VOL. 37, ISSUE 1, SPRING 2023 The Scientist

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THE CANCER CODE ONCE DISMISSED AS GENOMIC NOISE, SOME NONCODING SEQUENCES (AND THE MICROPROTEINS THEY ENCODE) PLAY IMPORTANT ROLES IN CANCER

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Contents SPRING 2023 | WWW.THE-SCIENTIST.COM | VOL. 37, ISSUE 1

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Features

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Cancer’s Hidden Codes

Cancer’s Jumping Genes

Noncoding RNAs and microproteins, once considered genomic noise, are turning out to be critical to the progression of some types of cancer.

Transposons may be key players in how tumors develop and spread—but they could also keep cancer at bay in some circumstances.

BY RACHAEL MOELLER GORMAN

BY DIANA KWON

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Department Contents 7

CROSSWORD

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CRITIC AT LARGE

Predicting Autoimmunity

A newly approved therapy for type 1 diabetes signals a paradigm shift to not only treating autoimmune diseases, but also preventing them. BY JANE BUCKNER, MD AND CARLA GREENBAUM, MD

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THE LITERATURE

Regrowing a Tail

A metabolic pathway used for tail regrowth may be critical for tissue regeneration in some cells.

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BY NATALIA MESA, PhD

NOTEBOOK

Researchers come closer to understanding why some soft tissues are more likely to be preserved as fossils; new techniques quantify what lived in and on preserved animals. BY IAN ROSE AND MARY BATES

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SCIENTIST TO WATCH

Alex Muir: Cancer’s Diet Detective

Assistant Professor, Ben May Department of Cancer Research, University of Chicago BY NATALIA MESA, PhD

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CAREERS

Extinction Calculus

How do scientists decide a species has gone extinct? BY ANDY CARSTENS

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READING FRAMES

The Skin Battery

The “wound current” has intrigued scientists for more than a century. It could turn out to be the key to healing catastrophic injuries.

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Bathing Through the Ages: 1300–1848

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Speaking of Science 1. Intervertebral cushion 5. Broadcast from a squad car, for short 8. Attachment of an infant’s mouth during breastfeeding 13. Really digging 14. March Madness org. 15. Slender, edible mushroom 16. Type of programmed cell death that is evaded by cancer cells 18. Glucose test unit 19. Feeds, as a baby mammal 20. Some plays in badminton or tennis: 2 words 22. Gene that, when mutated, can cause ataxia telangiectasia 23. End of a root branch 25. Suffix in the names of sugars 26. With 44-Across, genomic hallmark of cancer associated with DNA loss 31. Topic for debate 34. Item used to mount insect specimens 35. Nickname for the Field Museum’s T. rex skeleton 37. Flower bud used as a flavorful garnish 38. Special treatment, for short 39. Country that’s home to the Amami rabbit 41. Paleozoic, Mesozoic, or Cenozoic 42. Chicken ___ king: 2 words 43. Come next 44. See 26-Across 49. Delighted exclamation 50. Seargent’s superiors 51. Solemn promise 54. What an atmospheric purifier provides: 2 words 59. Cannabis ___ 61. “Filthy” money 62. Cellular structures that enable replicative immortality in cancer cells 64. Cause a mutation in, perhaps 65. Panache 66. Monthly expense for a tenant 67. Walks with a heavy step 68. Oncogene that regulates expression of ~15 percent of human genes 69. Smelling apparatus

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Roman equivalent of Artemis Enter, as data Atmospheric disturbance Police officers, informally Organization for practitioners of the “central science” 6. Result of nociception 7. Cytosine, for one 8. Not as much 9. Patriotic song 10. Bull, by another name 11. Proto-oncogene involved in mast cell development 12. Joints in mammals, or fruits from roses 14. Military denial: 2 words 17. Connection between a neutrophil and endothelium 21. Talking-___ (reprimands) 24. Opener on some soda cans 26. Signal’s passive counterpart 27. Inverse of kilo28. Available for work if needed: 2 words. 29. Egyptian cobras

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30. Party at which kalua pig might be served 31. Glacier’s makeup 32. Wedding garment, often 33. Life ___ (measure of longevity) 36. Hydrocarbon suffix 39. Habitat for some pelagic fish 40. One or more 45. Flew like an osprey 46. Powders used in printers 47. Exclamation when solving a puzzle 48. Exclamation before starting a fight: 2 words 51. Yellow-throated songbird 52. Appliances that might be used for sterilization 53. Guano, to bats 54. Thunderbolt’s sound 55. Quiet period 56. Prefix similar to exo57. Agenda component 58. Depend (on) 60. Seabird with a forked tail 63. Insect’s protective secretion Answer key on page 5 SPRING 2023 | T H E S C IE N T IST

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CRITIC AT LARGE

Predicting Autoimmunity A newly approved therapy for type 1 diabetes signals a paradigm shift to not only treating autoimmune diseases, but also preventing them. BY JANE BUCKNER, MD AND CARLA GREENBAUM, MD

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T H E SC I ENTI ST | the-scientist.com

long-running international studies demonstrating that is possible to identify individuals destined to develop T1D based on the presence of multiple T1D-associated autoantibodies. Strikingly, essentially all of these individuals will eventually develop T1D, but teplizumab delayed disease onset by a median of two years, and longer for some individuals.  To put those numbers in context, we routinely screen for high blood pressure. Left untreated, about 2 to 3 percent of people with high blood pressure will develop a stroke or heart attack within five years. This risk is considered a big enough problem that instead of merely telling patients they are at risk of future stroke or heart disease, we give high blood pressure a name: hypertension. We recognize the importance of screening to find people with hypertension because we know that early identification matters to those people who will experience the worst outcomes. This works because we have therapies that can prevent or reduce the risk of stroke and heart attacks. In the case of T1D, as essen-

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ighty million Americans have an autoimmune disease. Most such diseases are lifelong and often debilitating. They have no cure, and treating them is estimated to cost more than $100 billion annually, not counting lost productivity. Until recently, prediction and screening haven’t been a focus for these diseases because we had no way to slow or prevent them. But late last year, the US Food and Drug Administration (FDA) approved a therapy that could shift the paradigm of how we treat these diseases to earlier intervention—and ultimately, prevention. The first revolution in autoimmune disease treatment was the transition from treating symptoms to treating the underlying cause: the immune system attacking “self.” Rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis are all routinely treated with immunotherapy. These therapies are not curative but have improved the quality of life of those with these diseases. Now, another paradigm shift is underway. In type 1 diabetes (T1D), an autoimmune disease that most commonly begins during childhood in which the insulin-producing beta cells of the pancreas are destroyed, researchers have learned that pathogenic processes start long before any symptoms appear. Testing apparently healthy individuals allows us to identify T1D early— and now, potentially, to get better outcomes, thanks to the FDA’s November 2022 approval of teplizumab, the first therapy to prevent or delay any auto-immune disease. (One of us, Greenbaum, was chair of TrialNet, a US National Institutes of Health (NIH)–sponsored network that conducted the Teplizumab Prevention Trial). We are finally able to reap the benefits of 40 years of research and definitively state that early T1D detection is not only feasible but can support treatment decisions that could avert symptoms from ever developing. T1D is leading this new paradigm of early detection and prevention of autoimmune diseases. To this point, T1D care has been entirely symptom-focused; when people are diagnosed, we treat their high blood glucose with insulin, and this continues for the rest of a person’s life. While researchers have developed better and better ways to deliver insulin and monitor glucose, there has been no treatment of the aberrant immunity underlying the disease. Now, we are looking at a world where T1D is not only treated with immunotherapy, but where this happens early, during the asymptomatic stages of disease. The development of teplizumab and the results indicating that it can delay T1D diagnosis are founded on data from several

tially all of those at high risk will eventually develop T1D, everyone treated has the potential to benefit from therapy. Furthermore, with the FDA approval of teplizumab, there is now precedent for therapies to prevent other autoimmune diseases as well. The milestone demonstrates to the NIH that decades of funding natural history studies in T1D were worthwhile, and to the biotech and pharmaceutical industries that there is a pathway for FDA approval for therapies that prevent these diseases. Through work at our institution, the Benaroya Research Institute, and elsewhere, we know that T1D is not the only disease with an asymptomatic early stage. We are developing the tools to predict who is going to develop rheumatoid arthritis (RA), and early detection research for multiple sclerosis is accelerating. Critically, echoing T1D, prevention in RA is also moving ahead, including trials testing whether a therapy called abatacept can delay development of the disease in those who are flagged as being at risk primarily based on the presence of autoantibodies. While the approval of teplizumab is transformative, how rapidly it and similar therapies will change clinical practice remains an open question. Here, the complicated dynamics of the US healthcare system play a role. Clinical practice standards will need to change such that screening for, and early detection of, autoimmune diseases becomes routine. Insurers will need

to pay for screening, monitoring, and the new preventive treatments. Clinicians will need to be educated about this new paradigm, and patients will need to understand their new diagnoses and their treatment options. Sadly, paradigm shifts in clinical practice are slow, and clinicians are hard pressed for the time to discuss screening and prevention in a standard 15-minute appointment. Advancements in preventive care may end up being driven by individuals living with autoimmune diseases since personal experience with the disease can lead to advocacy for screening and early detection. Teplizumab’s approval is just the first critically important step toward screening, early detection, and prevention of autoimmune diseases. These paradigm shifts will, with time, change the way physicians think about autoimmunity and, more importantly, change the lives of people living with, and at risk for, these diseases. J Jane Buckner, MD is the president of Benaroya Research Institute (BRI). Carla Greenbaum, MD is the director of the Center for Interventional Immunology and of BRI’s Diabetes Research Program and was the chair of Diabetes TrialNet, the NIH clinical trial network that conducted the Teplizumab Prevention Trial, and BRI was a clinical site for this and other trials using teplizumab to alter the course of type 1 diabetes.

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EDITOR’S CHOICE PAPERS

The Literature CELL BIOLOGY

Regrowing a Tail THE PAPER

Tadpole

© NICOLLE FULLER, SAYO STUDIO

J. H. Patel et al., “Elevated pentose phosphate pathway flux supports appendage regeneration,” Cell Rep, 41:111552, 2022. Frog tadpoles have a remarkable ability to regenerate their tissues—a process that requires a lot of energy, fast. In other animals, rapidly growing tissues, such as tumors and embryos, are thought to get this energy from glycolysis—the products of which can be used to build biomass more easily than other forms of ATP production. So when Andrea Wills, a biologist at the University of Washington, and her team started looking into what fuels tail regeneration in tadpoles, they expected to find that tadpoles shuttle glucose into this metabolic pathway too, Wills says. Their first experiment seemed to bear this out. After the team snipped the tails off tadpoles of the western clawed frog (Xenopus tropicalis), they observed increased glucose levels in the regenerating cells. “But then we ran into a problem,” says Wills. The researchers’ RNA-seq experiments didn’t show increased gene expression of any of the enzymes involved in glycolysis, except hexokinase, which performs the first step. Levels of lactate, a product of glycolysis, were also normal, and blocking glycolysis had no effect on tail regrowth. “We just kept putting on more of these inhibitors and they didn’t do anything,” she says. So the researchers started searching for alternative metabolic pathways that consume glucose. They looked for increased expression of other genes and found higher levels of all but one enzyme used in the pentose phosphate pathway (PPP). And blocking the PPP prevented tail regrowth, suggesting that it was this pathway, not glycolysis, fueling regeneration.

Q 1 Snip Q 5 Cell division Glucose transport genes

Nucleus DNA

Glucose transport protein

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Q 3 Pentose phosphate pathway

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Fatty acids

Tail regeneration

Regenerating cell

SUGAR COATED: Tadpole tails regenerate when lost (1). In this study, researchers found that to do

so, tadpoles increase the production of genes for proteins that shuttle glucose into the cell (2). Glucose feeds into the pentose phosphate pathway (PPP) (3). The PPP produces two molecules, NADPH and ribonucleotide-5-phosphate (R5P), which are precursors for fatty acids and nucleotides, respectively (4). The cells use fatty acids to build more cell membrane, and nucleotides to build more DNA as cells rapidly divide and increase their numbers (5). This leads to tissue growth, and eventually, the tadpole has its tail back.

The PPP makes NADPH, a molecule used to build fatty acids, and ribose-5phosphate, a nucleotide precursor. Wills says it makes sense that the pathway would be upregulated because “if you’re going to be a proliferative tissue, you will need to make lots of new membrane out of fatty acids, [and] you’re going to need a lot of nucleotides” for DNA. Caroline Beck, a developmental biologist at the University of Otago in New Zealand who was not involved in the work, says that the authors “have done a good job” presenting a compelling story, but cautions that “these pathways cross-

talk with each other all the time, so it’s quite difficult to rule [a pathway] in or out completely.” “One of the long-term goals,” Wills says, “is to be able to use this knowledge to enhance regenerative outcomes in people,” but she adds that by itself, switching on the PPP probably wouldn’t give cells regenerative capability. “You have to have growth . . . and patterning, and you also have to have inhibition of the scarring response. . . . One of the elements that we’ve been missing is how to enable growth.” The PPP may be critical to enabling that growth. —Natalia Mesa, PhD SPRING 2023 | T H E S C IE N T IST 1 1

Hidden Parasites

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ituated on the Seattle campus of the University of Washington, the Burke Museum hosts the largest repository of preserved fish in North America. More than 400,000 individuals representing 4,100 species line the shelves of the museum’s ichthyology collection. Preserved in ethanol, these specimens are a window into the marine and freshwater ecosystems of the past. For University of Washington (UW) ecologist Chelsea Wood, however, the most interesting things in those thousands of jars aren’t the fish themselves, but the parasites they carried in and on their bodies. Wood and her lab are studying these tiny creatures to answer a long-debated

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question: How has the abundance of parasites changed over time? “Until recently, I didn’t think that there was ever a way that we were going to find answers to that question,” Wood says. Many ecologists have worked under the assumption that parasite loads in the past were lower than today, she explains. That’s because parasite abundance is often seen as a sign of stress, as hosts may be less able to control their parasite loads when faced with stressors such as food shortages or pollution—conditions that have intensified in many regions in recent years. But that assumption has remained untested, with very little data to support or refute it. While previous studies have been able to detect parasites in preserved fish specimens—some of them years or centu-

SPRING 2023

HIDDEN RESIDENT: A monogenean flatworm, a common fish parasite, under magnification

ries old—those studies gave little insight into the parasites’ abundance. The problem, Wood explains, was that there was no way to verify whether the steps taken to preserve those specimens were affecting the number of parasites detectable on them. In 2020, Wood and her team found a way to collect those data. They took fresh fish of three species and preserved some of them in ethanol, the same method used by the Burke and other museums around the world. Several days later, the researchers compared the parasite counts from these experimentally preserved fish to counts

KATIE LESLIE

Notebook

IN STORAGE: Specimens of Alaska pollock in

the University of Washington’s Burke Museum

KATIE LESLIE

from fresh specimens they dissected right away and, for the first time, confirmed that the preservation process did not bias the numbers. This validation study meant that any fish preserved and stored this way— potentially millions of specimens in museums around the world—could be used to study the question of past parasite abundance, Wood says. To get accurate counts of these tiny creatures, Wood’s lab used a variation on an established methodology that entails cutting filets from preserved fish and flattening the muscle tissue between glass plates to detect parasites. Wood’s team made an incision and spread the body cavity open, then shone a powerful light through the fish’s side. Parasites showed up as shadows against the light background of the preserved muscle, allowing the researchers to remove and identify them under a magnifying glass or microscope. Over the past few years, the lab has continued to refine the technique and apply it to a greater number and variety of samples. In a recent paper pub-

lished in the Journal of Animal Ecology, Wood explores how such methodology could help researchers address ecological questions about not only fish but other aquatic taxa. The paper presents historical parasite ecology as a new subdiscipline addressing the abundance of parasites in the past as well as biotic and abiotic factors that act on their populations, both then and now. As the method is applied more widely, it is challenging, or at least complicating, the assumption that early

seas were less parasite-dense. Quite the opposite, “our research suggests that a lot of metazoan parasites are diminishing in abundance,” says Wood. Joshua Brian, a postdoctoral research associate at King’s College London who was not involved in the research, says the team put together “a brilliant paper.” Brian studies host-parasite interactions in freshwater mussels, with a focus on these small and unbeloved parasite species’ vulnerability to extinction. “These sorts of methods, to look back in the past at where parasites were and what their abundance was, and linking that with host variability, environmental variability, it’s just so important to start to build this picture about how parasites are changing,” adds Brian. The Wood lab is now working on its largest analysis of fish parasites yet, covering dozens of parasite species and spanning 130 years of the history of Puget Sound in the Pacific Northwest. Wood says she expects the upcoming study to provide much more insight into ecological change for both parasites and their hosts over the past century.

RESEARCH OPPORTUNITY: A specimen of

rockfish collected in the 1970s, in the University of Alaska Museum of the North SPRING 2023 | T H E S C IE N T IST 1 3

NOTEBOOK

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Detailing Decay

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aleontology typically conjures images of digging up dusty bones in the field. So why is paleontologist Thomas Clements watching fish rot in the laboratory? “People are always a bit surprised when I tell them what I do for a living,” Clements says. It might seem odd, he explains, but by better understanding the process of decay, he hopes to answer big questions about how living things become fossils in the first place. Clements specializes in taphonomy, a subfield within paleontology that deals with the process of fossilization. He’s especially interested in why some soft tissues, such as muscles and certain internal organs, are more likely to show up in the fossil record than others. It’s important that paleontologists get to the bottom of this mystery, he says, as only by understanding the processes of decay and preservation will they be able to correctly interpret that record. “If you’re looking at a fossil from 500 million years ago, it can be hard to know what tissues are absent because of decay and what tissues are absent because they were not evolutionarily present,” says

CUT FISH: Thomas Clements dissects fish as part

of his research on rot. The tally on his lab coat tracks the number of dissections he has completed.

Clements, currently a research fellow at the University of Birmingham in the UK. Only a very small percentage of anything that was ever alive becomes fossilized. While it’s typically the hard parts— bones, teeth, and shells—that get replaced by minerals and preserved, soft tissues also stand a chance in certain circumstances, says Orla Bath Enright, a paleontologist at the University of Lausanne in Switzerland. “These soft and squishy parts are much rarer in the fossil record because their preservation requires a particular set of environmental conditions,” she says, including low oxygen levels, availability of minerals, and rapid burial. Around the world, there are a small number of sites, known in the research literature as Konservat-Lagerstätten, where the conditions were just right. The mineral that yields the most exceptional fossils, and is most frequently observed at these sites, is calcium phosphate (also known as apatite). When soft tissues

THOMAS CLEMENTS

In addition to preserved fish, museumkept mammal specimens are also proving to be useful for the historical study of parasites and disease. In a recent paper, researchers from the University of Richmond extracted bacterial DNA from mammal skins to learn more about the spread of Lyme disease through the eastern United States. By testing mouse specimens—and the ticks they carried—from the mammalogy collection of the Virginia Museum of Natural History, the paper’s authors were able to trace the spread of the Lyme disease–causing bacterium (Borrelia burgdorferi) southward over the past few decades. “Specimens are collected and archived for one reason, and turn out to be incredibly valuable historical records for other studies,” explains coauthor Nancy Moncrief, the Virginia Museum’s curator of mammalogy. She collected some of the mouse specimens used in the study herself, for research that had nothing to do with Lyme disease. “We can’t predict what technology is going to be here in ten years, but we’ve archived the samples and documented them correctly, to be able to answer those questions.” Wood notes that this is an advantage of her lab’s less invasive methodology for dissecting fish: it minimizes damage to the irreplaceable original specimens. Everything used in the lab’s studies goes right back into the museum collections, down to the last tiny parasite (labeled and stored separately from the source specimen), leaving them available for future researchers to explore questions of their own. The only concrete limits on what can be done with museum specimens are what samples make it into a collection, and the survival of the collections themselves. “Not as much material is being archived,” says Moncrief, pointing to a decline in new museum collections and a worrying trend of smaller collections—particularly at struggling universities—closing their doors. “You can’t go back in time,” she adds, but museum specimens offer a rare glimpse into the past. As long as the collections survive, their applications should only continue to grow. —Ian Rose

ANDRZEJ KRAUZE

are replaced by this mineral—a process known as phosphatization—organic structures can be preserved with subcellular fidelity. Under a microscope, even individual muscle fibers and cell organelles may be visible. Muscles, stomachs, and intestines are more commonly captured by calcium phosphate than are the liver, gonads, and kidneys, says Clements. Some researchers think this is because different organs decay at different rates, creating distinct pH microenvironments around themselves. In this scenario, the pH of some organs falls below the critical threshold of 6.4, allowing fossilization to occur there but not elsewhere. An alternative hypothesis is that it’s not the way organs rot, but the tissue biochemistry specific to each organ that determines its likelihood of becoming a fossil. It was through trying to directly test these hypotheses several years ago that Clements found himself, as a doctoral student at the University of Leicester, monitoring how sea bass carcasses rot over the course of 70 days. Previously, researchers were limited by equipment that could only monitor pH fluctuations around a decay-

ing carcass, not inside it, he says. Technological improvements have since resulted in smaller, more powerful pH probes able to collect data automatically and internally. With fellow paleontologists Sarah Gabbott and Mark Purnell, Clements placed probes inside specific organs to document realtime pH gradients during the early stages of decay. “We were just fortunate that we could secure equipment that was robust enough to sit inside a rotting fish for 70-odd days multiple times a year,” he says. After inserting the probes, the team suspended each fish carcass in plastic mesh netting in a frame, placed the contraption in a container filled with artificial sea water, and left the carcass to rot. The result was just as revolting as you’d imagine, says Clements. “The smell is horrific and there’s bits of rotten fish everywhere,” he says. “It absolutely is disgusting work, but you get used to it. It helps to have a strong stomach, though.” Somewhat surprisingly, the results revealed that organs do not generate unique pH microenvironments during decay (Palaeontology, 65:e12617, 2022). Clements says the inside of the fish became soup-like rather quickly, with most inter-

nal organs becoming “unrecognizable” within just five days. This soup of decaying organs had a pervasive pH environment—below the phosphatization threshold—that persisted until the skin finally ruptured. “There was this long-standing idea that pH of the microenvironment is important in soft tissue preservation,” says Philip Wilby, who is paleontology lead at the British Geological Survey and was not involved in this study. “This experiment clearly puts that idea to bed and shows that pH microenvironment is not the important process here.” Clements and his colleagues instead conclude that, when conditions are amenable, it is tissue biochemistry that contributes to selective preservation. Tissues such as muscles, for example, are already rich in phosphates—which help kickstart phosphatization—as well as collagen fibers that can act as a substrate for the process to occur. Stomach and intestinal contents often also contain high levels of phosphates. Wilby says these findings are an important step forward, but questions remain. “Phosphatization requires particular conditions that we are still working to understand. It is not a simple process,” he says. “If it was, everything that ever died would be phosphatized.” Wilby points out that because these experiments used recently dead fish from a fishmonger, the researchers may have missed important decay processes that occur in the first few hours after death. There are also other potential factors that bear further investigation, such as the chemistry of the sediments in which fossils typically form. Bath Enright, who also was not involved in this study, agrees there is more to learn and says she is excited about how the technological advances used by Clements and colleagues are pushing the field of experimental taphonomy forward. “Decay is a process that occurs over a period of time until the carcass is gone,” she says. “This paper shows the necessity of tracking decay at the level of specific organs to understand what is happening internally in a carcass, because that’s where a lot of the action is happening.” —Mary Bates SPRING 2023 | T H E S C IE N T IST 1 5

Cancer’s Hidden Codes Noncoding RNAs and microproteins, once considered genomic noise, are turning out to be critical to the progression of some types of cancer. BY RACHAEL MOELLER GORMAN

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s a PhD student at the University of Münster in the early 2000s, Sven Diederichs would bike three kilometers each morning to a compact lab in a brick building on the hospital campus. There, he’d defrost small vials of lung cancer specimens from the lab’s deep freezer and extract a single microgram of RNA from each tube. Some of the specimens came from people whose cancers eventually metastasized, and some were from people whose cancers never spread. Diederichs pooled the RNA samples from each group and analyzed the differences in gene expression between the two. One gene quickly stood out because its RNA was three times more abundant in eventually metastasizing tumor samples than in non-metastasizing ones.1 Diederichs, his fellow

postdoc Ping Ji, and their colleagues dubbed this RNA transcript, which was more than 8,000 nucleotides long, MALAT-1 (metastasis-associated in lung adenocarcinoma transcript 1, also abbreviated as MALAT1). High expression of the RNA predicted a poor prognosis, the team found. But, Diederichs notes, “we didn’t find a remarkable open reading frame, and we didn’t find any protein expressed from it. We decided that it has to be something noncoding.” The scientific community was resistant to the idea that a long noncoding RNA (lncRNA) was linked to increased cancer metastasis, says Diederichs. Biology textbooks have long preached the primacy of proteins, with RNAs the intermediary between code and final product, and scientists considered most noncoding RNAs to be useless junk, he explains. “Not many people believed

that long noncoding RNAs would do something. They were still referred to as transcriptional noise. We had reviewer comments that said, ‘If it doesn’t encode for a protein, it doesn’t do anything. So why should we care?’” Diederichs says it took multiple attempts before his study was accepted for publication in Oncogene, where it appeared in 2003. Some two decades later, that paper has been cited by more than 1,500 others, and it’s clear that the team was on to something. Indeed, advances in technology have revealed that much of the noncoding genome gives rise to lncRNAs and other RNA molecules that affect all manner of cellular processes. “The vast majority of the human genome gets transcribed into RNA, between 70 and 90 percent,” says Diederichs. And as of 2022, more than 100,000 human lncRNAs have been discovered. Although no one knows how many of these are functional, at least 30, and perhaps more than 100 have a strong link to cancer, while others associate with conditions such as schizophrenia or heart disease, as well as with normal physiological functions, such as cell growth and metabolism. How these lncRNAs exert their influence is in many cases still an open question, but scientists have begun to uncover mechanisms that confirm that the RNA molecules themselves interact with DNA, other RNA, and proteins, regulating proliferation, growth suppression, angiogenesis, and cellular immortality. In ad dition, some purported lncRNAs may not be noncoding after all: Hidden within many of them live codes for peptides called microproteins that are proving to have powerful physiological functions as well as important roles in the development and progression of cancer.

LncRNAs take off Geneticist Howard Chang’s first foray into lncRNAs came shortly after he started his lab at Stanford University in the mid-2000s. He decided that, instead of looking at the 2 percent of the human genome that encodes proteins, he would focus on the noncoding elements. “We hybridized RNA samples to arrays and, to our surprise, in addition to the known mRNA coding sequences, we also got all kinds of signal,” he recalls. “So, either we had some kind of terrible background [noise] in our array technology, or we have all these new RNAs that we should try to figure out how to characterize.” (Chang helped found and advises several companies working on RNA therapeutics, although he’s not directly involved in lncRNA commercialization.) Noncoding RNAs (ncRNAs) are defined as RNAs that do not contain any open reading frames greater than 300 nucleotides (enough to code for 100 amino acids). Researchers once assumed “that proteins smaller than 100 amino acids are likely not functional,” explains geneticist Jin Chen of Altos Labs, a Californiaand UK-based biotech company focused on cellular rejuvenation programming. This definition of ncRNAs was intended to capture any RNA that didn’t encode a functional protein. Any ncRNA longer than 200 nucleotides was dubbed a long noncoding RNA. As Chang and others turned their attention to these molecules in the late 1990s and early 2000s, examples of lncRNAs’ 1 8 T H E SC I EN T I ST | the-scientist.com

physiological functions began to accumulate. A famous early example is Xist (X-inactive specific transcript). Researchers originally thought that it came from a protein-coding gene, but the protein turned out to be an artifact. Rather, the RNA transcript itself coats and inactivates one of the two X chromosomes in female embryos. Jeannie Lee, a geneticist at Harvard Medical School who advises RNA-targeting drug company Skyhawk Therapeutics, notes the importance of the RNA’s structure for its function. “It’s a very large RNA; each piece of the RNA will recruit a different cluster of proteins. So, the whole RNA is actually wrapped up in a large particle that consists of probably 100 or more proteins. The transcript scaffolds an entire family of proteins that will be required to silence the X chromosome.” John Mattick, a molecular biologist at the University of New South Wales in Australia who has worked on lncRNAs since 2000 and is on the scientific advisory boards of RNA therapeutics companies US-based NextRNA Therapeutics and UK-based e-therapeutics, says that this feature is common in lncRNAs. These RNAs can have complex secondary structures that contain both “a sequence that will target a place in the DNA, but also another sequence that binds the protein that brings it to that location,” he says. After discovering MALAT1’s involvement in metastasis, Diederichs wanted to know whether the RNA played a functional role in disease, or if it was just a marker of cancer’s spread. He started his own lab at the German Cancer Research Center and at the University of Heidelberg, and set out to determine just what MALAT1 was doing in the body. His team began by silencing the gene in human lung tumor cells and got its first clue. “There was this moment when we first saw that the cells that lacked MALAT1 did not migrate,” he says. “That was the moment where I was convinced that this was functionally relevant.” The team subsequently found that MALAT1 regulates

DEFINITIONS BOX: Long noncoding RNAs (lncRNAs) are RNAs longer than 200 nucleotides that do not contain any open reading frames greater than 300 nucleotides (100 codons). The DNA sequences they’re transcribed from can be found between genes (intergenic), within introns of known genes (intronic), or in the antisense strand of DNA, among other places. Microproteins/micropeptides are proteins translated from a short open reading frame (sORF) of less than or equal to 300 nucleotides, producing a protein of up to 100 amino acids. These sORFs are often found within lncRNAs.

expression of a set of metastasis-associated genes, and later studies showed that the RNA localizes to cellular structures called nuclear speckles. Many other groups have followed suit, finding numerous lncRNAs whose expression levels correlate with features of different cancers. Chang, for example, found that a lncRNA his team dubbed HOTAIR (HOX antisense intergenic RNA) is a scaffold that recognizes various targets and silences Hox genes involved in body patterning.2 His and other teams have identified HOTAIR as an oncogenic molecule that affects proliferation, apoptosis, invasion, aggression, and metastasis of cells. The lncRNA tethers two proteins—polycomb repressive complex 2 (PRC2) on one of its ends and lysine-specific demethylase 1 (LSD1) on the other—that inhibit expression of tumor and metastasis suppressor genes, among others.3 HOTAIR has so far been linked to lung, breast, pancreatic, and other cancers. “There’s literally hundreds of papers every year coming out with new long noncoding RNAs,” says Diederichs. “Some are very cancerspecific . . . some with a mechanism, some without a mechanism. So it’s really a jungle out there of long noncoding RNAs these days.”

Secret coders A few years ago, the idea that perhaps many of these lncRNAs actually encode proteins—and that the arbitrary threshold of 100 amino acids to define a protein was wrong—began rumbling through the research community. Last year alone, researchers published three major review papers on the resulting microproteins, extolling the mysteries of this dark proteome. “Recently we found out that a lot of these so-called long noncoding RNAs are not really noncoding; they can encode these small microproteins and these small microproteins really play a very diverse role,” says Chen. “Because [of ] this initial oversight and assumption, there’s this whole proteome that’s basically missing” from scientific knowledge. In fact, researchers have recognized that small, functional proteins exist, but these were thought to always be processed from larger proteins, such as is the case with peptide

hormones and neuropeptides. Insulin, for example, is only 51 amino acids long, but it is cleaved from the longer proinsulin polypeptide chain (which is cleaved from the even longer preproinsulin). Microproteins, on the other hand, are born small—they are directly translated from short open reading frames (sORFs) that were overlooked as a result of the 100-amino-acid threshold. To uncover these formerly ignored microproteins in the vast human genome, exploratory studies use computational approaches to search DNA for evolutionarily conserved sequences with few or no mutations that would change the coded amino acid—a feature that would suggest that the conserved sequence represents a true protein. Researchers also use ribosome profiling, in which RNAs actively translated by ribosomes are extracted and sequenced, as well as mass spectrometry to directly quantify the resulting protein. Combining these methods, scientists can confidently assess whether a sORF actually codes for a microprotein. Further experimentation can help determine what the microprotein does. As with lncRNAs, researchers are discovering functional roles for microproteins in health and disease. Studies have shown that the peptides act as signaling molecules, regulators of enzymes, ligands for receptors, and critical transmembrane components. And now, scientists are finding more and more microproteins that appear to stoke cancer in humans.

We had reviewer comments that said, ‘If it doesn’t encode for a protein, it doesn’t do anything. So why should we care? —Sven Diederichs, University of Heidelberg

In 2018, as Diederichs and graduate student Maria Polycarpou-Schwartz were searching for lncRNAs and mRNAs involved in breast cancer, their team homed in on one lncRNA that was expressed at levels six times higher in breast tumors than in normal tissue. Scanning a protein sequence database for the region, the researchers saw that the lncRNA contained a sORF that was highly conserved in mice and rats. In humans, it appears to be translated into a small protein of 83 amino acids. Knocking down the gene, dubbed CASIMO1 (cancer-associated small integral membrane ORF), in cultured human breast cancer cells caused actin cytoskeleton deregulation, such that the cells were unable to migrate and less likely to proliferate. The knockdown also suggested a role for CASIMO1 microprotein in cell cycle progression. “For a long time, we thought that it’s noncoding because many of the prediction programs told us so and the open reading frame was really short and so on,” says Diederichs. So the team was surprised to eventually find that “if we take out the start codon of the longest ORF, we suddenly lost the function.” Diederichs and his colleagues subsequently identified 12 proteins that appeared to interact with CASIMO1, including an SPRING 2 02 3 | T H E S C IE N T IST 1 9

lncRNAs AND MICROPROTEINS IN CANCER Several long noncoding RNAs and microproteins have been implicated in cancer. While some appear to stoke the development of disease, others keep cancer’s progression in check. A selection is described below.

NAME

IncRNA OR MICROPROTEIN

Discourages cancer

Encourages cancer

EFFECT Sustained proliferative signaling

HOTAIR

lncRNA

Activates the miR-126/CXCR4 axis and downstream signaling pathways; increases lactate production, glucose uptake, and ATP production, among other actions

CASI-MO1

microprotein

Increases phosphorylation of ERK, part of the MAPK pathway of cell proliferation

PINT

IncRNA

Regulated by p53, its overexpression inhibits proliferation of tumor cells (see its microprotein below)

Evasion of growth suppressors PINT-87aa

microprotein

Transcribed from the PINT lncRNA, it interacts with PAF1c and inhibits transcriptional elongation of multiple oncogenes and suppresses tumorigenic capabilities of glioblastoma cell lines Resistance to cell death

CASC9

IncRNA

Loss of CASC9 leads to apoptosis, likely through the AKT signaling pathway and possibly other pathways Activation of invasion and metastasis

MALAT1

IncRNA

Regulates expression of a set of metastasis-associated genes

HOTAIR

IncRNA

Promotes cell migration and invasion by increasing EMT; regulates insulin growth factor-binding protein 2 expression, among other functions

CIP2A-BP

microprotein

Competes with PP2A for CIP2A binding, leading to inhibition of oncogenic P13K/AKT/ NFkB pathway

MIAC

microprotein

Interacts with Aquaporin 2 to inhibit the actin cytoskeleton, suppressing tumor growth and metastasis Deregulating cellular energetics

HOXB-AS3

microprotein

Helps splice the PKM enzyme into a form that supports normal cell metabolism, inhibiting cancer

Genome instability and mutation PACMP

micropeptide

Modulates DNA damage repair

Tumor-promoting inflammation Aw112010

micropeptide

Promotes a proinflammatory immune response

enzyme involved in cholesterol biosynthesis called squalene epoxidase (SQLE), which also happens to be oncogenic. Their work showed that CASIMO1 overexpression in cultured breast cancer cells led to increased SQLE protein levels, while CASIMO1 knockdown led to reduced SQLE. CASIMO1 knockdown also reduced levels of active extracellular signal-regulated kinase, part of the MAPK pathway of cell proliferation that is often involved in cancer. This work, published in 2018, crowned CASIMO1 as the first microprotein discovered to have oncogenic activity.4 Discoveries of additional oncogenic microproteins followed. Last year, for example, researchers in China found that a lncRNA highly expressed in certain chemotherapy-resistant breast tumors encodes a 44-amino-acid-long micropeptide, which they named PACMP (PAR-amplifying and CtlP-maintaining micropeptide). PACMP modulates the DNA damage response, thereby regulating cancer progression and drug resistance.5 Depleting the peptide in cultured cells reduced tumor growth and sensitized tumor cells to several chemotherapies.

Microprotein brakes Some microproteins slow cancer progression. In 2017, researchers discovered that a lncRNA called HOXB-AS3 encodes a conserved 53-amino-acid peptide that suppresses colon cancer growth.6 It works via the enzyme pyruvate kinase M (PKM), which comes in two isoforms. The team showed that when the HOXB-AS3 micropeptide is present, it facilitates the splicing of the first isoform, PKM1, which supports normal cell metabolism and growth. In lab experiments, the team further found that when HOXB-AS3 is absent, cells make PKM2, triggering aerobic glycolysis, which allows the rapid proliferation of cancer cells. Previous studies have shown that PKM2 production confers selective advantage to tumor cells and is seen in most cancers. By examining tissues from patients with colon cancer, the scientists saw that individuals with low levels of HOXB-AS3 had more advanced cancers and poorer prognoses. Similar cancer-blocking microproteins have been found in breast cancer. In 2020, researchers showed that downregulation of a microprotein called CIP2A-BP was linked to increased metastasis and reduced survival in patients with triple negative breast cancer.7 In a mouse model of breast cancer, injecting the microprotein reduced lung metastases and improved survival. The team also showed that CIP2A-BP binds to the tumor oncogene CIP2A to inhibit migration and invasion of the breast cancer cells via the PI3K/AKT/NFkB pathway. That same year, another group of researchers studying microproteins from various noncoding RNAs discovered a microprotein they called MIAC (micropeptide inhibiting actin cytoskeleton) in head and neck squamous cell carcinoma.8 Their study showed that when MIAC levels are low, survival tends to be poor in patients with these cancers. The team further analyzed the RNA sequences of 9,657 other human tissues, including 32 different cancers, and found that MIAC is related to the progression of five other types of tumors. Diving deeper into possible mechanisms, the researchers found that MIAC interacts with aquaporin 2, a protein that normally functions in the kidney. This interaction inhibits the actin cytoskele-

ton and eventually suppresses tumor growth and metastasis. “MIAC . . . differential expression is significantly related to patient prognosis and the tumor state,” says lead investigator Hanmei Xu at China Pharmaceutical University in Nanjing. “So, the possibility of [its] application in cancer diagnosis and treatment [is worth] exploring.” Although the field is young, the list of microproteins linked to cancer goes on and on. “These microproteins are very important. . . . There’s potentially tens of thousands of them,” says Chen, although not all are likely to be cancer related. Many open questions remain. It’s not clear, for instance, how many are stable and functional. “There are so many unknowns. I think it’s a very exciting field to be in at the moment,” Xu agrees. “Micropeptide discovery has a great future,” she says. “According to recent -omics studies, many microproteins have not been characterized. [Genomes] contain a great quantity of undiscovered treasure.” The field is already revealing new levels of complexity, too. For example, as Diederichs points out, some lncRNAs both have functions of their own and serve as recipes for microproteins. In 2013, researchers reported on a lncRNA called Pint that is regulated by the tumor suppressor protein p53 and encourages cell proliferation and survival.9 In 2018, researchers discovered that this lncRNA, in its circular form, contains an ORF that encodes an 87-amino-acid microprotein called PINT87aa, which tamps down tumorigenesis in glioblastoma cell lines in vitro and in vivo.10 The team found that mice injected with anaplastic astrocytoma cells that were lacking functional copies of the microprotein grew larger tumors than animals injected with unmodified astrocytoma cells. “I think it’s less important whether it’s coding or noncoding,” Diederichs says. “Indeed, my firm belief is that many of the transcripts we are looking at will have coding functions as well as noncoding functions.” J Rachael Moeller Gorman is a freelance journalist based in Boston who covers science and health. References 1. P. Ji et al., “MALAT-1, a novel noncoding RNA, and thymosin `4 predict metastasis and survival in early-stage non-small cell lung cancer,” Oncogene, 22:8031–41, 2003. 2. M.-C. Tsai et al., “Long noncoding RNA as modular scaffold of histone modification complexes,” Science, 329:689–93, 2010. 3. M. Hajjari and A. Salavaty, “HOTAIR: an oncogenic long non-coding RNA in different cancers,” Cancer Biol Med, 12:1–9, 2015. 4. M. Polycarpou-Schwarz et al., “The cancer-associated microprotein CASIMO1 controls cell proliferation and interacts with squalene epoxidase modulating lipid droplet formation,” Oncogene, 37:4750–68, 2018. 5. C. Zhang et al, “Micropeptide PACMP inhibition elicits synthetic lethal effects by decreasing CtlP and poly(ADP-ribosyl)ation,” Mol Cell, 82:1297–1312.e8, 2022. 6. J.-Z. Huang et al., “A peptide encoded by a putative lncRNA HOXB-AS3 suppresses colon cancer growth,” Mol Cell, 68:171–84.e6, 2017. 7. B. Guo et al., “Micropeptide CIP2A-BP encoded by LINC00665 inhibits triple-negative breast cancer progression,” EMBO J, 39:e102190, 2020. 8. M. Li et al., “Micropeptide MIAC inhibits HNSCC progression by interacting with aquaporin 2,” J Am Chem Soc, 142:6708–16, 2020. 9. O. Marín-Béjar et al., “Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2,” Genome Biol, 14:R104, 2013. 10. M. Zhang et al., “A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma,” Nat Commun, 9:4475, 2018.

SPRING 2023 | T H E S C IE N T IST 2 1

Cancer’s Jumping Genes

Transposons may be key players in how tumors develop and spread, but they also keep cancer at bay in some circumstances. BY DIANA KWON

MODIFIED FROM © ISTOCK.COM, NATROT, KOTO_FEJA CREDIT

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round 2015, Katherine Chiappinelli was investigating the mechanism behind a group of drugs approved to treat blood cancers1—and showing promise against other cancers— when she made a puzzling discovery. Earlier studies had suggested that these drugs, known as DNA methyltransferase inhibitors (DNMTis), worked by triggering the expression of tumor suppressor genes. But Chiappinelli, then a postdoctoral fellow in Stephen Baylin’s lab at Johns Hopkins University, also saw an upregulation in genes involved in innate immunity. It turned out that the drugs, which reduce the number of methyl groups that attach to and block transcription of certain segments of DNA, had removed the methylation suppressing expression of sequences within the genome that closely resemble and likely originated from retroviruses. These so-called endogenous retroviruses could then be transcribed into virus-like RNA that incited an immune response in cultured ovarian cancer cells. “The cell acts like it has a virus,” Chiappinelli, now at the George Washington University, explains. This immune response could be harnessed to help fight malignancies. In a mouse model of cancer, the team found that DNMTis enhanced the effect of immunotherapy. The finding was yet another example of the outsized effects that endogenous retroviruses and other “jumping genes”—fragments of DNA composed largely of repeating sequences that are capable of hopping around from one part of the genome to another—can have in diseases such as cancer. Transposable elements are part of the noncoding genome, the more than 98 percent of our DNA that doesn’t hold recipes for proteins, and they make up around half of our DNA. Decades of research point to their role in shaping the

The big question is are these elements just associated with cancer progression, or are they helping to drive it? —Katherine Chiappinelli, George Washington University

The study of transposable elements is a “fast-growing” subfield of cancer research, says Benjamin Greenbaum, a computational biologist at Memorial Sloan Kettering Cancer Center. Researchers have been approaching this topic from multiple angles, he adds, and “it’s really just starting to all come together.”

A flood of discovery In 1988, researchers at Johns Hopkins University were examining the genes of people with hemophilia—a heritable bleeding disorder characterized by blood that does not clot properly—when they spotted insertions of a transposable element, long-interspersed nuclear element-1 (LINE-1), in several sites along the gene for the coagulation protein factor VIII. At the time, transposable elements were largely seen as “junk DNA,” the term once given to the noncoding portion of the genome. But this study revealed that these elements can exert an effect: When a transposon landed in a gene, it could lead to disease. Researchers have since found more than 100 types of LINE-1 insertions that induce some sort of pathology. LINE-1 sequences make up approximately one-fifth of the human genome. They belong to a subset of transposable elements known as retrotransposons—DNA fragments that can replicate themselves via an RNA intermediate and reinsert into the genome. LINE-1 also encodes a reverse transcriptase protein, which it uses to generate the RNA intermediate. (Other transposable elements, such as short interspersed nuclear elements, or SINEs, can co-opt this reverse transcriptase for replication.) Only around 100–150 of the hundreds of thousands of LINE-1 sequences in the human genome are capable of activation, and these are usually suppressed by methylation. But over the years, studies from several groups have revealed that in the genomes of cancer cells, this methylation is sometimes removed.2 24 T H E SC I EN T I ST | the-scientist.com

In 2012, researchers at Harvard Medical School reported that LINE-1 could insert copies of itself into the middle of genes that are commonly mutated in tumors, possibly contributing to cancer formation. Shortly after, other groups published reports corroborating these findings. For example, Kathleen Burns, a pathologist at the Dana-Farber Cancer Institute, and her team reported that the expression of proteins encoded by LINE-1 was a key feature of many human cancers, including colon, lung, and breast cancer.3 “It was a really exciting time in the field,” Burns says. “I think a lot of gratification came to all of us by using different tools and different approaches and having a few different labs converging on some of the same conclusions.” In parallel, other groups, such as Chiappinelli’s, were gathering evidence for the importance of endogenous retroviruses. (Unlike LINE-1, endogenous retroviruses in humans can only generate virus like molecules such as RNA; these elements are mere relics of previous jumping events and can no longer move around the genome.) Around the same time that Chiappinelli and her colleagues were investigating endogenous retroviruses and the immune response their transcripts provoked in cultured ovarian cancer cells, another group discovered something similar in another cancer type. According to Chiappinelli, her advisor, Baylin, had been giving a talk about their findings when a researcher at the University of Toronto, Daniel De Carvalho, told Baylin that he had made similar observations in colorectal cancer cells. The two teams decided to cooperate by sharing their data. Baylin’s team went on to find exactly the same immune-activating effect of endogenous retroviruses in colorectal cancer, and the teams submitted their papers to the journal Cell at the same time.4 “I think that it was probably more easily accepted by the field because you had two different groups discovering the same thing,” Chiappinelli says.

Double-edged swords Scientists are still working out the extent to which transposable elements may promote and, in other cases, hold back cancer growth. Broadly, researchers know that transposable elements are expressed at much higher levels in cancer cells than in normal cells, although it isn’t clear why. According to Chiappinelli, one hypothesis is that this expression occurs in the context of global dysregulation of the epigenetic landscape of tumor cells, where disruptions to methylation and other epigenetic processes are common. “The big question is are these elements just associated with cancer progression, or are they helping to drive it?” Chiappinelli says. Transposable elements may, in rare instances, directly give rise to cancer-driving mutations. For example, in 2016, researchers reported a case where LINE-1 inserted itself into a tumor suppressor gene, initiating colorectal cancer.5 But it appears to be much more common for transposable elements to insert themselves into the cancer genome with no immediate cancer-boosting effect. In fact, some scientists suspect that in early cancer development, the insertion of these elements may be detrimental to a growing cancer cell.

© ISTOCK.COM, NATROT

evolution of genomes, and more recent work has revealed that these elements may also influence a wide range of diseases. In particular, over the past decade or so, researchers such as Chiappinelli have been amassing a body of evidence that these elements can both help and hinder the growth of cancer. Studies show that transposable elements are expressed in multiple cancers and influence how the disease develops in a variety of ways. Buoyed by this growing body of research, some investigators have already begun devising new therapeutics specifically targeting transposable elements, with a handful of new companies dedicated to this goal. Chiappinelli consults for one such company called ROME Therapeutics.

TRANSPOSABLE ELEMENTS IN CANCER In healthy cells, transposable elements (TEs) are typically inactivated by methylation. But in cancer cells, these elements can become demethylated, enabling them to be expressed (1). Some transposable elements code for proteins such as endonuclease and reverse transcriptase (2). The activity of these proteins enables transposable elements to reinsert themselves into DNA, causing damage and mutations (3). TEs that are unable to reverse transcribe may still be translated into antigens, which are can subsequently be expressed on the cell surface (4). The expression of transposable elements can also activate the cell’s innate immune response in various ways, such as through DNA damage or via the presence of TE-derived RNA in the cytoplasm (5), an effect that may subsequently alter a cancer’s ability to spread.

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In cells that have turned cancerous, expression of LINE-1 and other transposable elements typically increases. This increase in transposable element expression may inhibit cancer growth by triggering an immune response. “The moment a cancer cell allows LINE-1 elements to replicate, it turns into a big disaster,” says Andrei Gudkov, a professor of oncology at the Roswell Park Comprehensive Cancer Center. These elements can create new copies of themselves, which reinsert into the genome and produce mutations and chromosomal rearrangements. They can be expressed to make proteins such as reverse transcriptase, which can generate products triggering inflammation, and endonuclease, which cleaves DNA—changes that can harm a cell’s viability. “Cells with active LINE-1 elements behave as if they have a piece of uranium inside them. They are living under conditions of constant mutational pressure,” Gudkov explains. “It’s a massive genome and epigenome destabilizing factor.” Yet while this type of activity might be detrimental to some cancer cells, the flip side of LINE-1 activity is that it creates a huge amount of genetic diversity, which may ultimately endow some cancerous cells with characteristics that enable them to thrive. People don’t typically succumb to primary tumors, Gudkov says; it’s the tumors that have evaded all lines of therapy and metastasized that are more dangerous. “We die from cancer because cancer is creative.” Gudkov and others posit that, within cancer cells, transposable elements generate genetic diversity in a manner akin to that seen throughout the course of evolution within populations—albeit in an accelerated manner. To test whether targeting transposable elements could help treat cancer, Gudkov, Greenbaum, and others have examined the effect of giving cancer cells a so-called reverse transcriptase inhibitor that halts the activity of LINE-1. In animals, Gudkov’s team demonstrated that administering this inhibitor made cancer cells less capable of adapting to treatment and extended progression-free survival in mice. In a study published last summer, Greenbaum, Burns, and their colleagues found that a reverse transcriptase inhibitor slowed the progression of metastatic colorectal cancer in 9 of 32 patients with the disease.6

drugs to induce elements to activate an immune response, as Chiappinelli unexpectedly discovered several years ago, or engineering CAR-T cells to express the RNA from transposable elements to enhance these cells’ immunotherapeutic activity. Some researchers, including Burns, are also examining whether these elements might be useful for cancer diagnosis. If signs of changes in the epigenetic activation of these elements can be found in tissue samples of body fluids such as blood, they could be used as biomarkers of the disease, says Burns, who has consulted for ROME and other companies developing transposon-based cancer therapeutics. Still, many open questions remain. To what degree do these elements contribute to cancer evolution or to the immune activation that might help fight cancers? At which specific location on the genome are these transposable elements being activated, and how does this differ across cancer types? Another key consideration, notes John Coffin, a molecular virologist at Tufts University and a shareholder and member of the scientific advisory board of ROME, is what these elements might be doing in healthy individuals. In a recent study, he and his colleagues discovered that endogenous retroviruses are expressed in multiple human tissues despite being thought to be largely silenced by methylation in healthy cells.7 “It’s important to have some idea of what the normal expression of these is, and in which tissue, because that’s a possible site of off-target effects of your therapy.” All the same, the field of transposable elements is now overcoming earlier challenges as more researchers acknowledge that noncoding DNA is far from irrelevant, and as new technologies make it easier to study repetitive sequences. In particular, “we’re becoming more rigorous and building the right types of tools to approach the questions that we want to ask,” Burns says. “There’s been a complete paradigm shift in how we’re thinking about transposable elements and their possible functions in cancers. . . . There’s a lot of cool stuff on the horizon.” J Diana Kwon is a freelance science journalist based in Berlin, Germany.

To the clinic 

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

References 1. A. Mullard, “FDA approves an inhibitor of a novel ‘epigenetic writer’,” Nat Rev Drug Disc, 19:156, 2020. 2. D.T. Ting et al., “Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers,” Science, 331:593–6, 2011. 3. N. Rodiü et al., “Long interspersed element-1 protein expression is a hallmark of many human cancers,” Am J Pathol, 184:1280–6, 2014. 4. D. Roulois et al., “DNA-demethylating agents target colorectal cancer cells by inducing viral mimicry by endogenous transcripts,” Cell, 162:961–73, 2015. K.B. Chiappinelli et al., “Inhibiting DNA methylation causes an interferon response in cancer via dsRNA including endogenous retroviruses” Cell, 162:974–86, 2015. 5. E.C. Scott et al., “A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer,” Genome Res, 26:745–55, 2016. 6. M. Rajurkar et al., “Reverse transcriptase inhibition disrupts repeat element life cycle in colorectal cancer,” Cancer Discov, 12:1462–81, 2022. 7. A. Burn et al., “Widespread expression of the ancient HERV-K (HML-2) provirus group in normal human tissues,” PLoS Biol, 20: e3001826, 2022.

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Researchers have already begun developing anticancer drugs targeting transposable elements. These elements have “definitely gotten the biotech and pharma interest over the last two or three years in a way that would have been quite shocking five to ten years ago,” says Greenbaum, who cofounded ROME Therapeutics to carry out this task. Gudkov, too, has founded a company, Genome Protection Inc, to develop such treatments. Some of this work has already spurred clinical trials. For example, Gudkov says that his team recently launched a study examining the effect of a reverse transcriptase inhibitor in small cell lung cancer patients. Companies can choose from a variety of ways to target these elements. According to Nicolas Vabret, an immunologist and virologist at Mount Sinai’s Icahn School of Medicine who consulted for ROME on one occasion, these include using epigenetic

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Alex Muir: Cancer’s Diet Detective Assistant Professor, Ben May Department of Cancer Research, University of Chicago BY NATALIA MESA, PhD

JEAN LACHAT UCHICAGO CREATIVE

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lex Muir never thought he would become a cell biologist. Instead, when he enrolled in the University of Chicago as an undergraduate in 2006, he was set on becoming an archaeologist. His parents were history buffs, and he fondly remembers sitting on the couch eating gelato and watching the History Channel with his dad. But at the end of his sophomore year, Ilaria Rebay, a cell and molecular biologist at the university, presented him with an opportunity for a summer job in her lab and he said yes. Rebay’s team had recently discovered a new Drosophila transcription factor involved in eye development that was also a phosphatase, but they didn’t know if or how the two functions were related. Muir was tasked with finding the protein’s substrates. As an undergrad without much of a science background, he now says he didn’t have a prayer of completing the project, but he was hooked by the opportunity to ask big picture questions and “the freedom to explore ideas.” He continued to the University of California, Berkeley, where in 2010, he joined Jeremy Thorner’s lab to pursue a doctorate in biochemistry, biophysics, and structural biology. There, Muir studied the signaling pathways that eukaryotic cells turn on to cope with mechanical challenges. Thorner had discovered a group of yeast cell kinases called Ypk1 that respond to excessive membrane compression or stretch, and Muir developed a genetic screen to pinpoint the kinase’s substrates (eLife 3:e03779, 2014). Of the potential hits the screen identified, Muir found that two are essential to cell survival in times of stress. They form part of an enzyme complex called ceramide synthase, which produces a family of waxy lipid molecules that get deposited to the membrane to recover membrane tension. Later in his time as a graduate student, as he was exploring his next steps, Muir took a class on metabolism that spurred his interest in the area. “It felt like a lot of the old par-

adigms [in the field of metabolism] were changing, and that seemed really, really fascinating to me,” he says. Wanting to move into more translational research, he joined molecular biologist Matthew Vander Heiden’s lab at MIT’s Koch Institute for Integrative Cancer Research as a postdoc in 2016. Vander Heiden’s lab, which studies tumor metabolism, was wrestling with a puzzling discovery. The dogma in the field up to that point, based purely on results from in vitro studies, was that tumor cells consume glutamine. But Vander Heiden’s research had shown that when the same cells were transferred to a mouse, they stopped metabolizing glutamine. “Clearly, [if you’re a tumor] there’s something about having physiology around you that changes your metabolism. All of those glutamine metabolic pathways don’t become as important,” Muir explains. So he set out to study what exactly cancer cells consume in vivo. He and his colleagues began by developing a new technique that combined mass spectrometry and RNA-seq to measure the components of the interstitial fluid surrounding lung and pancreas tumors in mice (eLife 8:e44235, 2019). The method allowed them to compare the relative levels of nutrients and gene expression among samples. They found that nutrients around tumors differ from those throughout the body, and that animal diet, tumor type, and tumor location affects these nutrients. “He had a ton of impact. I probably get [the most] collaboration requests still today to go and help isolate what nutrients are available in various biological fluids,” says Vander Heiden. “Alex is one of the most thoughtful people that I’ve ever worked with,” says Mark Sullivan, a postdoctoral researcher who worked alongside Muir as a PhD student in Vander Heiden’s lab. “He thinks deeply about everything,” Sullivan says, including “How should we go about science? How should we go about mentorship?”

In 2019, Muir joined the University of Chicago as an assistant professor. His team researches what nutrients cancers consume and which metabolic pathways and genes they use to consume them. Muir describes this as figuring out what’s typically on the “menu” for pancreatic cancer cells and how these molecules either promote or inhibit growth. Currently, he’s particularly interested in the role of the amino acid arginine, which is almost completely depleted in the microenvironment of pancreatic cancers in mice. Muir’s team investigates how cancer learns to live without arginine, how its absence affects cancer metabolism, and whether the affected cellular pathways can be exploited to combat tumors. “Alex is a very special individual,” Vander Heiden says. “I think he’s going to do really great things in his career.” J

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Extinction Calculus How do scientists decide a species has gone extinct? BY ANDY CARSTENS

ARTHUR A. ALLEN AND THE MACAULAY LIBRARY AT THE CORNELL LAB OF ORNITHOLOGY

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n an overcast October morning in 2021, self-taught ornithologist Mark A. Michaels was staking out a sweetgum tree in a Louisiana forest when he spotted a bird flying below the canopy about 50 yards away. Based on the bird’s size and long neck, Michaels first presumed he was looking at a duck. But then he noticed it intermittently tucking its wings during flight, something he says that ducks don’t do—but woodpeckers do. “Ivory-bill!” he recalls shouting, now certain that the bird was an ivory-billed woodpecker (Campephilus principalis), a species that hasn’t been definitively identified in the wild since 1944, and one that he’d been seeking for about 15 years. Michaels, a research associate at the National Aviary, has had several possible ivory-bill encounters, including at that same tree, but he couldn’t fully convince himself of their authenticity. This sighting was different. “That was really the first time I was absolutely sure of what I saw,” he tells The Scientist. Despite his certainty, not everyone agrees this woodpecker remains in the wild. In September 2021, the US Fish and Wildlife Service (USFWS) proposed a ruling that would declare the ivorybilled woodpecker, along with 22 other species, extinct. The decision would remove the bird from the federal list of endangered species and, therefore, formally eliminate conservation protections required under the US Endangered Species Act, such as preserving habitat and taking other steps to try to increase population size. However, the data supporting the proposal are debated. Conflicting evidence presented at a public hearing in January 2022 convinced USFWS that disagreement among scientists as to the ivory-billed woodpecker’s status was substantial enough to warrant further review. As a result, on July 6, the agency issued a stay of extinction: it gave the woodpecker six more months before deciding whether to strip away its protected status as an extant, endangered species. The ongoing case highlights some of the challenges researchers face in determining whether a species has actually gone extinct. It’s “difficult to prove the absence of something,” says H. Resit Akçakaya, an ecologist at Stony Brook University, and so a lack of verifiable sightings is not necessarily evidence of extinction. According to guidelines issued by the International Union for Conservation of Nature (IUCN), an organization that tracks species’ conservation statuses on the basis of surveys, modeling, and expert opinion, “A taxon is Extinct when there is no reasonable doubt that the last individual has died.” But researchers typically don’t know when or if that last death has occurred, Akçakaya adds.

GLAMOUR SHOT: An ivory-billed woodpecker (Campephilus principalis) in 1932

Moreover, there are costs to making the wrong call about a species’ existence in the wild, he says. Continuing to classify an actually extinct species as endangered can lead to underestimating extinction rates, and obscuring the bigger conservation picture, as well as misdirecting financial resources away from protecting vulnerable species to searching for ones that no longer exist. On the other hand, declaring something extinct when it really isn’t can inflict further harm on a struggling species.

The price of mistakes The Cebu flowerpecker (Dicaeum quadricolor), a small, colorful songbird native to the Philippine island of Cebu, was presumed to have been eradicated around 1906. Ornithologists in the early-to mid-20th century failed to find any trace of it, and suggested that deforestation had left the bird with insufficient habitat to survive, SPRING 2023 | T H E S C IE N T IST 3 1

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according to a 1998 article by conservation biologist Nigel Collar with BirdLife International (Oryx 32: 239–44, 1998). This erroneous conclusion failed to spark the sort of conservation movement that might have protected the area, so logging continued to afflict what little forest remained. By the 1990s, when the bird was rediscovered, only a few islets of forest dappled the surface of Cebu, equating to a scant 0.03 percent of its original coverage. And while Cebu flowerpeckers persist today, their future appears grim: A species assessment published in 2021 put its population at around 60 to 70 birds and declining.

In addition, he notes, scads of well-meaning people trying to spot one could disrupt the woodpeckers’ habitat. People’s efforts to prove the birds exist have ended with casualties before: in the 1930s, after hunting and logging had reduced the woodpeckers’ population so much that people disagreed about whether they still existed, Louisiana state representative Mason Spencer attempted to settle the debate by shooting one and using its carcass as proof. For those reasons, Michaels says, he and others are not disclosing where they’re searching for the bird.

Probability of extinction

This is one of the biggest problems with incorrectly declaring a species extinct: Doing so can actually spell its demise. “The main fear that we have is that because we think [a species is] extinct, we no longer have to protect it,” Akçakaya tells The Scientist, adding that “it may actually go extinct, because we stop protecting it.” This, he says, is called the Romeo Error because of the ending of Romeo and Juliet: He, thinking she’s dead, takes his own life, which prompts her to do the same. “It’s a self-fulfilling prophecy.” If USFWS decides to declare the ivory-billed woodpecker extinct, could that result in the Romeo Error? “Possibly,” says Michaels, but he’s optimistic it won’t, not least because the agency has for decades been operating under the assumption that the bird is already extinct, he says. USFWS’s ivory woodpecker recovery plan, a type of document required for each endangered species, states that conservation efforts will focus on searching for the bird and will only protect habitat areas if a population is found. Despite no formal requirement to keep the woodpecker’s forested habitat healthy, Michaels says, good management practices have done just that. He’s hopeful those practices would continue even after an extinction declaration, he says, although it’s possible that such a declaration would instead undermine the incentive to continue that work—something that could harm other species that share the ivory-bill’s habitat. Additional issues can arise if a species that’s been declared extinct is later found. Discovery of such a “Lazarus species” can cause the public to lose faith in scientists, according to Akçakaya, and may increase poaching demand in some cases. (See “Secret Resurrections” on page 34.) Although poaching may not be an immediate concern for the elusive ivory-billed woodpecker, demand for the bird as a collectible could explode if the species is found, Michaels says. HAZY SIGHTING: The outline of a bird at the center of this photo, taken

in Louisiana in 2021, is one of the pieces of evidence for the existence of the ivory-billed woodpecked cited in a preprint coauthored by ornithologist Mark Michaels. 32 T H E SC I EN T I ST | the-scientist.com

To aid consequential extinction decisions, IUCN has developed a methodology to help scientists make the best use of available data, says Akçakaya, who also chairs the organization’s Standards and Petitions Committee as a volunteer. Although government agencies such as USFWS make their own determinations while following the requirements laid out in specific laws or regulations, the IUCN methodology is intended to help shepherd discussions about whether to further invest in conserving a particular species, he adds. The framework, published in 2017 and adopted into IUCN’s guidelines in 2019, infers a species’ status by combining two approaches for estimating the probability that it has died out (Biol Conserv, 214:336–42). One approach uses so-called exhaustive surveys conducted throughout the species’ historic range during times and seasons when it’s expected to be present. The more surveys conducted and the wider an area searched, the higher the confidence in the assessment. The second estimates extinction probability based on the extent and severity of threats that a species faces. The more habitat loss that occurs across a missing species’ range, for example, the more likely that the species no longer exists. The methodology has value, according to Stuart Butchart, an ornithologist at BirdLife International who was one of the first scientists to test it. “It forces the user to consider explicitly all the different sources of relevant information,” he writes in an email to The Scientist. “It should also help increase con-

PROJECT PRINCIPALIS

Decisions ultimately come down to the verdict of a jury of experts.

© MARCOS GUERRA, THE SMITHSONIAN INSTITUTE FOR TROPICAL RESEARCH

sistency in assessments.” He and his coauthors went through the procedure in 2018 for dozens of bird species (Biol Conserv, 227:9–18). If both the threat-based and survey-based methods estimated greater than 50 percent extinction probability, the team suggested that the species be considered both “critically endangered” and “possibly extinct.” Only species with probabilities higher than 90 percent using both approaches were recommended to be categorized as extinct. For the ivory-billed woodpecker, Butchart’s analysis estimated a 75 percent probability of extinction using the threatbased method, a result primarily due to habitat loss. From surveys and recorded sightings, the odds of its extinction were lower—around 20 percent. This latter approach tries to account for the possibility that some reported ivorybilled woodpecker sightings were cases of mistaken identity, but Butchart says that if multiple records made this error, that could have led to an underestimate of the bird’s extinction probability.

DEFUNCT AMPHIBIANS: A male and female Chiriqui harlequin

frog (Atelopus chiriquiensis) photographed in 2010. The species was declared extinct in 2019.

Based on their results, Butchart and his colleagues recommended classifying the woodpecker as critically endangered, although their estimates might be different today considering more-recent evidence that Michaels and others have assembled.

Qualitative sentencing Although data-driven methods can aid a decision about whether to declare a species extinct, they’re not always enough to make a final call. In some cases, there simply isn’t sufficient information on a species to produce a reliable prediction, says Kelsey Neam, a conservationist with the nonprofit Re:wild. She has tested IUCN’s framework on amphibians, although she hasn’t yet used it to recommend the extinction status of a species, in part due to

the dearth of information. “It’d be nice to have a lot of data for amphibians, but we don’t.” Whatever the level of available data, decisions ultimately come down to the verdict of a jury of experts. As an assessment facilitator for IUCN’s Amphibian Specialist Group, Neam leads working groups of experts from particular regions in reviewing species’ status. “Sometimes it’s unanimous,” she says. “Everyone goes, ‘Of course, this is totally extinct.’ Other times, there’s a lot of debate.” Local experts who love the species sometimes hesitate to pronounce it gone forever, Neam says. They may fear the Romeo Error, or get caught up in a species’ taxonomic importance, or simply think it “looks cool,” she says. Her job as an expert in using the IUCN criteria is to remain unbiased. “I often do feel like I’m the head juror,” she says. “It’s a lot of pressure.”

Declaring something extinct when it really isn’t can inflict further harm on a struggling species. Determining whether the surveys conducted are exhaustive is one of the most important factors, she says, but she acknowledges “it’s kind of a qualitative term.” Sitting in a room with the people who’ve conducted them helps gauge how extensive they were. When people waver, or mention that the species could be in another area, “that, for me, is a red flag,” she says. “You know, right away it’s not smart to declare something extinct in that moment.” In those cases, the outcome is prioritizing where future searches should occur. In cases when everyone agrees that the species no longer exists, there’s always a quiet moment, she says. “You get goosebumps because it’s not just something you do and [then] go to the next species. We sit there and we actually kind of mourn the loss of the species in that moment.” Michaels says he hopes it won’t come to that for the ivory-billed woodpecker, but he’s concerned that an unattainable quality of evidence will be required to prove its survival. Last July, while presenting evidence to USFWS that the bird still exists, he mentioned his opinion that a series of photographs taken in the 1930s created an unrealistic burden of proof. “Most of them were taken from a blind at nest height, close to the birds,” he tells The Scientist. “They’re glamour shots.” In contrast, he’s trying to make a case with blurry images and video clips extracted and enhanced from drone footage. “It’s a very, very difficult situation,” he says, “because you’re shooting up into the sky, it’s all backlit, birds move fast.” During his presentation, often on a frame-by-frame basis, he pointed to distinguishing ivory-bill features such as white secondary wing feathers, a long neck, and dorsal stripes that reach over the bird’s shoulders up onto its face. “It would be lovely to have a picture that clearly shows its big white bill,” he says. But the photos he has don’t. Now, he can do nothing but await USFWS’s ruling, expected this month. “I have no idea what the outcome will be, and I wouldn’t hazard a guess,” says Michaels. But he says SPRING 2023 | T H E S C IE N T IST 3 3

he’s encouraged that the agency gave the extension, which to him suggests it is seriously considering the possibility that the ivory-billed woodpecker remains extant. Michaels, for his part, has no doubt. “I’m personally 100 percent sure that it’s out there.” J

BIRD SEARCH: Mark Michaels (left) and Steve Latta of the National

Aviary look at signs of woodpecker foraging on a tree.

Andy Carstens is a freelance science journalist and past intern at The Scientist.

SECRET RESURRECTIONS Ecologists and others who come across a Lazarus species—a creature thought to have died out but then found in the wild—often face a dilemma. Sharing the discovery can rally public support around conserving the species, but it can also incite poachers to go after it. Animals can end up in an extinction vortex, where every time one is killed, the value of those remaining ticks up, says Vincent Nijman, a wildlife trade specialist at Oxford Brookes University in the UK. That ever-increasing demand can lead to poachers snuffing out a species for profit—a situation that Nijman says contributed the local extinction of the Javan rhinoceros (Rhinoceros sondaicus) in Vietnam in 2010 (Biol Conserv, 174:21–29, 2014). Something similar happened years later to a population of the Sumatran rhinoceros (Dicerorhinus sumatrensis) after it was rediscovered in 2013 on Kalimantan, the Indonesian side of the island of Borneo. The species had been abundant across Southeast Asia until the early 20th century, but poachers after the rhinos’ treasured horns drove them to near extinction by the 1970s (Biol Conserv, 175:21–24, 2014). While many people believed the Kalimantan population had in fact been wiped out, Nijman says, hunters didn’t. He and a colleague were in contact with poachers who believed the rhino still lived in remote areas but weren’t willing to invest in the months-long expeditions needed to find, capture, and kill one of the few remaining. After the rhino’s rediscovery made global headlines, the economics shifted, Nijman says. “We know that immediately afterwards, rhino hunters from Sumatra went over to Kalimantan.” Nijman argues that keeping the discovery a secret would have been the smarter play. That, he says, would have enabled more resources to be devoted to conservation efforts to help the species recover. “Instead, by announcing it, a substantial amount of money, now—overnight—has to go into protecting them” from poachers, which he says is an enormous challenge in a rainforest with exposed edges. Even delaying such announcements by several years, Nijman says, could give Lazarus species much-needed time to recover.

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READING FRAMES

The Skin Battery The “wound current” has intrigued scientists for more than a century. It could turn out to be the key to healing catastrophic injuries. BY SALLY ADEE

T

ake a hard bite on the inside of your cheek. You’ll feel a tingling sensation. That tingling is the wound current, whose existence has frustrated and misdirected scientists since the 18th century. It intruded on Luigi Galvani’s seminal attempts to demonstrate the existence of animal electricity in frogs; it got in Emil du Bois-Reymond’s way as he tried to pin down the action potential in the 19th century. In 1843, du Bois-Reymond, whose famously obsessive approach to creating tools shaped the emerging science of electrophysiology, built his own galvanometer to measure this weird electrical interloper. Painstakingly created from at least a mile of coiled wire, the device revealed that leaking out of a cut in his finger, alongside the blood, was about one micro-ampere of electrical current. Nearly two centuries later, the modern tools helping us pry open the secrets of bioelectricity would be all but unrecognizable to du Bois-Reymond. At last, we are gaining the understanding and technology to enlist the wound current in the fight against septic and chronic wounds and to speed the healing process for everything else. Most biologists are aware that every cell has a membrane potential: This voltage is the result of an imbalance of ions on either side of the membrane, turning the cell into a tiny battery. Probably the best known membrane voltage is neurons’ -70 mV resting potential, disruption of which is essential to passing the neural impulse. But skin cells also have a resting potential. These cells are connected to one another by little doors called gap junctions, meaning epithelial tissue connects tightly to encapsulate our skin, rather like the membrane around the cell. And just as an individual cell has a voltage, so does this epithelial tissue as a whole. This is the skin battery. Cut or tear this tissue, and the battery short-circuits, creating a “leakage current” that gushes out of the wound. The resulting electric field attracts and guides a host of helper cells, including keratinocytes and fibroblasts, to come and patch up the damage. This self-generated bioelectric field also affects cell division and proliferation, which are important components of the healing process. We have been steadily gaining insights into these intriguing phenomena for decades, but they have been difficult to harness for medical purposes. One milestone came in 2006, when physiologist Min Zhao, then at the University of Aberdeen in the UK, and geneticist Josef Penninger, who was at the Institute of Molecular Biotechnology in Austria, first identified some of the genetic migration machinery that is switched on when electric fields are applied to wounds. In sheets of human skin cells grown in culture, these fields changed gene expression in several types of repair cells, including neutrophils and fibroblasts. Zhao and Penninger

36 T H E SC I EN T I ST | the-scientist.com

Hachette Books, February 2023

also found that augmenting or damping the naturally occurring electricity could accelerate or completely halt the healing process. A few years after that milestone, physicist and biologist Richard Nuccitelli, a former professor at the University of California, Davis, who had founded his own company, BioElectroMed, created the Dermacorder, the first device that could measure minute changes in wound current across space and time. He found that the current’s magnitude changed as healing progressed, peaking at injury and then decreasing until the wound was totally healed. The device also yielded other interesting insights: As they age, people generate progressively weaker wound currents—a shift associated

with slower healing from injury. With this improved understanding, Nuccitelli and his collaborator Christine Pullar of the University of Leicester were able to use targeted electrical stimulation to coax new blood vessels to form in the wounded tissue of 40 human volunteers. It wasn’t just electrical simulation that could increase the speed of wound healing—other studies in animals used drugs. The idea of accelerating wound healing is now taking off. In 2020, the US Department of Defense launched a $16 million program, helmed by Zhao and several other researchers, to devise a next-generation wound healing system for major traumatic wounds. The goal, says Zhao, now at the University of California, Davis, is a “closed loop system involving a tiny camera, electrical stimulation and delivery, and wound monitoring using multiple sensors that take visual and chemical measurements.” The system would determine how far healing had progressed and then use targeted stimulation to trigger bioelectric healing of multiple tissues at once—not only skin—and double the speed of recovery. The project is now transitioning from rodent studies into large animals such as pigs, whose skin is more similar to human epithelium. Human trials should begin next year. Where the DOD project is ambitious in translating what we already know, the Air Force Office of Scientific Research is chiseling away at “a more fundamental understanding of bioelectricity, integrating from the head to the toe the genes and molecules involved in the electrical activity of each system,” says Zhao, who

is also part of a consortium that studies this deeper science, which includes scientists from Johns Hopkins University, Arizona State University, and the University of Maryland. Deciphering bioelectricity’s role in healing our injuries and manipulating it in our favor is only one of many ways this research could change how we treat our most common ailments. Similar voltages and currents are at work in bacterial communication networks and allow microbes to form antibiotic-resistant biofilms. New research targeting these networks could lead to new ways around the growing problem of antibiotic resistance. As today’s major research projects bear fruit, we will begin to grasp the outline of the electrome. Our ability to harness the power of electricity in the 19th century lent it the moniker “the electric century”—being able to finally understand the electricity in our own bodies could put the 21st into the history books as the “bioelectric century.” J Sally is a freelance science and technology journalist based in London. She reports on the places where electricity meets biology, whether that’s future neurotech, the body horror of 19th-century electrophysiology experiments, or the role internet protocols play in geopolitics. She previously edited technology news and features at New Scientist and IEEE Spectrum. Her book about the electrical signals generated by life, We Are Electric is out now in the UK and the US.

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Bathing Through the Ages: 1300–1848 BY CLARE WATSON

I

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

BLOODLETTING AND BATHING: In urban bathhouses in Germany and the surrounding low countries, bathhouse proprietors, known as baders, provided visitors with basic medical care. To draw blood, baders would scratch the skin before placing a heated cupping glass over the incision to extract blood and purge the body. Other tools associated with baders, including dental forceps and an amputation saw, hint at further services they provided.

larger cities as they grew and health conditions deteriorated. As Coomans and fellow historian Guy Geltner argue in their treatise on medieval urban hygiene, local authorities sought to promote health and curb disease, devising policies that they likely gleaned from medical practitioners and popular attitudes toward hygiene. Dismissing the role of bathhouses in early attempts to design healthier cities and rid city streets of foul stenches therefore “obscures their immense contribution,” Coomans and Geltner write, as bathhouses likely served as a “vector transporting medical theory into urban policy.” Their popularity over the next few centuries tracked advances in medicine and public health. By the 16th century, urban bathhouses started to disappear as Europe was ravaged by plague, smallpox, and syphilis, and as theories of contagion became more widely discussed in medi-

cal circles. People were advised to avoid public baths, along with other crowded places, although it would take another three centuries before Louis Pasteur and Robert Koch solidified the germ theory of disease in the 1880s. By then, public baths had seen another change in fortune, with the emergence of free-to-use facilities in Britain. Sanitation reformers argued that making bathing facilities available to the poorest classes of society offered an “affordable and immediate way” of improving public cleanliness and health, University of Liverpool historian Sally Sheard writes. Yet as Sheard notes, many historians have since overlooked the provision of public baths, focusing instead on the installation of waterworks and sewage systems that together laid the foundations for the UK Public Health Act, which passed in 1848. J

GERMANISCHES NATIONALMUSEUM, NUREMBERG

n medieval times, long before there were bathrooms in private homes, bathing was a social affair. Visitors to Dutch and German bathhouses in the late Middle Ages emerged from such spaces cleansed of more than just their grime: They also received basic medical care from “baders,” bathhouse proprietors who were also licensed health practitioners, skilled at lancing abscesses and pulling teeth. Steam rooms, mineral baths, cupping, and herbal concoctions were also commonly used to alleviate ailments from scabies and leprosy to migraines and miscarriages. Not until Victorian times would bathhouses become a little noticed but influential item in public health policies. But according to Janna Coomans, a historian at Utrecht University in the Netherlands who studies public health in medieval cities, bathhouses provided access to more affordable medical care as baders charged less than doctors for their services. Sweating, bloodletting, and lancing were attempts to correct an imbalance in the four “humors”—phlegm, blood, yellow bile and black bile—that were thought to cause ill health. While these practices may seem unhygienic by modern standards, bathhouses enabled “a large part of the population to maintain health and hygienic norms, according to their ideas of health,” Coomans says. Historical records show that baders held a stained reputation, however. Even though many were trained in and approved by guilds, surgeons scorned them as incompetent, akin to quacks and travelling shamans, Coomans says. “There was a strong wish [among the medical elite] to control who gets to practice medical procedures,” she says. Despite these professional squabbles, bathhouses still held a strong social place in communities, and their popularity throughout the 14th and 15th centuries helped shape the public health services of

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