTING SUICIDE Medical professionals consider suicide a preventable public health problem. In the US, agencies such as the Centers for Disease Control and Prevention and the Substance Abuse and Mental Health Services Administration oversee initiatives designed to help assess and respond to suicide risk in the general population, and particularly in communities considered to be at high risk, including among people with mood disorders, substance abuse problems, or a family history of suicide. Many nonprofit organizations also work to raise awareness of the problem, fund research on suicide, and provide resources for people affected by suicide. Find information about suicide warning signs, treatment, and other resources at the American Foundation for Suicide Prevention website, www.afsp.org. For help, call the confidential, free 24/7 National Suicide Prevention Lifeline at 1-800-273-8255.

Alternative Endings It’s now clear that cells can carve up transcripts in different ways to generate a variety of proteins from the same gene. Yet many questions remain about alternative splicing and its effects.

BY GABRIELLE M. GENTILE, HANNAH J. WIEDNER, EMMA R. HINKLE, AND JIMENA GIUDICE

S

eventeen years ago, the completion of the Human Genome Project revealed that there are around 20,000 protein-coding genes in the human genome—a puzzling result, given our intricate biology. Thanks to the advancement of large-scale proteomic studies over the decade following that milestone, researchers realized that some human cells contain billions of different polypeptides. Researchers realized that each gene can encode an array of proteins. The process of alternative splicing, which had first been observed 26 years before the Human Genome Project was finished, allows a cell to generate different RNAs, and ultimately different proteins, from the same gene. Since its discovery, it has become clear that alternative splicing is common and that the phenomenon helps explain how limited numbers of genes can encode organisms of staggering complexity. While fewer than 40 percent of the genes in a fruit fly undergo alternative splicing, more than 90 percent of genes are alternatively spliced in humans.

38 T H E SC I EN TIST | the-scientist.com

Astoundingly, some genes can be alternatively spliced to generate up to 38,000 different transcript isoforms, and each of the proteins they produce has a unique function. Like the chapters of a book, coding segments of the genome, known as exons, appear in series, and alternative splicing works by including or leaving out some of these genomic passages. Some chapters are required—that is, they are found in every transcript—and some are optional, so-called alternative exons. The differential splicing of these regions from an RNA transcript creates customized and condensed genetic messages. Molecular editors control the complicated flurry of exon selection by recognizing the chapters needed for a given protein and discarding the others. The final arrangement of exons in a spliced RNA molecule shapes the resulting protein’s structure and function. Although much remains to be learned about how these molecular editors work, it is now clear that they can have serious consequences

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The discovery of RNA splicing In 1941, George Beadle and Edward Tatum established the field of molecular biology with their one gene–one enzyme hypothesis, which was later refined to one gene–one polypeptide. Yet exactly how a gene encoded a protein was still unclear. In the late 1950s, Francis Crick presented his central dogma of molecular biology, a unifying paradigm in which genetic information flows from DNA to RNA to protein. According to this model, RNA serves as an intermediate, suggesting

HOW ALTERNATIVE SPLICING WORKS While some details of the mechanisms of splicing remain to be worked out, it’s known that mature, edited mRNAs result from an interplay between multiple factors within and outside the transcript itself. Among these is the spliceosome, the machinery that carries out the splicing. Each splicing event requires three components: the splice donor, a GU nucleotide sequence at one end of the intron; a splice acceptor, an AG nucleotide sequence at the opposite end; and a branch point, an A approximately 20–40 nucleotides away from the splice acceptor. These three “splice sites” are recognized by two core small nuclear RNAs (snRNAs) of the spliceosome, U1 and U2, followed by a protein, U2AF. The binding of these molecules to a transcript recruits a complex of three more snRNAs—U4, U5, and U6—which facilitates the splicing reaction. A variety of factors affect how transcripts from a particular gene are spliced. Exon recognition by the spliceosome can be influenced by RNA binding proteins (RBPs), which bind to enhancer and silencer motifs within the mRNA and help or hinder spliceosome recognition of the splice sites. And because premRNAs are frequently spliced as they’re transcribed, the speed of transcription by RNA polymerase II further tunes the window of opportunity for splice site recognition by the spliceosome. Binding motif Splice sites DNA RNA binding proteins Spliceosome

U4

Exon 1

U6

U2

RNA polymerase II

AF

U5

U2

U1

Exon 2

that the molecule is simply a disposable DNA copy. Yet RNA’s role would turn out to be far more complex and important than that of a middleman. In a series of experiments in 1977, Sue Berget, then a postdoc in Phil Sharp’s lab at MIT, demonstrated that viral messenger RNA (mRNA) is split—that is, it’s discontinuous relative to the original DNA sequence.1 Berget garnered this insight by isolating a viral gene and its corresponding mRNA and then combining the two molecules so that, with some chemical encouragement, the complementary sequences would base pair. Any noncomplementary sequences would be excluded, forming loops of single-stranded DNA that protruded from the double-stranded molecule. Berget, Sharp, and their colleagues used electron microscopy, the highestresolution technique at the time, to visualize the RNA-DNA hybrid, and observed many such loops. That same year, Rich Roberts and colleagues at Cold Spring Harbor Laboratory independently made the same finding.2 Sharp and Roberts would later be jointly awarded the Nobel Prize in Physiology or Medicine for the discovery of split genes. In 1978, Wally Gilbert, a colleague of Sharp, coined the terms intron (intragenic region) and exon (expressed region) to describe this novel concept of “genes in pieces.”3 This was not exclusive to viruses, either. The process of removing introns and joining coding regions together appeared to be conserved in virtually all organisms in the animal kingdom. The discovery of this basic mechanism, known as RNA splicing, introduced an important additional step to the central dogma and raised questions about how cells coordinate this process. Biochemists in the 1980s tried to tackle this question. Using gradient sedimentation and chromatography techniques, they purified large splicing complexes and combined them in vitro to reconstitute the RNA-snipping process. The burgeoning popularity of mass spectrometry throughout the 1990s, paired with the growing number of genomes uploaded in sequence repositories, enabled the identification of individual splicing components. These days, we know that the assembled complex, the spliceosome, is a massive molecular machine composed of five small nuclear RNAs (snRNAs) at the core, which may be aided by an array of more than 80 accessory proteins. Together, these snRNA-protein complexes form small nuclear ribonucleoproteins (snRNPs, pronounced “snurps”) that comprise the spliceosome. As an mRNA’s molecular editor, the spliceosome discriminates introns from exons and catalyzes their removal to link exons and assemble a protein. (See illustration at left.) Still, from an evolutionary perspective, the idea of RNA splicing seemed bizarre to some researchers. In September of 2003, the Encyclopedia of DNA Elements (ENCODE) project was launched to identify the functional elements in the human genome, and the effort ignited controversies as to whether introns were genetic “junk” that the cell invested precious energy and resources to transcribe only to trash prior to translation. Alternative splicing gave these seemingly nonfunctional elements an essential role in gene expression, as evidence emerged over the next few years that there are sequences housed within introns that can help or hinder splicing activity. These enhancer and silencer sequences are recognized by RNA-binding

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for a protein’s story, influencing whether it leads to healthy development or to disease.

proteins (RBPs) whose presence affects spliceosome docking and assembly. The RBPs allow exons or portions of exons to be combined or skipped in unique patterns, such that a single transcript can be spliced into several possible mature mRNA isoforms, or splice variants, each translated into proteins with potentially diverse functions. This overturned Beadle and Tatum’s hypothesis and illustrated that there was perhaps much more to the splicing story than had thus far been discovered. Not long after the biochemical mechanism underlying RNA splicing was pieced together, more scientists jumped onto the splicing bandwagon and set out to study its functional consequences. Some of the earliest accounts came in the late 1980s, when several groups studying Drosophila melanogaster development independently noted that the genes involved in the fly’s sex determination cascade have female- and male-specific splice isoforms that determine the fly’s sexual fate.4–7 The field then began to recognize that alternative splicing wields extraordinary power in shaping development and tissue identity. Over the following decade, researchers published isolated examples featuring the functional roles of splice isoforms in other model organisms, from yeast and worms to mice and rats. Then, the race was on to study splicing regulation in humans. In late 2008, three separate teams led by Tom Cooper at Baylor College of Medicine, Chris Burge at MIT, and Ben Blencowe at the University of Toronto published landmark papers on genome-wide splicing patterns across a host of human tissues and cell lines. Collectively, their studies revealed that every tissue in the body is characterized by a unique set of splicing events.8–10 Four years later, the Burge lab took an evolutionary approach to compare alternative splicing among higher-order vertebrate species, including the rhesus macaque and cow. They found that brain, heart, and skeletal muscle present with the most highly conserved and tissue-specific alternative splicing patterns,11 further underscoring the functional importance of tissuespecific alternative splicing.

New developments In general, splicing patterns change during development. Intriguingly, genes that are spliced are, more often than not, expressed at similar levels in all organs and across all developmental stages. This suggests that splicing can tune the production of proteins that result from these uniformly expressed genes to different contexts with regulators that modulate splicing depending on tissue type and stage of development. Indeed, RNA-binding proteins come and go as development unfolds, and they assume the role of molecular switches of alternative splicing events. The vast number of potential interaction combinations between enhancer and silencer sequences and the RBPs that recognize them inspired the field to adopt the idea of a splicing code—that certain RBPs bind to certain RNA motifs to produce a given edit. Current efforts are focused on cracking that code. But defining a set of RBP targets is exceedingly complex, as RBPs can recognize multiple motifs depending on the biological context. The intricate and precise action of RBPs controls alternative splicing networks, groups of transcripts from different genes that are each

targeted by one or more of the same RBPs. A network can coordinate a specific cellular function that contributes to development or to tissue homeostasis. In recent years, groups of researchers have concentrated on unraveling these splicing networks. Among other researchers, the Burge and Cooper labs continued their long-standing collaboration to tackle this task in mice. The two groups sequenced RNA to track gene expression and the abundance of the various transcript isoforms during cardiac muscle development, and they observed that the conversion from fetal to adult heart cell function parallels a transition from fetal to adult splicing profiles. As a postdoc in the Cooper lab, one of us, Jimena Giudice, found that numerous differentially spliced genes encode proteins involved in intracellular trafficking, and these splicing events are controlled by two RBPs: CELF and MBNL.12 All signs pointed to a splicing network. Follow-up work revealed that the expression levels of CELF and MBNL are inversely tied to one another during muscle development, and that they antagonistically regulate more than 1,000 pre-mRNA transcripts, some of which are translated into proteins critical for muscle contraction.13

Alternative splicing helps to explain how limited numbers of genes can encode organisms of staggering complexity. Since the early efforts to describe splicing, the textbook view of the process has been that it occurs post-transcriptionally. However, researchers are challenging this view by demonstrating that RNA polymerase II (RNAPII) dynamics have the potential to influence spliceosome assembly, perhaps coupling transcription to splicing. Karla Neugebauer and her lab at Yale University champion this model and use biochemical and computational approaches to study the phenomenon. Recently, they developed a single-molecule intron tracking (SMIT) technique to measure splicing kinetics and found that introns are spliced as soon as they emerge from RNAPII.14 Last year, an international team of researchers published on the in vivo consequences of such co-transcriptional splicing, showing that mouse embryonic stem cells with a knocked-in gene for a slow-transcribing version of RNAPII exhibit neuronal differentiation defects due to the failure to properly splice genes involved in synapse signaling.15 This suggested that the rate at which RNAPII transcribes RNA affects how that RNA is spliced. Researchers are also exploring the possibility that chromatin architecture and epigenetics serve as another layer of splicing regulation by modulating the rate of RNAPII transcription. Despite a collection of cases teasing apart the mechanism of alternative splicing and highlighting its functional consequences, the number of uncharacterized splicing events is immense, and the pages documenting the physiological importance of alternative splicing largely remain blank.

Mis-splicing in disease More than one-third of disease-causing mutations map to sites bound by the spliceosome or RBPs, or to RBP-encoding gene regions. 01 /02. 202 0 | T H E S C IE N T IST 41

SPLICING MATTERS Titin, which codes for a protein in muscle, is one example of a gene whose pre-mRNA transcript can be spliced in multiple ways to yield different protein isoforms. During development of the fetal heart, more exons are left in during splicing, which produces a relatively long, springy protein. In adult hearts, an RNA-binding protein called RBM20 associates with long stretches of the mRNA transcript during splicing, forcing the spliceosome to cut out those bits of DNA. The result is a relatively short, stiff protein. If RBM20 is missing or defective in adult hearts, these hearts will produce more fetal, springy titin protein relative to the stiff adult version. This is thought to reduce the capacity of the heart to contract, contributing to a condition known as dilated cardiomyopathy. FETAL SPLICING SCENARIO

ADULT SPLICING SCENARIO

Titin gene

Transcription

Transcription

RBM20

When low levels of RBM20 are present, the titin gene is predominantly spliced as the longer, springy isoform.

Splicing

Translation

Long, springy titin protein

High ratio of fetal to adult titin

High ratio of fetal to adult titin

Pre-mRNA

High levels of RBM20 force the splicing machinery to leave out a number of exons from the final titin mRNA transcript.

Splicing

Translation

Short, stiff titin protein

Low ratio of fetal to adult titin

Healthy fetal heart A higher-than-normal proportion of fetal titin in an adult heart can contribute to a heart that’s too elastic, a condition called dilated cardiomyopathy.

Healthy adult heart

THE SCIENTIST STAFF

Alternative exons

Pre-mRNA

Therefore, mis-splicing has a strong potential to be implicated in disease. Parkinson’s, progeria, cardiomyopathy, spinal muscular atrophy, myotonic dystrophy, breast cancer, ovarian cancer—these devastating illnesses and many more appear to be caused partly by defects in RNA splicing, emphasizing the range of crippling effects that can stem from even slightly tipping the balance of protein isoform expression.

Many devastating illnesses appear to be caused partly by defects in RNA splicing. One scenario involves the titin (TTN) gene, which holds the record for the highest number of exons—a whopping 363—among all mammalian genes and encodes the largest known protein in the human body, weighing in at 4.2 megadaltons. The TTN protein is a molecular spring that contributes to the elasticity of heart muscle. Over the course of cardiac development, there is a gradual increase in the frequency of TTN exon skipping by the spliceosome, and these exons are thus spliced out from the mRNA. (See illustration on opposite page.) The transition from the fetal to the adult cardiac titin isoform is part of the normal developmental program and is orchestrated by an RBP called RBM20. That RBP promotes skipping where it’s found, thus inducing a shift in protein expression from a long, elastic TTN isoform to a short, stiff isoform. Using a rodent model, an international cohort of researchers and physicians demonstrated that the absence of RBM20 causes TTN missplicing, leading to the buildup of long, elastic TTN and phenotypes resembling the decreased heart contractility seen in humans with dilated cardiomyopathy induced by mutations in RBM20.16 The results strongly suggest that TTN mis-splicing contributes to RBM20-linked cardiomyopathy. Another example is DMD, the gene that encodes the dystrophin protein, which is important for muscle integrity and force transmission. Mutational variants in DMD are notoriously associated with Duchenne muscular dystrophy, a disease that severely impairs muscle function. (See “Mending Muscle,” September 2018.) One diseasecausing DMD mutation is a multiexon deletion that commonly results in a frameshift starting at exon 51. Splicing the remaining exons together results in a shortened dystrophin protein with compromised function. The lack of fully functioning dystrophin protein causes muscle weakness and atrophy, which drastically limit the physical abilities of people suffering from this disease. Some therapies currently in development for Duchenne muscular dystrophy and other diseases aim to correct defects in splicing to alleviate symptoms. For example, researchers have been able to partially recover dystrophin function by using antisense oligonucleotides that prevent the spliceosome from recognizing exons downstream of the deletion. By hiding these regions from the spliceosome, the exons will be skipped. This can then restore the reading frame and produce a near-full-length protein. Two years ago, scientists at Japan’s National Center of Neurology and Psychiatry

reported results from a Phase 1 trial hinting at the oligonucleotides’ safety and capacity to induce DMD exon-skipping in patients with Duchenne muscular dystrophy.17 Understanding the story behind each protein in our bodies has turned out to be far more complex than reading our DNA. Although the basic splicing mechanism was uncovered more than 40 years ago, working out the interplay between splicing and physiology continues to fascinate us. We hope that advanced knowledge of how alternative splicing is regulated and the functional role of each protein isoform during development and disease will lay the groundwork for the success of future translational therapies. g Gabrielle M. Gentile, Hannah J. Wiedner, and Emma R. Hinkle are graduate students in the Curriculum in Genetics and Molecular Biology at the University of North Carolina at Chapel Hill, where Jimena Giudice is an assistant professor in the Department of Cell Biology and Physiology. Giudice is also a member of the university’s McAllister Heart Institute in the School of Medicine.

References 1. S.M. Berget et al., “Spliced segments at the 5' terminus of adenovirus 2 late mRNA,” PNAS, 74:3171–75, 1977. 2. L.T. Chow et al., “An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA,” Cell, 12:1–8, 1977. 3. W. Gilbert, “Why genes in pieces?” Nature, 271:501, 1978. 4. R.T. Boggs et al., “Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene,” Cell, 50:739–47, 1987. 5. L.R. Bell et al., “Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins,” Cell, 55:1037–46, 1988. 6. B.S. Baker, M.F. Wolfner, “A molecular analysis of doublesex, a bifunctional gene that controls both male and female sexual differentiation in Drosophila melanogaster,” Genes Dev, 2:477–89, 1988. 7. K.C. Burtis, B.S. Baker, “Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sexspecific polypeptides,” Cell, 56:997–1010, 1989. 8. Q. Pan et al., “Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing,” Nat Genet, 40:1413–15, 2008. 9. J.C. Castle et al., “Expression of 24,426 human alternative splicing events and predicted cis regulation in 48 tissues and cell lines,” Nat Genet, 40:1416–25, 2008. 10. E.T. Wang et al., “Alternative isoform regulation in human tissue transcriptomes,” Nature, 456:470–76, 2008. 11. J. Merkin et al., “Evolutionary dynamics of gene and isoform regulation in mammalian tissues,” Science 338:1593–99, 2012. 12. J. Giudice et al., “Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development,” Nat Commun, 5:3603, 2014. 13. E.T. Wang et al., “Antagonistic regulation of mRNA expression and splicing by CELF and MBNL proteins,” Genome Res, 25:858–71, 2015. 14. F. Carrillo Oesterreich et al., “Splicing of nascent RNA coincides with intron exit from RNA polymerase II,” Cell, 165:372–81, 2016. 15. M.M. Maslon et al., “A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes,” EMBO J, 38:e101244, 2019. 16. W. Guo et al., “RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing,” Nat Med, 18:766–73, 2012. 17. H. Komaki et al., “Systemic administration of the antisense oligonucleotide NS-065/ NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy,” Sci Transl Med, 10:eaan0713, 2018.

01 /02. 202 0 | T H E S C IE N T IST 4 3

EDITOR’S CHOICE PAPERS

The Literature MICROBIOLOGY

THE PAPER

Ankyphage containing code for ankyrin

M. Jahn et al., “A phage protein aids bacterial symbionts in eukaryote immune evasion,” Cell Host Microbe, 26:542–50, 2019. While studying sponges and their endosymbiotic microbes for his PhD, Martin Jahn found himself pondering where viruses fit into the mix. “We didn’t know anything about the viruses associated with sponges,” says Jahn, now wrapping up his doctorate at the GEOMAR Helmholtz Centre for Ocean Research Kiel in Germany. To investigate, Jahn and his colleagues sampled four sponge species off the coast of northern Spain and analyzed both the sponges and samples of the surrounding seawater for the presence of viruses. Not only did the researchers find viruses living in sponges that weren’t in the seawater, they discovered substantial diversity in the viromes of different species, and even among conspecifics. Digging further into the genomic data, the team noticed one group of previously unidentified bacteriophages that were particularly abundant in sponge viromes. To Jahn’s surprise, these phages contained genetic sequences for so-called ankyrin repeats, protein motifs usually studied in bacteria that help pathogenic or commensal microbes infect and manipulate eukaryotic hosts. He wondered if the viruses, which the team dubbed ankyphages, might facilitate interactions between sponges and their resident bacteria. Both sponge cells and their endosymbiotic bacteria are difficult to culture, so to test Jahn’s idea, the team set up an experiment with mouse cell lines and E. coli. The researchers first cultured E. coli with ankyrin protein synthesized from the viral sequences. Then they added the bacte44 T H E SC I EN TIST | the-scientist.com

Mouse macrophage

Endosymbiotic bacteria

Marine sponge

E. coli displaying synthesized ankyrin peptides

Recombinant E. coli carrying ankyphagederived sequence, allowing the bacteria to secrete ankyrin proteins

BETTER TOGETHER: Researchers have discovered a diverse set of bacteriophages in tissue samples

from marine sponges, which are known to host abundant endosymbiotic bacteria  A . In vitro experiments revealed that a protein made by a subset of the sponge-borne viruses, known as ankyphages, appears to help suppress immune responses in murine macrophages when taken up and displayed, or expressed and secreted, by E. coli  B —bacteria with the protein, ankyrin, were less likely to be consumed by the immune cells than were controls. The results suggest that ankyphages could facilitate the cohabitation of commensal bacteria with their eukaryotic hosts.

ria, which displayed the protein on their cell surfaces, to murine immune cells. Sure enough, the E. coli that had been cultured with ankyrin protein were better at surviving exposure to mouse immune cells: they escaped being engulfed by macrophages more often than control bacteria did. E. coli engineered to produce and secrete the phage proteins themselves also survived macrophage exposure. The team ran further experiments to confirm that the protein wasn’t toxic to either the bacterial or murine cells, and concluded that phage-derived ankyrin was indeed helping to suppress macrophage responses toward the bacteria. “I was quite impressed by these . . . proteins being associated with spongespecific phage communities,” says Breck

Duerkop, a microbiologist at the University of Colorado School of Medicine who wasn’t involved in the work. That phages might moderate host immunity is a “really interesting idea,” he adds, although the team’s experiments don’t quite establish that such three-way interactions are playing an important role in sponges. Scanning genome databases for other phyla, Jahn and his colleagues found evidence that ankyphages are also present in the microbiomes of other eukaryotic organisms, including humans. The findings hint at the importance of bacteriophages in eukaryotic function, says Jahn. Far from being incidental stowaways in eukaryotic organisms, phages “are central elements,” he says. “It opens a lot of perspective for further research.” —Catherine Offord

© KELLY FINAN

Three’s Company

©ISTOCK.COM/ LUISMMOLINA; JOHN GOULD

ANEUPLOID PROBLEMS: Eggs from girls and from older women show elevated rates of errors in chromosome number.

ONSITE BUFFET: Diving beetle eggs (circled in red) laid on frog spawn hatch

CELL BIOLOGY

ECOLOGY AND ENVIRONMENT

Errors in the Egg

Tadpole Snacks

THE PAPER

THE PAPER

J.R. Gruhn et al., “Chromosome errors in human eggs shape natural fertility over reproductive life span,” Science, 365:1466–69, 2019.

J. Gould et al., “Diving beetle offspring oviposited in amphibian spawn prey on the tadpoles upon hatching,” Entomol Sci, 22:393–97, 2019.

Females of most mammalian species are fertile throughout their adult life. But humans are different, says University of Copenhagen molecular geneticist Eva Hoffmann. A woman’s fertility follows a curve, increasing from puberty, peaking in her 20s, and falling rapidly starting in her mid-30s. Researchers attribute this decline partly to a rise in egg aneuploidy, or incorrect chromosome number, which can lead to pregnancy failure. Hoffmann and colleagues wanted to know more about how aneuploidy occurs in human eggs, and whether it’s connected to female fertility from a young age. The team collected more than 3,000 eggs from women between 9 and 43 years old through a collaboration with IVF clinics and Danish hospitals that preserve ovarian tissue from cancer patients about to undergo chemotherapy. The researchers found, as expected, that older women’s eggs showed higher-than-normal rates of aneuploidy. Surprisingly, eggs from young girls showed high rates too, resulting in a U-shape aneuploidy curve—the inverse of the relationship between fertility and age. This egg aneuploidy has to do with the molecular glue that holds sister chromatids together, Hoffmann says. In older women, the team showed, the glue fails prematurely during cell division; in girls, it’s overeffective, releasing chromatids later than usual. Both abnormalities can influence chromatid segregation and result in aneuploidy. The study is “Herculean in its efforts,” says Karen Schindler, a reproductive biologist at Rutgers University who was not involved in the work. Using girls’ eggs was a “unique and important approach” that “really fleshes out [what’s happening at] the younger age.” However, she notes, there could be other mechanisms behind agerelated changes in aneuploidy rates. The findings have implications for researchers’ understanding and treatment of infertility, not just in older women but in young girls who freeze their eggs for health reasons, Schindler adds. A girl’s eggs “clearly behave differently than they do when she’s reproductive age.” —Catherine Offord

While studying the conservation of endangered amphibians during his PhD at the University of Newcastle in Australia, Jose Valdez spent a lot of time peering into ponds looking for tadpoles. One night a few years ago, he noticed a group of predaceous diving beetles (family: Dytiscidae) ripping into a tadpole. Both larval and adult diving beetles are known predators of tadpoles, but witnessing the act himself, Valdez began to wonder about the influence of these invertebrates on amphibian survival. “These predators perhaps are overlooked,” he says. When Valdez surveyed ponds in a half-acre area, he found that 80 percent of the tadpoles he observed were living in ponds free of diving beetles, suggesting the beetles might be influencing tadpole survival (Aust J Zool, doi:10.1071/ZO19039, 2019). He and University of Newcastle biologist John Gould decided to investigate further, and soon discovered that not only did diving beetles feed on the tadpoles, they laid eggs directly on the frog spawn from which tadpoles would emerge. “It’s a buffet,” says Valdez, now a postdoc at Aarhus University in Denmark. “There’s food everywhere.” When the researchers collected frog spawn containing diving beetle eggs for observation in the lab, they found that beetle larvae hatched within 24 hours of the tadpoles emerging. “The fact that they were hatching right at the right time to prey upon the tadpoles was pretty cool,” says Corinne RichardsZawacki, an ecologist at the University of Pittsburgh who wasn’t involved in the work. “It begs more experiments to see whether or not this is something they do as an adaptation.” Either way, notes Valdez, the findings suggest that invertebrates such as diving beetles may be more important predators of amphibians than previously thought. The study is a prime example of “good natural history,” Richards-Zawacki adds. “Kudos for paying attention and taking the time to tease apart what was going on.” —Catherine Offord

within hours of the emergence of the tadpoles the beetle larvae feed on.

01 /02. 202 0 | T H E S C IE N T IST 4 5

PROFILE

Switch Master University of California, Berkeley molecular biologist Barbara Meyer’s work with bacteriophages and nematodes exposed the role of genetic switches in early development. BY DIANA KWON

AN UNEXPECTED PATH Growing up in Stockton, a small city in California’s Central Valley, in the 1950s, Meyer never thought she’d become a scientist. She was fascinated by the sciences, which she encountered both through interactions with an uncle who was an aeronautical engineer and via a wide selection of nonfiction books. But when asked in high school to write an essay about what she envisioned as her future career, Meyer wrote that she would become a medical lab tech. “[I wasn’t] even dreaming that I’d be able to have a PhD,” Meyer recalls. In her town, “there was no model for being a scientist . . . certainly not for a girl.” 46 T H E SC I EN TIST | the-scientist.com

Meyer longed to escape Stockton, which she describes as a rather isolated place. She imagined traveling the world like another of her uncles, a globetrotter who would visit with exotic gifts from faraway places. She got her first travel opportunity after she was accepted into Stanford University, which allowed her to study abroad. She spent a semester at the Stanford campus in Stuttgart, West Germany, in 1969. On one of her visits to Berlin, where the infamous wall split the city in two, Meyer remembers encountering a distressing sight: soldiers shooting at people who were trying to escape across the border into the West. “When you see these kinds of things, it makes you happier thinking about science because there’s some logic to it, rather than watching people get shot,” Meyer says. “That compelled me even more [to go into science].” After completing her undergraduate studies, Meyer joined the lab of David Clayton, a developmental biologist at Stanford’s California campus. There, she used herpes simplex virus and mouse mitochondria to investigate a nucleotide-forming enzyme called thymidine kinase. “That convinced me that I really wanted to do [research],” she says. “Whether I could or not was another question, but I really wanted it.” While working in Clayton’s lab, Meyer decided she wanted to pursue medical research and applied to medical school. During the application process, several interviewers told her that they saw her more as a researcher than a physician, or that if they offered her a position in a medical program, she would simply get married, pregnant, and drop out, Meyer recalls. She was ultimately rejected. That was a demoralizing experience, she says, but it turned out for the best. She had also applied to graduate school, and soon after her med school rejections, she learned she’d been accepted into a program at the University of California, Berkeley—where she would not only earn her PhD, but eventually go on to become a leader in the study of genetic switches.

A FATEFUL SWITCH In graduate school, Meyer focused on viruses that infect bacteria. One such bacteriophage, the famous lambda phage, can live one of two lifestyles: a peaceful one, where it stays dormant in a host’s DNA, or a violent one, where it hijacks the host’s cellular machinery to replicate, then releases its progeny into the environment. When Meyer started at Berkeley, she was intrigued by the factors that influenced how the lambda phage ended up on

DEBORAH STALFORD

T

he tiny, transparent worm Caenorhabditis elegans can be born either as a male, typically with one X chromosome, or as a hermaphrodite, with two. When Barbara Meyer began a professorship at MIT in the 1980s, she wanted to pinpoint the genetic pathway that determined the worm’s sexual fate. There was evidence that this process was tied to dosage compensation, a delicate balancing act that ensures X-chromosome expression is matched between the two forms—a condition necessary for survival not only in worms, but also in humans, fruit flies, and other animals in which one sex carries two X chromosomes, while the other carries only one. Meyer suspected that the gene responsible would control the levels of X chromosome expression in only one form of the worm—either it would limit expression in XX C. elegans, or it would boost expression in individuals with only a single X—and failure to do so would result in the animal’s death. Her team, however, struggled to find such a gene. Then, one day, a “eureka moment” almost got thrown down the drain. In one experiment, her team was growing hermaphrodites with a mutation in a sex-specific gene they knew was involved in X dosage compensation. At first, as expected, the nematodes were growing poorly and dying, but then a lab technician noticed that they suddenly looked healthy and started multiplying. Thinking that there was a problem with the culture, he started to discard the samples in the sink. From across the room, Meyer shouted, “Stop!” She suspected that those healthy worms might contain the answer to her question, and she was right. There had been a spontaneous mutation in what turned out to be the master sexswitch gene, responsible for both determining sex and repressing the expression of X chromosomes in hermaphroditic worms (Cell, 48:25–37, 1987). “In that [culture], through serendipity, was the long-sought-after gene that I wanted,” Meyer says.

CAREER TITLES/AWARDS Professor, Department of Molecular and Cell Biology, University of California, Berkeley Investigator, Howard Hughes Medical Institute Elected Member, National Academy of Medicine (2018) E.B. Wilson Medal, American Society for Cell Biology (2018) Thomas Hunt Morgan Medal, Genetics Society of America (2018)

Greatest Hits • Identified a genetic switch that determines whether bacteriophage lambda becomes virulent or lives dormant in a host’s genome • Found the “master sex-switch gene” that determined whether C. elegans becomes male or hermaphrodite and controlled dosage compensation—the process of balancing X chromosome expression that is crucial to an organism’s survival • Discovered that parts of the protein complex involved in dosage compensation are co-opted from the machinery used to segregate chromosomes in mitosis and meiosis • Revealed that the size of a nematode’s genome differs based on its mode of reproduction (sexual vs. self-fertilization)

one of these two paths. At the time, scientists had discovered that the phage itself carried a repressor, a protein that inhibits gene expression by binding to DNA. They knew this protein was involved in determining a phage’s fate, but the mechanism behind its function was an open question. “The fact that there was a genetic switch that enabled that decision—I really wanted to understand that,” Meyer says. Her advisor, Harrison Echols—a biologist who conducted pioneering work on bacteriophage lambda—thought that trying to understand the genetic switch was too difficult for her to pursue, Meyer recalls; as an alternative, he suggested she map out all the promoter sequences for RNA polymerases in the lambda phage’s DNA. So, she put her passion project on hold and began mapping polymerase promoters. A few months later, she sat in on a seminar by molecular biologist Mark Ptashne, then a professor at Harvard University. Ptashne had been the first to isolate the lambda repressor protein, and his lab was in the process of investigating how it controlled the phage’s fate. According to Meyer, he too discouraged her from trying to tackle the question of the bacteriophage lambda’s genetic switch. “He said, you can’t possibly compete with us, so forget about it,” she recalls. But when she ran into him again a year later at a conference at Cold Spring Harbor Laboratory in New York, Ptashne asked her to sit in on meeting with a few members of his team, where he surprised Meyer by inviting her to his lab at Harvard to conduct the experiments on bacteriophage lambda that she was dying to do. “I had been dreaming about this for a whole year, and then this miracle happened,” she says. After Echols approved the transfer, Meyer packed her bags and moved to Cambridge, Massachusetts. Within a few weeks of joining Ptashne’s lab, she had demonstrated that the lambda repressor regulated the transcription of its own genes in a test tube (PNAS, 72:4785–89, 1975). For her, this was an “aha” moment that led to a cascade of follow-up experiments and around a dozen papers during her PhD studies alone. Working with Ptashne taught Meyer to be both a rigorous scientist and an expert communicator, she says. Ptashne is “a master at figuring out how to get messages across. . . . Plus, he had little patience, so your experiments had to be perfect for him to believe them,” she tells The Scientist. “I think that was all part of my education.”

SEX AND DEATH DECISIONS After completing her doctoral studies in 1979, Meyer wanted to find a research question that she could make her own—and 01 /02. 202 0 | T H E S C IE N T IST 47

PROFILE a more complex organism in which to study developmental biology. For her postdoc, she moved across the Atlantic to Cambridge, England, to work at the MRC Laboratory of Molecular Biology. There, she joined the lab of Sydney Brenner, a biologist who later would win the Nobel Prize for his groundbreaking work on C. elegans. Meyer chose to study the nematode because she was drawn by the question of how its sex is determined. “I was fascinated again by a binary developmental decision,” Meyer says. “It was the same idea [as with the lambda phage], but on a much huger scale.” Jonathan Hodgkin, a nematode biologist who had been a graduate student in Brenner’s lab, had already identified a mutation that caused genetic males to become hermaphrodites, and vice versa. But Meyer wasn’t convinced that this mutation explained the sex switch, because there had been hints from previous research that the switch might be linked to dosage compensation of X-linked genes. Too much or too little X expression is lethal, so “I thought I couldn’t just look for sex reversal, because the animal I’m looking for might be dead,” Meyer explains. Years earlier, biologists Victor Nigon and Robert Herman had shown that C. elegans embryos were sensitive to the number of X chromosomes relative to sets of non-sex chromosomes called autosomes in their genomes. By studying animals that carried two, three, and four sets of autosomes, they discovered that worms born with an X chromosome:autosome ratio between 0.5 and 0.67 would be male, while ratios between 0.75 and 1 would be hermaphrodites. (Other ratios would either be lethal or impossible to generate.) There had also been work by Thomas Cline, a geneticist who was then at Princeton University, that revealed a link between sex determination and dosage compensation in fruit flies. Meyer decided to work backwards in the worms, screening for genes that were involved in dosage compensation. Eventually she found autosomal genes that, when disrupted by mutation, led to abnormal levels of X-chromosome expression (Cell, 47:871–81, 1986). “Barbara is a brilliant and creative scientist,” says Cynthia Kenyon, the vice president of Calico, a San Francisco-based biotech company. Kenyon, who met Meyer while the two were both graduate students at Harvard in the 1970s, says that Meyer will “stop at nothing to figure out how to dissect a system of incredible complexity.” Meyer continued her work on C. elegans at MIT in Cambridge, Massachusetts, where she started a professorship in the early 1980s. Anne Villeneuve, a Stanford University geneticist who was one of Meyer’s first graduate students at MIT, says she was inspired by Meyer’s boldness in the early days of her lab. “She had to have confidence in the system that she was building from scratch, and she had to believe that it was going to work out,” Villeneuve says. “Once it was built, everyone could see how cool it was.” Her worm system led to the drain-dumping-turned-eureka moment, as well as to many other important insights into the 48 T H E SC I EN TIST | the-scientist.com

molecular machinery involved both in dosage compensation and in other fundamental cellular processes, such as meiosis.

A BRUSH WITH FATE During Meyer’s early days of working on C. elegans, Princeton’s Cline was but a faceless author on papers she’d read with great interest. She was amazed by the quality of the manuscripts, and because he was the only author on many of them, she suspected that he must have had a long career in academia—and thus was many decades older than her. Meyer later learned that this was not the case: Cline was, in fact, only a few years her senior. The two met for the first time at a developmental biology conference in 1981 and bonded over their shared scientific interests. They started dating several years later, in 1986, and once their romance began, things moved quickly— within a month of getting together, they were married. “I fell in love with him from reading his papers,” Meyer says. When the couple got married, they were professors in different states: Cline was in New Jersey, and Meyer in Massachusetts. Cline, like Meyer, was from California, and he wanted to return. Although Meyer loved MIT and the fast-paced lifestyle of the East Coast, managing her father’s health care from across the country was proving to be a challenge. After her father had a heart attack, Meyer and Cline moved back to their home state, where they both obtained faculty positions at Berkeley. One of the defining features of the couple’s now decades-long relationship has been a common passion for science. Together, they have coauthored a handful of review articles, and to this day, they edit each other’s papers. “We’re each other’s best critics,” Meyer says. The two also share a love of hiking and have trekked along numerous, sometimes-treacherous trails around the world. On one evening outing with Cline on Costa Rica’s Osa Peninsula in 1999, Meyer took a wrong step in the dark and fell off a 12-foot-high cliff. She landed on her back and shattered her ankle, but she considers herself lucky: there was a block of concrete right by her head, and iron rods jutting out from the space between her legs. “It makes me realize that anything can happen at any moment, and you better live your life well,” she says. “It made me think really hard about what could happen in the future—so I’m very good at troubleshooting in advance.” That preparedness has served Meyer well both inside and outside of the lab. Throughout her career, she has juggled many tasks, from running her lab and mentoring countless students to organizing scientific meetings and serving on numerous advisory boards for universities, professional societies, and both governmental and nonprofit organizations. And, she’s still determined to crack more scientific mysteries—for example, to further unravel the biochemical mechanisms underlying dosage compensation and to understand how chromosome structure affects gene expression. “There are quite number of big questions left,” she says. g

SCIENTIST TO WATCH

Oded Rechavi: Epigenetic Expressionist Associate Professor, Tel Aviv University, Age: 39 BY EMILY MAKOWSKI

© YADID LEVY PHOTOGRAPHY

A

lthough neurobiologist Oded Rechavi comes from a family of doctors and researchers, it was not his original plan to go into science. “It wasn’t something that I had thought about,” he tells The Scientist. Instead, he went to Paris after high school to train as an artist and exhibited his work in his home country of Israel before enrolling at Tel Aviv University. “I still didn’t know exactly what I’d do, whether I’d be an artist or something else,” he says. At university, Rechavi became interested in studying psychology, philosophy, and biology—specifically, the biology of the brain, which fascinated him. He earned a bachelor’s degree in neuroscience in 2006 and then went on to do a PhD, also at Tel Aviv. For his graduate work, he pivoted his focus to immune cells and found that when T and B cells connect with each other, they exchange macromolecules such as small interfering RNAs (siRNAs) that can break down messenger RNA molecules, preventing them from being translated into proteins—a process known as RNA interference, or RNAi (Genes Dev, 23:1971–79, 2009). “Now we know that small RNAs are exchanged in many different organs,” he says. Rechavi continued to study RNA as a postdoc in Oliver Hobert’s lab at Columbia University in New York. “It was pretty clear when he came to visit the lab that he was really very special—incredibly thoughtful, creative, and very excited and engaged about the projects that we discussed,” Hobert says. One of those projects was investigating siRNAs that C. elegans produces as a defense against viral infection. In 2011, Rechavi and colleagues demonstrated that the worms passed down those siRNAs from parent to offspring (Cell, 147:1248–56). “There’s no virus that infects C. elegans efficiently,”

Rechavi explains. “Only mutants that are defective in RNA [interference] are infected with viruses, and this could be in part because [nematodes] inherit siRNAs.” In 2012, Rechavi moved back to Israel and established his own lab at his alma mater. Inspired by human epidemiological studies, which have suggested that famine is associated with an increased risk of diabetes, heart disease, and obesity in subsequent generations, he wanted to see if changes in siRNAs caused by an environmental stressor could be inherited several generations down the line in nematodes. Sure enough, Rechavi’s group, in collaboration with Hobert, showed that starving nematodes passed down siRNAs that cause silencing of genes involved with fat regulation and stress resistance (Cell, 158:277–87, 2014). “We showed that small RNAs leave a mark that’s perceived for multiple generations after starvation,” Rechavi says. Additional studies from his group showed that inherited siRNAs can also have effects on movement and even decision making in the worms. “He’s got a great model system in C. elegans. . . . It was very impactful, and it shed a new light on these problems,” says Michael Levin, a systems biologist at Tufts University who studies planarian flatworms and has written review papers with Rechavi on RNA inheritance. “I always thought that his work was particularly creative and rigorous, and I think he has a very unique kind of mind.” Although Rechavi ended up focusing his career on science instead of art, he often looks for ways to combine the two. He’s a research associate in an interdisciplinary group of artists and scientists that focuses on culture, society, and philosophy at the Van Leer Jerusalem Institute. And he’s also using Twitter to

organize a February 2020 conference in Tel Aviv informally called “The Woodstock of Biology,” which will feature a collaborative art exhibition focusing on natural resources. “He has a lot of research interests and an infectious enthusiasm for things,” Hobert says. “The extent of his creativity really knows no bounds.” g

01 /02. 2020 | T H E S C IE N T IST 49

NOT ALL CONFERENCES AND EXPOSITIONS ARE EQUAL. IF YOU ARE ONLY GOING TO ATTEND ONE, PITTCON 2020 IS THE CLEAR ADVANTAGE. Investigate, examine, touch, and test the latest scientific instrumentation at Pittcon, all while enriching your professional expertise. Join peers from around the world at the vibrant exposition, at the conference’s informative technical sessions and skill-building short courses, and at distinctively scientific events. Collaborate, learn, share ideas, compare best practices, and find solutions to your most demanding laboratory challenges at Pittcon 2020.

McCormick Place | Chicago, IL | March 1-5, 2020 | www.pittcon.org

BIO BUSINESS

Ohio, Gene Factory The state is emerging as a nascent gene therapy hub. BY SHAWNA WILLIAMS

S

arepta Therapeutics’s outpost in Ohio occupies a collection of offices and labs on the second floor of a large, squat structure known as “Building 4” in a business park outside of Columbus. Despite the facility’s un assuming exterior, the start of research onsite here in spring 2019 marked a milestone in Ohio’s twodecade-long march toward becoming a gene therapy hub. “We’re making a very significant commitment and investment in Columbus,” Doug Ingram, the CEO of Cambridge, Massachusetts–based Sarepta, told Columbus Business First in an article about the new division. “There is a real chance Columbus, Ohio, could become the most important place in the world for gene therapy development.” With its Ohio research center, Sarepta joins a local gene therapy ecosystem. Bolstered by academic research at Columbus’s Nationwide Children’s Hospital and Cincinnati Children’s Hospital along with public and private funding, the state has spawned several gene therapy startups in the past decade, and bioscience firms currently employ about 75,000 people across Ohio as a whole, according to the industry group BioOhio. But Sarepta’s move marked the first time an established biotech has opened a gene therapy research center in the state, attracting attention and, observers hope, future investment in Ohio’s nascent gene therapy industry.

© ISTOCK.COM, PAWEL.GAUL

Getting in on the ground floor Most of Ohio’s commercial gene therapy research programs got their start at Columbus’s Nationwide Children’s Hospital, which launched its Center for Gene Therapy in 2002. It was a risky decision, given the death of 18-yearold Jesse Gelsinger just three years earlier, after the teenager had a severe

immune response to a gene therapy for a rare metabolic disorder during a clinical trial run by the University of Pennsylvania. The incident prompted many institutions to put the brakes on their gene therapy programs, but Nationwide opted to bolster its investment, developing treatments that use vectors other than adenoviruses—which had triggered Gelsinger’s immune reaction—to deliver the gene-modifying machinery. A pivotal figure in the center’s research is neurologist Jerry Mendell, who has for decades worked to develop adeno-associated viruses (AAVs) as vectors for gene therapies that would treat muscular dystrophies and other neuro-

THE CENTER OF IT ALL: Columbus is home

to Nationwide Children’s Hospital, whose Center for Gene Therapy has provided the starting point for many new gene therapy products in the last decade.

muscular diseases—first at Ohio State University, an affiliate of Nationwide, and then, from 2004, at Nationwide itself. Mendell and Nationwide’s leadership shared a vision “for investing in and building a true bench-to-bedside translational infrastructure for gene therapy,” says Matt McFarland, Nationwide’s vice president of commercialization and industry relations. To that end, Nationwide went on to recruit other top gene therapy researchers and to build its own 01 /02. 2020 | T H E S C IE N T IST 51

BIO BUSINESS

In the last decade, Ohio has spawned several gene therapy startups as well as outposts of more-established biotech companies such as Sarepta, all of which benefit from the proximity to academic centers of gene therapy research and sources of research funding.

CLEVELAND Milo Biotechnology Abeona Therapeutics Case Western Reserve University

COLUMBUS Sarepta Therapeutics Gene Therapy Center Myonexus* Nationwide Children’s Hospital Ohio State University Rev1 Ventures CINCINNATI Aruvant University of Cincinnati Cincytech

*Myonexus was acquired by Sarepta in 2019.

clinical manufacturing facility for making gene therapy treatments that could be used in clinical trials. With the ability to perform its own early-stage trials, thanks to treatments made in-house and staff dedicated to regulatory tasks such as filing investigational drug applications, Nationwide was able to “de-risk” its discoveries, McFarland says, by gathering preliminary clinical data and thus moving discoveries closer to market. This derisking makes the prospect of licensing Nationwide’s products for commercialization more attractive to companies. “We’ve helped prove the concept that gene therapy can actually go from 52 T H E SC I EN TIST | the-scientist.com

bench to bedside,” says Dennis Durbin, Nationwide’s chief scientific officer. “We are one of a small number of leading institutions that have really taken it across the finish line,” he adds, referring to the 2019 approval of Novartis’s Zolgensma, a gene therapy for spinal muscular atrophy that stemmed from findings made by Mendell’s team. So far, five startups have been launched in Ohio and elsewhere based on prospective AAV-based therapies developed at Nationwide. Collectively, the companies are developing treatments for 16 conditions, McFarland says. The technology transfer office he heads, which tries to find companies to license

Full speed ahead Ohio’s first gene therapy startup, Milo Biotechnology, launched in 2012 in Cleveland, just a couple of hours’ drive north from Columbus. The company, whose sole employee is its chief executive officer, Al Hawkins, is collaborating with Mendell and others at Nationwide on a few small clinical trials testing a treatment, licensed from the hospital, for a form of muscular dystrophy. In 2013, another startup, Abeona Therapeutics, also launched in Cleveland based on technologies developed at Nationwide. Abeona has clinical-stage gene and cell therapies for the connective tissue disorder recessive dystrophic epidermolysis bullosa and for Sanfilippo syndrome, in which a buildup of toxic sugars in the body causes neurodegeneration and death. The company is also conducting preclinical studies on treatments for several other disorders. Abeona opened its own manufacturing center in Cleveland last spring to produce the therapies needed for its clinical trials. However, the US Food and Drug Administration (FDA) has asked

THE SCIENTIST STAFF

GENE THERAPY IN OHIO

and commercialize therapies currently in development, has expanded from 3 staffers to 11 since he joined the hospital in 2012, he adds. According to entrepreneur Mike Triplett, who helped launch the Columbusbased startup Myonexus in 2017 based on technology licensed from Nationwide, the hospital has grown into one of the “foundational pillars” of gene therapy research, along with the University of Pennsylvania and its affiliated Children’s Hospital of Philadelphia, and the University of North Carolina School of Medicine. McFarland says the hospital’s current position owes much to its earlier decision to stick with gene therapy when the technology’s future was far from certain. “Because we had made all those investments when gene therapy was still a potentially risky proposition . . . it put us in a place where now we have the responsibility and the opportunity to be leaders in the gene therapy space.”

for more data before allowing a Phase 3 clinical trial of Abeona’s treatment for recessive dystrophic epidermolysis bullosa to proceed. Shareholders filed a lawsuit late last year alleging that the company misled them by continuing to state that the trial would begin in mid-2019 even after becoming aware of problems likely to stall FDA approval of the study. In 2017, a few years after Abeona’s founding, Triplett and Nationwide researcher Louise Rodino-Klapac cofounded Columbus-based Myonexus based on gene therapies that RodinoKlapac’s team had developed for five different forms of a neuromuscular disease called limb-girdle muscular dystrophy. In mid-2018, Rodino-Klapac left Nationwide to join Sarepta as vice president for gene therapy. The biotech had partnered with Myonexus from its inception and last year bought the startup for $165 million. While Rodino-Klapac had personal reasons for wanting to stay in the Columbus area after leaving Nationwide, she says it was also “what’s best for the technology,” given the site’s proximity to the hospital and her ability to recruit members of her old research team to Sarepta’s new gene therapy research center, which she heads. Three of the company’s limb-girdle treatments are now in small Phase 1 clinical trials, and a Duchenne muscular dystrophy therapy that Sarepta licensed in 2017, also developed by Rodino-Klapac’s team while she was at Nationwide, is being tested in a Phase 1/2 trial with 40 patients. An earlier, four-patient trial of that therapy “showed significant promise,” Rodino-Klapac says. She adds that there’s preclinical research in the works on other treatments that the company isn’t yet ready to talk about. The relatively humble officepark home of Sarepta’s gene therapy research center is temporary; the company is building a new 85,000-squarefoot facility, expected to be ready later this year, in another Columbus suburb. Rodino-Klapac says she hopes that the company’s Columbus outpost will help

seed an expanded local gene therapy hub. Speaking with people at companies in other states, she is often asked: “‘Sarepta’s in Ohio—what are we missing?’” She takes it as a good sign. “I think [Sarepta’s presence is] going to help create an ecosystem here . . . for the gene therapy and biotech space and maybe beyond that.”

There is a real chance Columbus, Ohio, could become the most important place in the world for gene therapy development. —Doug Ingram, Sarepta Therapeutics

Looking forward Nationwide’s McFarland says he thinks Ohio has much to offer companies. For example, state government–fostered initiatives such as Rev1 Ventures offer services to entrepreneurs in the Columbus area, and Nationwide has partnered with the state to offer seed-funding programs for early-stage companies. The low costs to operate in Ohio, relative to more established, coastal hubs such as Boston, are another plus, says Severina Kraner, the director for health care at JobsOhio, a nonprofit company working to boost industry in the state. Moreover, Kraner adds, the state is home to large research institutions such as Case Western Reserve University in Cleveland, Ohio State in Columbus, and the University of Cincinnati, providing a rich source of researchers to be recruited to up-andcoming biotechs. The growth of gene therapy in Ohio is also aided by changes in the industry more generally. From an investor’s point of view, the outlook for gene therapy itself has changed, says John Rice, the director of life sciences at Ohio-based venture capital firm Cincytech, making it less important that particular therapies be de-risked by academic institu-

tions before being picked up by industry. His company provided start-up funding for Myonexus in 2017, and having clinical-stage technologies developed at Nationwide “made a big difference,” he says. “We could get in with some confidence that we’re going to see some value for our investment.” But now his firm is more willing to consider projects still at the preclinical stage, Rice says—in fact, in order to invest in novel gene therapies, investors have to do so. Today, “anything that was clinical stage, here or anywhere else, has already been partnered out—a company’s created it and is moving along.” Developments outside Ohio have of course contributed to gene therapy’s maturity as a therapeutic strategy, and not all treatments created in the state have stayed there. Zolgensma (onasemnogene abeparvovec), for example, a therapy for spinal muscular atrophy that received FDA approval in May 2019, originated in research at Nationwide and was developed in the Chicago area by a startup and later by Novartis. And Amicus Therapeutics, which acquired several of Nationwide’s gene therapy technologies for rare metabolic conditions in 2018, is based in New Jersey and has a gene therapy center in Philadelphia. For McFarland, having commercialization proceed in other states is not a problem. When considering potential licensees for each of the hospital’s technologies, he says, his team asks, “What is the best for this asset? And what gives this asset the best chance of having an impact on patients? And every once in a while, that’s not going to be here.” Yet McFarland and others think that in the coming years, many gene therapy companies will find that Ohio offers them the conditions they need to thrive. Based on confidential conversations she’s had with business leaders, Kraner says she hopes to see more companies crop up in the state within the next year. Looking forward, she says, “I think there’s going to be continued investment and opportunities to attract and grow [gene therapy] companies.” g 01 /02. 2020 | T H E S C IE N T IST 53

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READING FRAMES

Slip Sliding Away Navigating a wintry landscape forces the mind and body to come to a constructive equilibrium and reveals the fascinating dialogue between the two elements of a human being. BY SCOTT GRAFTON

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hen people ask me about the “mind-body connection,” I typically suggest walking on an icy sidewalk. Skip the yoga, mindfulness, or meditation, and head to the corner on a cold, windy, snowy day. Every winter, much of North America becomes exceedingly slippery with ice. Emergency rooms across the continent see a sharp uptick in fractured limbs and hips as people confidently trudge outside in such conditions, unveiling a profound disconnection between what people believe and what they can actually do with their bodies. One might think that a person could call on experience from years past to adjust their movement or provide a little insight or caution. But the truth is that the body forgets what it takes to stay upright in these perilous conditions. Why is there so much forgetting and relearning on an annual basis? We remember how to ride a bike. Why can’t we remember how to walk on ice? I attempt to answer this and other questions concerning the connection (or lack thereof ) between motion in the mind and motion by the body in my new book, Physical Intelligence: The Science of How the Body and the Mind Guide Each Other Through Life. Falling on ice reveals a delicate tradeoff that the brain must reconcile as it pilots the body. On the one hand, it needs to build refined motor programs to execute skills such as walking, running, and throwing. On the other hand, those programs can’t be too specific. There is a constant need to tweak motor plans to account for dynamic conditions. When I throw a backpack on, my legs don’t walk in the same way as they do without the pack: my stance widens, my stride shortens. Often, the tweaking needs to happen in

moments. As I pick the pack up, I need to lean in or I could tip myself over. Just as importantly, as soon as I put it down, I need to forget I ever held it in the first place. In my lab at the University of California, Santa Barbara, we investigate the mechanisms the brain employs to make these super-fast adaptations. We have participants pick up tippy objects the size of a desk stapler that are weighted to roll to the left or right. After only a few trials with one object, subjects typically succeed without any mistakes. Then, we switch the object. The tipping is back, but in the opposite direction. The subjects learn yet again. We know from neurophysiologic recordings and brain imaging that the skills necessary to pick up objects are controlled by motor areas of the cortex, the brain stem, and the spinal cord. From fMRI scans we know the cerebellum is a key area for acquiring and forgetting these rapid adaptations of finger forces. Pattern analysis of the brain scans suggests that when a participant must learn how to successfully lift a new object, the brain can completely erase the pattern previously used to lift the one that rolled in the opposite direction. The brain falls back to a generic pattern for lift forces that it applies whenever it has to refamiliarize itself with the dynamic properties of the object. Fast adaptation like this occurs whenever a person’s body is in motion, whether it is an adjustment of the fingers to pick up an object or of the legs to walk down a slippery sidewalk. We are usually unaware of the countless small tweaks we make to our movement patterns as we adapt to changing conditions. It is one of the many aspects of physical intelligence that we take for

Pantheon, January 2020

granted. A lack of insight into the forgetting in particular is one explanation for why people fall on ice. As winter sets in, the tweaks necessary to stay upright on the ice are long since forgotten. We simply don’t retain this kind of dynamic knowledge. The only way to adapt to a slippery surface is to experience it anew through direct contact. A marathon of virtual reality exposure wouldn’t help a bit. Only by shuffling, sliding, and feeling the grip of our shoes can we adapt and learn what is physically possible in those specific conditions. As this crosstalk between mind and body helps us attain equilibrium, beliefs and motions are brought into harmony. And the ice walker trods steadily on. g Scott Grafton is a neuroscientist at the University of California, Santa Barbara. Read an excerpt of Physical Intelligence at the-scientist.com. 01 /02. 2020 | T H E S C IE N T IST 5 5

COMINGSOON

From Ancient Recipes to the Blood of Komodo Dragons: The Quest for a Solution to the Current Onslaught of Superbugs

The search for solutions to the antibiotic resistance problem is moving beyond traditional antibiotic drugs, leading researchers to some unexpected places. The Scientist is bringing together a panel of experts in the field of antibiotic development to present their research on novel solutions for overcoming superbugs that have developed resistance to traditional antimicrobials.

STEVE DIGGLE, PhD Associate Professor Center for Microbial Dynamics & Infection School of Biological Sciences Georgia Institute of Technology

THURSDAY, JANUARY 9, 2020 2:30 - 4:00 PM EST REGISTER NOW! www.the-scientist.com/ ancient-recipes-komodo-dragons

The webinar video will also be available at this link. BARNEY BISHOP, PhD Associate Professor Department of Chemistry and Biochemistry George Mason University

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CAR T Cell Therapy: Overcoming Toxicities

Following the success of the first checkpoint inhibitors for treating cancer, many researchers are now turning their attention to chimeric antigen receptor (CAR) T cell therapy. This novel treatment makes use of a cancer patient’s own T cells, which are removed and modified so that they can better recognize and attack cancer cells. These modified cells are then infused back into the patient’s body. While this approach has shown some success for certain cancers, severe toxicities have limited its widespread application. Researchers are attempting different methods to overcome these toxic effects, including using gene-editing tools such as CRISPR to modify CAR T cell protein production and employing antibody therapy to potentially block toxicity-inducing proteins. Will CAR T cell therapy emerge as a game changer for cancer treatment? Or are the hurdles too high? Join The Scientist for an educational webinar, sponsored by Enzo, Nanostring, Sartorius, and IsoPlexis, as we explore CAR T cell therapy research and gain insight into the next steps. DAVID T. TEACHEY, MD Associate Professor of Pediatrics Children’s Hospital of Philadelphia Department of Pediatrics Perelman School of Medicine at the University of Pennsylvania

ORIGINALLY AIRED MONDAY, DECEMBER 2, 2019 WATCH NOW! www.the-scientist.com/car-t-toxicities TOPICS COVERED:

RAWAN FARAMAND, MD Assistant Member, Blood and Marrow Transplant & Cellular Immunotherapy H. Lee Moffitt Cancer Center and Research Institute

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Research depends on samples from patients and healthy volunteers. But obtaining biospecimens from human subjects can be a challenge for researchers and patients alike. Home-based specimen collection directly connects patients to researchers, while alleviating the burden of travel to secondary sites. Using this approach, researchers efficiently collect patient samples in the comfort of their homes. This webinar, sponsored by Sanguine, details research experiences from researcher and patient perspectives.

CHRISTINE VON RAESFELD Independent Patient Advocate Board member, More Than Lupus Patient Advisor, Aurinia Pharmaceuticals Patient Advisor, PatientsLikeMe Participant in the Stanford Humanwide Program

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STEPHANIE CULLER, PhD Co-Founder and CEO Persephone Biome, Inc.

• Development of an AI drug discovery platform for microbial-based immunotherapies using patient samples • Research from a patient’s perspective • Practical considerations for engaging patients in research • The direct-to-patient trial model WEBINAR SPONSORED BY

ONDEMAND

How Centrifugal Devices Facilitate Molecular Biology Research

Biomolecule purification involves a complex series of steps in which target purity and recovery are vital. Many common nucleic acid and protein handling procedures can benefit from the use of centrifugal devices outfitted with microfiltration or ultrafiltration membranes. But finding the right products can be challenging. This webinar, sponsored by Pall Laboratory, highlights ways to simplify purification in the molecular biology lab.

JORGEN DE HAAN, PhD Senior Application Scientist Pall Laboratory

ORIGINALLY AIRED THURSDAY, NOVEMBER 21, 2019 WATCH NOW! www.the-scientist.com/centrifugal_devices_pall TOPICS COVERED: • The interplay between membrane selection, sample composition, and centrifugation conditions • Device choices available for processing nucleic acid and protein samples ranging in volume from 50 μL to 60 mL • Nucleic acid applications • Cleanup of DNA fragments, such as PCR products, prior to restriction, digestion, and labeling reactions

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01/02.2020 | T H E S C IE N T IST 59

FOUNDATIONS

A Woman of Firsts, Early 20th Century BY EMILY MAKOWSKI

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6 0 T H E SC I EN TIST | the-scientist.com

PEEP THIS: Sabin at the Rockefeller Institute (now Rockefeller University), where she worked from 1925–1938. During her time in New York, Sabin raised money for women in science, including $10,000 (more than $183,000 in today’s dollars) for a scholarship in advanced mathematics that was established at Bryn Mawr College in 1936.

vessels’ development occurred the other way around. After accepting a position at Rockefeller, she studied how white blood cells attack tuberculosis bacteria (Mycobacterium tuberculosis). Sabin retired from research in 1938 and moved back to Colorado. She became a publichealth activist in Denver, and in the late 1940s, helped launch a TB prevention and screening campaign that lowered the incidence of the disease in the city by almost half in two years. She died in 1953, at the age of 81. Sabin’s accomplishments are all the more notable considering the obstacles women faced in science during this time.

Although medical schools had started to admit women, their enrollment numbers were limited so as not to deter men, and research funding opportunities for women were scarce. “I hope my studies may be an encouragement to other women, especially to young women, to devote their lives to the larger interests of the mind,” Sabin said after receiving an achievement award from the women’s magazine Pictorial Review in 1929. She had the intended effect on at least some of her contemporaries. “Your great work has been an inspiration not only to women in medicine, but to women in all walks of life,” Helen Keller wrote to Sabin in a 1922 letter. g

© BACHRACH

s a child growing up in Colorado, Illinois, and Vermont in the late 19th century, Florence Sabin lacked musical talent—but that ended up being a good thing. After a classmate told her that her piano-playing ability was “merely average,” Sabin gave up on n her dream of becoming a pianist and started to study science. What followed was a long and illustrious career that spawned landmark discoveries and helped inspire women in STEM at a time when they faced many challenges. After graduating in 1900 from Johns Hopkins School of Medicine, Sabin became the first woman appointed to the faculty of the university in 1903, specializing in anatomy and physiology. It was 17 years, however, before she was promoted to the rank of full professor. Later, she became the first woman to head a department at Rockefeller University (then the Rockefeller Institute for Medical Research). She was also the first woman president of the American Association of Anatomists and the first woman elected to the National Academy of Sciences. She took a special interest in embryology and developmental biology, an emerging field of study at the time. Sabin’s 1901 book, An Atlas of the Medulla and Midbrain, a detailed account of the structure of the brainstem of a newborn infant, became an important reference work for developmental biologists. “She had unbounded curiosity and a deep love for research,” retired historian Patricia Rosof, formerly an adjunct instructor at New York University and the Fashion Institute of Technology, tells The Scientist. “She pursued her goals relentlessly and had an independence of approach and spirit.” At Johns Hopkins, Sabin showed that mammalian embryos’ lymphatic vessels grow from veins out into surrounding tissues, in contrast to the then–commonly accepted belief among researchers that the

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THE MOMENT YOUR RESEARCH MOVES FROM BENCH TO BEDSIDE_

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