Volume 35, Issue 3. June 2021 The Scientist

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JUNE 2021 | VOL. 35, ISSUE 3 | WWW.THE-SCIENTIST.COM

THE RECENTLY DISCOVERED STRUCTURES ARE MAKING WAVES IN MICROBIOLOGY

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Contents

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THE SCIENTIST

THE-SCIENTIST.COM

VOLUME 35 ISSUE 3

HURT BY NOISE

Features

ON THE COVER: © SCIENCE PHOTO LIBRARY, STEVE GSCHMEISSNER

26

34

Sound from cars, aircraft, trains, and other man-made machines is more than just annoying. It increases the risk of cardiovascular disease.

Several labs have reported the formation of bacterial nanotubes under different, often contrasting conditions. What are these structures and why are they so hard to reproduce?

BY THOMAS MÜNZEL AND OMAR HAHAD

BY SRUTHI S. BALAKRISHNAN

Hurt by Noise

What’s the Deal with Bacterial Nanotubes?



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JUNE 2021

Department Contents 18

11

FROM THE EDITOR

The Psychology of Panic

The recent news of consumers hoarding gasoline in the face of a brief closure of one of the world’s biggest petroleum pipelines is just the latest episode of panic buying since the COVID-19 pandemic started. BY BOB GRANT

16 CRITIC AT LARGE Comparing Coronaviruses

In addition to continued scrutiny of SARS-CoV-2, research on similar pathogens could aid in the fight against the COVID-19 pandemic and future disease outbreaks. BY NICOLA PETROSILLO

xx

18 NOTEBOOKS As firefly tourism grows, so do threats to the flashy insects; projects aimed at collecting big data about the ocean expand our view of marine life

42

42 THE LITERATURE

at City of Hope Orange County. Opening in 2022.

RADIM SCHREIBER; © JULIA MOORE, WWW.MOOREILLUSTRATIONS.COM; COURTESY OF OPENBIOME

Lennar Foundation Cancer Center

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Fungi squeezed through microchannels offer clues to cell growth; broken heart syndrome linked to the brain; mucosal vaccines protect mice from viruses, cancer

45 SCIENTIST TO WATCH Adriana L. Romero-Olivares: Fungus Tracker BY AMANDA HEIDT

46 BIOBIZ Making Poop Profitable

With multiple microbiota therapeutics in the pipeline for recurrent Clostridium difficile infection, clinicians foresee a shift in treatment options for the condition.

xx xx  46

BY SHAWNA WILLIAMS

51

ANSWER

READING FRAMES

The Brains Behind the Brain

Improving the function of inhibitory neurons could be key to developing more-effective treatments for a variety of brain disorders. BY LAUREN AGUIRRE

52 FOUNDATIONS Leader of the Pack, 1903–1994 BY LISA WINTER IN EVERY ISSUE

9 13

CONTRIBUTORS SPEAKING OF SCIENCE



PUZZLE ON PAGE 13

S P UR S E P H P E RM I A A I N L A V A J E B P E R I OD E X L O T SW I A Y O GE C KO I H D CROS S E

A M N I O T I C F L U I D

D A R M Y H A N C T S E Z N E R O

P T E D U E A R S H I I NN E S C A B L E I OV UM O O QU A L A T RC A S

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JUNE 2021

ONLINE AT THE-SCIENTIST.COM:

Hybrid Animals Are Not Nature’s Misfits

Can Single Cells Learn?

In the 20th century, animals such as mules and ligers that had parents of different species were considered biological flukes, but genetic sequencing is beginning to unravel the critical role of hybridization in evolution.

A controversial idea from the mid-20th century is attracting renewed attention from researchers developing theories for how cognition arises with or without a brain.

AS ALWAYS, FIND BREAKING NEWS EVERY DAY ON OUR WEBSITE.

Coming next month • Tired of dancing to the tunes of international funders, and doubtful that long-promised national grants will come, a handful of African biomedical scientists have turned to private investors to bankroll their dreams of research autonomy. • Evolutionary biologists are tackling the complicated question of how bacteria living inside the digestive tract could drive adaptive genetic changes in their hosts. • One hundred years after BCG was developed, it’s still the only vaccine for tuberculosis, the infectious disease that has killed more people than any other. But there are new vaccines for this wily foe on the horizon. AND MUCH MORE

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

Pandemic Accelerates Trend Toward Remote Clinical Trials

Now more than ever before, recruiting patients for a research study doesn’t have to mean getting them to leave their homes.

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JUNE 2021

Contributors

PETER PULKOWSKI; OLIVER RÜTHER; ANNIE VALVA

Out of high school in the south of Germany, Thomas Münzel entered the medical field as a nurse. But a couple years later, after his father died of a heart attack at the age of 55, Münzel decided he wanted to become a doctor. He was 30 years old when he completed med school at Albert Ludwig University of Freiburg in 1985—too old, colleagues told him, to embark on a research career. But he got a stipend to spend two years at the University of Freiburg studying the regulation of coronary artery tone, and then another stipend to travel to David Harrison’s lab in the division of cardiology at Emory University in Atlanta. There, he studied nitrate tolerance and endothelial function, which became the focus of his research when he returned home to Germany a few years later. He became as assistant professor at the University Medical Center Hamburg-Eppendorf in 1995. Nearly a decade later, when he accepted a position as chief of cardiology at the University Medical Center at Johannes Gutenberg University of Mainz, he moved very close to Frankfurt Airport and had his “first experience with this incredible aircraft noise.” This got him thinking about the associations that had been made between noise and cardiovascular disease, among other health ailments. A few years later, when the airport opened a new runway that resulted in planes flying directly over his house, Münzel decided to refocus his research on the topic. “I said [to myself]: ‘I have to start studying noise and explain to people why noise is endangering us.’” Münzel’s postdoc, Omar Hahad, used to study psychology, earning a master’s in the subject in 2016. But his exposure to basic science as part of his psychology studies drew him in, and in 2017, he joined Münzel’s lab for his PhD studying noise annoyance and its effect on vascular function. “Today, we know that mental stress plays a fundamental role in literally every disease,” Hahad says. “Thus the leap from psychology to biology was not that big for me.” He finished his doctorate degree last year but stayed on in Münzel’s lab, and also started to work at the Leibniz Institute for Resilience Research, a multidisciplinary center focused on examining mechanisms of resilience that allow people to be resistant to stress. On page 26, Münzel and Hahad write about the link between noise and coronary heart disease, and specifically, the mechanisms by which exposure to noise can cause changes in the vasculature that lead to cardiovascular dysfunction. Nicola Petrosillo is no stranger to the front lines of an infectious disease outbreak. Serving in his post as director of the clinical and research department at Italy’s Lazzaro Spallanzani National Institute for Infectious Diseases, he was responsible for the care of SARS patients in 2004, and in 2014 ran an Ebola hospital in Lagos, Nigeria. Most recently, he has attended to patients suffering from COVID-19 in Italy. At the same time, he is studying SARSCoV-2, the virus that causes the disease. He says that his experiences with patients over the past year and a half have left an indelible mark on him as a healthcare provider. “The most difficult thing was to accept that aged patients would die alone without comfort, without their son, without their daughter, without anyone during their last minutes,” he tells The Scientist. “So [doctors and nurses] were everything. We were father, mother, son, daughter, priest, psychologist.” Petrosillo writes on page 16 that in addition to learning from the intense research focus on SARS-CoV-2, scientists can draw lessons from studying other coronaviruses, namely SARS-CoV and MERS-CoV, to help combat our current and future pandemics. Considering structural similarities and differences between viral agents as well as the epidemiology of past viral disease outbreaks, he says, will help researchers hone treatment, vaccination, and public health measures and ultimately save lives.

It was her freshman year of college when Lauren Aguirre realized she wanted to be a science communicator. In talking to a science reporter and editor for The New York Times, “I thought, ‘What a great job. You just get to talk to scientists and sort of get the highlights reel of their life,’” she recalls. Aguirre transferred from the University of Massachusetts, Amherst, to MIT, which had a science writing program. After graduating in 1986, she went on to tell science stories as a producer at the PBS program NOVA. In 2017, Aguirre began to dig deeper into the strange story of a cluster of cases of memory loss in people who had survived opioid overdoses. Intrigued, she quit her job to write a book about the phenomenon and what it reveals about memory more generally and about another memory-robbing condition, Alzheimer’s disease. On page 51, she writes about the role of inhibitory neurons in coordinating brain activity. Her favorite part of reporting the book, The Memory Thief, was interviewing scientists and “going deeper than the highlights reel. And they’re giving you the outtakes and the bloopers, the blind alleys that they went down, and the obstacles and the things that they had to do again and again and again,” she says. “And that is just much more interesting.” 06.2021 | T H E S C IE N T IST

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FROM THE EDITOR

The Psychology of Panic The recent news of consumers hoarding gasoline in the face of a brief closure of one of the world’s biggest petroleum pipelines is just the latest episode of panic buying since the COVID-19 pandemic started. BY BOB GRANT

ANDRZEJ KRAUZE

A

s I penned this editorial in mid-May, the internet was abuzz with news and images of people in some parts of the US East Coast panic-buying large volumes of gasoline. The behavior was driven by the closure of the Colonial Pipeline, the largest petroleum artery in the US, after its operator was targeted by a ransomware attack perpetrated by Russian hackers. Although the pipeline was closed for less than a week, individuals filled both approved gasoline containers and improvised ones with gas in nervous anticipation of a dearth of the crucial resource. Last year, in the early days of the COVID-19 pandemic, shoppers similarly rushed to hoard obscene amounts of toilet paper and cleaning products, their consumer drive shifted into high gear by a looming catastrophe. As COVID-19 swept through communities and workplaces, infecting tens and then hundreds of thousands around the world and killing thousands every day, high among citizens’ and retailers’ worries was that the pandemic would disrupt supply chains. History is littered with similar examples of panic buying, large and small. Humans have a tendency to gorge themselves on critical resources as crises approach, even if facts on the ground don’t necessarily warrant such panic. What is it about our psychology that makes us vulnerable to such behavioral swings? Sadly, the scientific literature surrounding this phenomenon is somewhat lacking. Most of the papers that do address it were spawned by panic buying that resulted from the start of the COVID-19 pandemic. According to a review paper published in May of last year, four factors likely drive this impulse: the perception of a coming crisis and corresponding resource scarcity, fear of the unknown, coping behavior triggered by control deprivation, and socialpsychological factors including social network dynamics. While it is easy to deride as illogical the people seen in viral images stocking up on toilet paper, gasoline, or hand sanitizer, the urges undergirding this behavior are intimately familiar to anyone who has lived through the past 18 months. Ironically, it is panic buying that can break the very supply chains that are perceived to be imperiled by crises such as pandemics. In the COVID-19 era, there have been shortages of particular goods, and some businesses have suffered immensely due to the disruption the outbreak has wrought. But arguably, panic buying has been more central to breaking supply chains and causing some shortages than the actual disease spurring the disruptions.

As the authors of the aforementioned review suggest, better understanding of the biology behind panic buying “offers some implications for health professionals, policy makers, and retailers on implementing appropriate policies and strategies to manage panic buying.” Putting an end to this practice can help to insulate the supply chains that COVID-19 has revealed as more fragile than they should be. Watching recent events unfold, another thought that occurred to me was how impressively some of the people navigating our new reality have avoided panic, maintaining a calm and focused course. One of these people is Nicola Petrosillo, this month’s Critic at Large. A veteran of infectious disease outbreaks—attending to patients in Italy during the SARS epidemic of the early 2000s and running an Ebola hospital in Nigeria as that viral scourge ravaged western Africa in the past decade— he has seemingly never wavered from a logical and scholarly approach. Even as Petrosillo, an infectious disease physician and researcher at Rome’s Lazzaro Spallanzani National Institute for Infectious Diseases, cared for COVID-19 patients last year and saw his hospital fill to bursting with sick and dying people, he retained his trust in science. On page 16, he writes about the utility of studying not only SARS-CoV-2 but also other coronaviruses that have sparked deadly outbreaks in order to get a more complete sense of how to end this pandemic and avoid the next one. It’s this devotion to logic, even in the face of intense emotional turmoil, that I feel is key to steering humanity in the right direction now and in the future. Here’s hoping that we find a way, as a society, to make such calm reason widespread as we stare down crises that come our way. g

Editor-in-Chief [email protected] 06.2021 | T H E S C IE N T IST 1 1

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Combating climate change— it’s not optional. It’s essential at EPA. We will move with a sense of urgency because we know what’s at stake.

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—US Environmental Protection Agency (EPA) Administrator Michael Regan, announcing the resumption of the agency’s focus on climate change (March 17). Last month, the EPA updated its Climate Change Indicators website with data from a delayed 2017 report, which states for the first time in the agency’s history that humans are, at least in part, driving climate change.

It is thinkable that the investigation of the behaviour of migratory birds and carrier pigeons may some day lead to the understanding of some physical process which is not yet known. —Albert Einstein, in a recently rediscovered letter dated October 18, 1949, to radar researcher/actor Glyn Davys. The letter was published in full on May 13.

© JONNY HAWKINS

BY EMILY COX AND HENRY RATHVON

ACROSS

DOWN

1. Calcaneal outgrowths 4. Avoided extinction? 8. Last period of the Paleozoic 9. Site of paludal events 10. Output of Karangetang 11. German astronomer Kepler 13. Conception of Mendeleev (2 wds.) 15. Figure ultimately associated with NaCl (2 wds.) 17. Female gamete 20. Sticky-footed lizard 21. Like sides of a scalene triangle 22. Hybridized 23. Predators in black and white

1. Component of a calyx 2. Where salmon go to spawn 3. Tibia’s setting 4. Liquid in which a fetus is cushioned (2 wds.) 5. Aggressive predatory foragers (2 wds.) 6. City with a carbon-dated shroud 7. Split along a natural line, as seedpods 12. Shrubs often used in hedging 13. Occurring in the open sea 14. Nest of live bodies made by 5-Down 16. Danish astronomer Brahe 18. Undergoes ecdysis 19. What Fibonacci called “zephyrum” Answer key on page 5

06.2021 | T H E S C IE N T IST 1 3

ONDEMAND

ORIGINALLY AIRED

TUESDAY, APRIL 27, 2021 TOPICS COVERED • Targeting non-small cell lung cancer using a cell carrier system to deliver an oncolytic and immunostimulatory adenovirus • Efficacy of oncolytic virotherapy in 3D cell culture and in vivo

Chimeric antigen receptor (CAR) T cells hold great promise for targeting and fighting cancer. However, their efficacy in solid tumors has been below expectations, as the tumor microenvironment (TME) limits their infiltration and produces a range of immune-inhibitory molecules to deactivate T cells. Researchers are developing new strategies to enhance CAR T cell therapy for solid tumors by disrupting the TME and promoting T cell persistence. In this webinar brought to you by IsoPlexis, Katie McKenna will discuss how she uses CAR T cells in combination with mesenchymal stromal cells that deliver engineered adenoviruses to stimulate the immune response in the TME. WATCH NOW! www.the-scientist.com/IsoPlexis-solid-tumor-adenovirus-therapy

• Assessing T cell functionality with singlecell proteomics

KATIE MCKENNA, PHD Postdoctoral Fellow Center for Cell and Gene Therapy Baylor College of Medicine Texas Children’s Hospital Methodist Hospital

TUESDAY, MAY 18, 2021 TOPICS COVERED • How single cell approaches help researchers characterize human cerebral cortex development • The role of ionic flux in in utero developmental processes

WATCH NOW!

• The functional role for a sodium channel subtype and a sodium/potassium ATPase pump in human neocortical folding

www.the-scientist.com/brain-development-using-single-cell-technologies-10x

WEDNESDAY, APRIL 28, 2021 TOPICS COVERED • The scientific questions that FACS can help answer • How to prepare cellular samples for optimal FACS results

Fluorescence-activated cell sorting (FACS) is an important tool for gaining deep biological insights. By gathering specific cells of interest, scientists can elucidate critical cellular functions through downstream assays. However, to obtain viable cells for subsequent assays that still maintain their physiological properties, cells need to be prepared properly and handled carefully. In this webinar, brought to you by BD Biosciences, flow cytometry core laboratory directors Dagna Sheerar and David Haviland will discuss the key factors impacting flow cytometry sorting experiments and how to obtain the best cell samples possible.

www.the-scientist.com/optimizing-fluorescence-activated-cell-sorting-bd

ORIGINALLY AIRED

WEDNESDAY, MAY 19, 2021 TOPICS COVERED • Securing the antibody supply chain

Scientific Liaison Research Solutions at BD Biosciences

DAVID HAVILAND, PHD Director Flow Cytometry Core Houston Methodist Research Institute

Manager Carbone Cancer Center Flow Cytometry Laboratory University of Wisconsin – Madison

For scientific and diagnostic laboratories, a secure supply chain for high-quality reagents is critical for weathering unforeseen circumstances such as the COVID-19 pandemic. Historically, recombinant antibody technology has been reserved for companies developing novel biologics. The technology mitigates manufacturing and supply risks by “digitizing” antibody assets and guaranteeing long-term reproducible production. Recombinant technology also increases the ability to engineer existing antibodies for any specific biophysical requirements that might arise, as producing antibodies by transient transfection enables rapid reformatting.

• The importance of recombinant antibody technology during the COVID-19 pandemic

In this webinar, brought to you by Sartorius, Nicholas Hutchings will describe how recombinant antibody technology came to the rescue during the COVID-19 pandemic. COVID-19 antibodies were quickly produced and tested in a variety of formats so that researchers would have the tools they needed to investigate the virus and develop diagnostic assays and treatments. This process can be mimicked for future emergency scenarios to speed up pandemic responses.

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Comparing Coronaviruses

SARS-CoV-2 belongs to a family of viruses that includes pathogens responsible for recent and ongoing epidemics.

In addition to continued scrutiny of SARS-CoV-2, research on similar pathogens could aid in the fight against the COVID-19 pandemic and future disease outbreaks.

C

OVID-19 is not humanity’s first brush with a coronavirus outbreak. A related pathogen, SARS-CoV, first emerged in Foshan, China, in November 2002. In February 2003 the virus was transported to Hong Kong, and from there, severe acute respiratory syndrome (SARS), the disease it causes, spread globally. By May 2004, that epidemic was quelled. Almost a decade later, in April 2012, the first cases of Middle East respiratory syndrome (MERS) occurred in Jordan. Countries in the region hosted persistent epidemics, and cases of MERS popped up in countries outside the Middle East. We can learn a lot about SARS-CoV-2 by comparing and studying the characteristics of these similar coronaviruses and the outbreaks they fueled. SARS-CoV-2 belongs to the diverse family of coronaviruses that are enveloped, single-stranded RNA viruses. Among the four genera (alpha, beta, gamma, and delta), alpha and beta coronaviruses are the most relevant to public health due to their propensity to cross animal-human barriers, thus becom-

1 6 T H E SC I EN T I ST | the-scientist.com

ing human pathogens. SARS-CoV, SARS-CoV-2, and MERSCoV are all beta coronaviruses with high morbidity, mortality, and transmissibility. Other human coronaviruses, of both the alpha and beta variety, are responsible for up to one-third of common cold cases and sometimes cause gastroenteritis. SARS-CoV, SARS-CoV-2, and MERS-CoV all consist of nonstructural replicase proteins and four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The N protein stabilizes the RNA genome, and the S, E, and M proteins together create the viral envelope. Phylogenetic analysis shows that SARS-CoV-2 belongs, together with SARS-CoV and SARS-like coronaviruses isolated in China from horseshoe bats between 2015 and 2018, to a different clade from MERS-CoV, and it is more closely related to the bat SARS-like coronaviruses than to SARS-CoV. Accumulating evidence based on genomic analyses suggests that SARS-CoV-2 shares with SARS-CoV the same human cell receptor, angiotensin-converting enzyme 2

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(ACE2)—analysis of receptor affinity shows that SARS-CoV-2 binds ACE2 more efficiently than SARS-CoV does—while MERS-CoV uses dipeptidyl peptidase 4 (DPP4) to enter host cells. But pathogenicity is different among the three viruses. The pathogenesis of SARS-CoV-2 and SARS-CoV is related to an immune system phenomenon involving a sharp increase in inflammatory proteins called a cytokine storm, whereas MERS-CoV’s proteins target host interferons to inactivate natural killer cells. In addition to initiating cytokine storms, SARS-CoV-2 promotes various cell death programs, such as pyroptosis, apoptosis, and necrosis, which may contribute to COVID-19 pathogenesis. In the immediate future, an indepth study of these peculiarities of SARS-CoV-2 requires novel approaches—i.e., omnigenetics, network immunological and biological approaches, etc.—to identify intrinsic factors (genetic risks, immune response kinetics, and other determinants) and biomarkers associated with COVID-19 severity. Although it changes rapidly, as per a July 2020 paper, the COVID-19 case fatality rate was 4.4 percent, compared with 9.5 percent and 34.4 percent for SARS and MERS, respectively. This can be partly explained by the fact that the case fatality rates for both MERS-CoV and SARS-CoV infections may be overestimates of the true mortality rates, as mild cases of SARS and especially MERS may have been missed by surveillance systems at the time. Regarding differences in transmissibility, a metric used to describe this spread is the basic reproductive rate (R0), defined as the average number of secondary transmissions from one infected person. According to a paper published last year, the R0 estimates for SARS-CoV-2, SARS-CoV, and MERS-CoV are on average 2.5, 2.4, and 0.69, respectively. The incubation periods range from 4 to 11, 2 to 7, and 2 to 14 days for SARS-CoV-2, SARS-CoV and MERS-CoV, respectively. Unlike SARS-CoV and MERS-CoV, SARS-CoV-2 uses multiple modes of transmission, and its structure is optimized for different environmental conditions. During the course of the pandemic, SARS-CoV-2 has undergone genomic rearrangements, resulting in the new variants that have appeared in patients around the world—an important means of immunological escape. For the past year and a half, much scientific research has been devoted to COVID-19, but many questions remain unanswered: Why is COVID-19 transmitted so quickly? Do some specific features of SARS-CoV-2’s structure play a role in its rapid spread? Are any host or environmental factors responsible for the different course of COVID-19 compared to other coronavirus diseases? Has the knowledge accumulated during

the pandemic advanced our efforts to fight COVID-19? Researchers can answer these crucial questions by continuing to study the intricacies of SARS-CoV-2. But scientists should also consider the structure and behavior of the virus’s deadly cousins and the outbreaks they sparked. Already, the emergence of SARS-CoV-2 variants has increased transmissibility of the virus up to 50 percent above that of the original strain that emerged in Wuhan. Concurrently, hospitalizations and death rates associated with these variants have risen across all age groups, particularly in older patients with comorbidities. A revised R0 estimate for these variants has increased to around 3.5. Intensively studying SARS-CoV-2 is crucial, especially in light of the fact that the virus continues to change and adapt as it lingers in human populations around the globe. But scientists should keep in mind that SARS-CoV-2 belongs to a family of viruses that includes pathogens responsible for recent and ongoing epidemics. Understanding the evolutionary, structural, and functional relationships among coronaviruses can facilitate the prevention or mitigation of the next pathogen poised to cross over from animals to humans. g Nicola Petrosillo is director of the clinical and research department at Italy’s Lazzaro Spallanzani National Institute for Infectious Diseases in Rome. Reach out to him at [email protected].



11.2020 | THE SCIEN TIST 1 7

An hour’s drive southwest of Bangkok, Thailand, tucked into a curve of the Mae Klong River, lies the village of Amphawa. Until recently, tourists flocked here to watch a spectacular evening light show. Thousands of male Pteroptyx malaccae fireflies would gather in the three-storytall mangrove trees that line the Mae Klong and flash in synchrony. “It looks like a big Christmas tree with lots of tiny lights,” says Anchana Thancharoen, an entomologist at Kasetsart University in Thailand who has studied fireflies for more than two decades. The district government started promoting firefly tourism in Amphawa back 1 8 T H E SC I EN T I ST | the-scientist.com

in 2004. Within just a few years, hundreds of motorboats were zooming up and down the river each night. New hotels, restaurants, and roads transformed the “quiet, peaceful province into an urban area,” says Thancharoen. By 2014, due to light pollution and loss of habitat, firefly numbers had fallen by about 80 percent, all but extinguishing the dazzling displays. These days, most tourists visit Amphawa not for fireflies, but to shop at the floating markets for food and souvenirs. It’s a pattern that Thancharoen and other firefly researchers worry could be repeated as the popularity of firefly watching grows worldwide. Thancharoen says she hopes Amphawa’s mistakes serve as a lesson for other sites looking to capi-

LIGHT SHOW: A group of tropical fireflies (Pteroptyx malaccae) illuminate a tree in Thailand.

talize on local invertebrate fauna—before it’s too late. Fireflies—or lightning bugs, depending on where you’re from—are actually beetles in the family Lampyridae. Generated by a chemical reaction in light-producing organs called lanterns, the green or yellow flickers are the insects’ elaborate courtship displays. It’s “the love language of fireflies,” explains Thancharoen. Although the females and larvae of some species produce light, it’s usually the males that put on the flashiest shows. The practice of watching this spectacle has a long history in some countries such

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Mind the Fireflies

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Notebook

as Japan, says Sara Lewis, an evolutionary ecologist at Tufts University who studies the sex lives of fireflies. But in recent years, “firefly tourism really seems to be taking off, partly driven by the popularity of the images that people are taking” and sharing on social media, she says. The phenomenon is part of a larger trend of insect-related tourism—or entomotourism. “There’s been a tremendous growth in insect festivals, some of them are amazingly large,” says Glen Hvenegaard, an environmental scientist at the University of Alberta. Every year, tens or hundreds of thousands of tourists swarm to monarch butterfly migratory sites in Mexico, glowworm caves in New Zealand and Australia, woolly bear caterpillar festivals in the US, and insectariums across the world. Through interviews, surveys, and internet searches, Lewis, Thancharoen, and their colleagues recently quantified global tourism for fireflies, specifically. The researchers found that firefly tourist destinations are dotted across 13 countries in North America, Asia, and Europe. At smaller sites such as the Pennsylvania Firefly Festival, just 1,000 or so people come to watch Photinus carolinus displays, while some places in Taiwan and South Korea draw as many as 200,000 tourists each season. In 2013, about 51,000 tourists visited the tiny town of Nanacamilpa in southeastern Mexico to watch the synchronous spectacle of Photinus palaciosi that occurs for just two weeks each year. By 2019, that number had grown to more than 120,000, says study coauthor Tania López Palafox, a graduate student in the department of evolutionary biology at the Instituto de Ecología of the Universidad Nacional Autónoma de México who works on this species. The researchers’ study is a “timely” effort to understand threats to the beetles and encourage sustainable practices, notes David Merritt, an entomologist at the University of Queensland who was not involved in the work. “It gives managers of tourism and environmental managers something to work from,” he adds. All in all, the researchers estimate that more than 1 million people traveled to

HANGING ON: The females of many firefly species lack wings, making them especially vulnerable to trampling in areas with lots of human activity.

watch fireflies around the world in 2019. “That really knocked our socks off,” says Lewis. “It’s great for the tourists—they get this amazing experience—and it’s great for local communities, which in many cases are getting a substantial economic boost.” But tourism isn’t necessarily great for the beetles, which, like many insects, are facing global declines. “We would love to make it a win-win-win situation, including a win for the fireflies,” she adds. To ensure that firefly populations thrive even as tourist numbers grow, it’s crucial to protect fireflies at all stages of

The other main threat to the bioluminescent beetles at tourist sites is light pollution, which interferes with the fireflies’ courtship displays, spoiling their chances of finding mates, says Thancharoen. This means that artificial lights from buildings, streetlights, and cars should be minimized at firefly sites, and tourists should refrain from using cellphones, flash photography, and flashlights. “We know enough about . . . the things that fireflies need to survive to be able to protect species against some of the threats associated with tour-

There’s been a tremendous growth in insect festivals, some of them are amazingly large. —Glen Hvenegaard, University of Alberta

the insect lifecycle Lewis, Thancharoen, and their colleagues say. In Amphawa, motor oil polluted the river, and waves generated by the boat traffic washed away the riverbanks, destroying habitat for P. malaccae larvae. The researchers suggest that tours use nonmotorized or electric boats to minimize the impacts on species with water-dwelling larvae. At sites with species that have subterranean larvae, visitors should stick to designated paths or walkways to avoid compacting soil and trampling the insects.

ism,” says Lewis. But, she adds, protecting the insects can be complicated by the social and economic factors unique to each location. In Amphawa, “there were a lot of conflicts between what fireflies needed, what the local community needed, and what the tour operators were doing.” During the peak of Amphawa’s popularity, 200 motorboats zipped tourists up and down the river for hours each night, sometimes until midnight, prompting one fed-up resident to cut down a firefly display tree, says Thancha06.2021 | THE SCIEN TIST 1 9

Swashbuckling Science Stéphane Pesant was on deck finishing up processing samples and preparing gear after a long day when he heard it: the exhalation of a sperm whale. It was a calm night back in 2011 in the waters off Peru, remembers Pesant, a marine biologist, and he was one of just two people still on deck when the giant animal surfaced. “We couldn’t see it at all, it was pitchdark,” he says. “But we could hear it, and it would dive and come back up.”

Speaking from his office on land in the UK, where he works for the European Bioinformatics Institute, Pesant tells The Scientist that he’s had many such moments of wonder aboard Tara, a 36-meter research sailboat now on its latest in a series of multi-year expeditions to learn more about the world’s oceans. Managed by the Paris-based nonprofit Tara Ocean Foundation with help from multiple scientific partners and corporate sponsors, the craft collects data that are made freely available to researchers. Samples collected aboard Tara over the past decade-plus have helped researchers identify hundreds of thousands of new viruses and catalog tens of millions of microbial genes. Tara’s latest mission, which began in December last year and is slated to run through 2022, is collecting water samples along the Pacific and Atlantic coasts of South America, and it will collect more en route back to France, in an effort to better understand microbes’ roles in the marine ecosystem. Pesant first became involved with the projects in 2009, with Tara Ocean, the first Tara mission to focus on microbes, and much of his work takes place from a desk back in Europe rather than on the ship. He manages how samples are cataloged on the boat and flow to the various labs on land that analyze data. That’s no small task: to be

OCEAN TRIP: Tara carries a team of scientists,

crew, writers, and artists around the world’s seas.

useful, each sample must be cataloged with metadata on the location and depth at which it was collected, along with other parameters. That involves barcodes and notes taken on paper, which must be scanned to PDFs. Then, the results of analyses by different labs need to be made accessible so that researchers can use others’ results to validate or otherwise build on their own. To Pesant, this centralized sample collection and tracking is a key advantage of Tara over large oceanographic excursions that carry multiple research teams, each with their own sample-collecting and data-handling protocols. “Although we are split as different labs, we act as one lab,” he explains. In 2015, for example, genomics researchers on one team identified different types of plankton that were frequently found together in water samples—and were able to use imaging results from another group to confirm the organisms’ symbiotic relationships with one another. Tara’s relatively small size—compared to, say, the US National Oceanic and Atmospheric Administration’s Okeanos Explorer, which measures 68 meters— does pose a challenge when it comes to finding space for samples, Pesant notes. The solution involves offloading the sam-

© ELOI STICHELBAUT; POLARYSE; TARA OCEAN FOUNDATION

roen. Although some locals reaped economic benefits from tourists, many of the new businesses were run by people from outside the community. To minimize these types of conflicts and ensure that local residents benefit from firefly tourism, it’s important to involve communities in designing, planning, and operating tourist sites, says study coauthor Harvey Lemelin, a social scientist at Lakehead University in Canada who says he became interested in insects after attending a dragonfly symposium. “I looked into those big multifaceted eyes . . . and I fell in love with them,” he recalls. He says that “inclusion of local people in terms of their stories, their narratives, their experience, their traditional knowledge is an essential component [of sustainable tourism].” By bringing these perspectives, local tour guides can help make entomotourism not just an entertaining activity, but an experience that teaches visitors to care about insects and their conservation, he says. In Amphawa, Thancharoen and others have installed educational displays about firefly biology and conservation and run training programs for tour operators, local residents, and children. Now that just a few boat tours shuttle tourists along the Mae Klong each night, firefly populations are slowly rekindling, says Thancharoen. “Fireflies have started to come back.”  —Asher Jones

© MAÉVA BARDY; TARA OCEAN FOUNDATION

NOTEBOOK

ples every three months and shipping them to the European Molecular Biology Laboratory in Heidelberg, Germany, via a specialized courier service. Crew members also rotate about every three months, while scientists tend to stay on the boat for about one month. The boat has writers and artists on board, too, for shorter stints. Tara Ocean wasn’t the first project to deploy a smaller vessel and uniform sampling and analysis protocols over diverse swaths of the world’s seas. The Global Ocean Sampling (GOS) Expedition, launched in 2004 by J. Craig Venter in the wake of his team’s work on a draft sequence of the human genome, took place on Venter’s 95-foot (29-meter) yacht. Yu-Hui Rogers, who ran the J. Craig Venter Institute’s genome center at the time, says GOS was driven by curiosity about “who’s out there” in the ocean, and by a more practical aim. Venter “really wanted to know what kind of new organisms out there actually have biological functions that can potentially be used in other areas,”

MISSION MICROBIOMES: The Tara team passes Isla Faro, in Patagonia, Chile, on its latest expedition, this time focusing on marine microorganisms.

such as bioremediation or energy production, she says. Like Pesant, Rogers, now at the nonprofit Jackson Laboratory, sees consistent sample-handling protocols as a plus of the single-boat approach. In GOS’s case, the samples were all analyzed onsite at

the Venter Institute. The work also carries risks, however. For instance, “[on] certain legs, we had our director of security on board with actual firearms to make sure that we didn’t get caught in a pirate situation,” she recalls. Sailing from Bermuda to the US Virgin Islands, “we had

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nine-foot waves the entire time. . . . By the time we got there, I had so many bruises on me, it’s unbelievable.” Other ways of collecting big data on ocean microbes have their own advantages and drawbacks. Working with multiple sample-collecting partners rather than a single vessel can cover more ground— or in this case, water, says Mitchell Sogin of the Marine Biological Laboratory at Woods Hole, who organized a different large-scale microbe-collecting project at around the same time GOS began. For that initiative, the International Census of Marine Microbes (ICoMM), “my notion was . . . we should just collect samples from as many different labs who were working with different cruises from around the world, so we engaged thirty or forty different labs as opposed to a single vessel,” and this

enabled a more comprehensive picture of the ocean than do projects carried out with a single boat, he says. On the other hand, with multiple teams involved, collecting standardized metadata to go along with each sample proved a challenge. “You can’t imagine how many different ways people can mess up longitude, latitude parameters.” ICoMM and GOS showed, Sogin says, that “diversity in the oceans was enormously greater than anyone had predicted.” These days, Tara’s current mission continues to uncover a wealth of new organisms, but it’s also aiming to address specific questions, says Eric Pelletier, a genomics researcher who spoke with The Scientist from aboard the ship. Those include assessing the influence of upwelling, in which nutrient-rich water flows from the ocean depths to the surface, on plankton communities.

Pelletier says Tara’s smaller size is an advantage when studying the mixing of different waters in the ocean, including upwellings, because it can be navigated to the locations of such events as identified by satellite images—something that wouldn’t be feasible with less-maneuverable oceanographic vessels. On a personal level, with only 14 people on board, he also enjoys getting to know everyone—scientists and crew alike. The work is constant, he says, with mariners pitching in with scientific work and scientists helping to sail the boat. Tara’s petite size is also one of the boat’s charms for Pesant. Thinking back to what made the experience with the sperm whale in 2011 so special, he explains, “Tara is very low on the water so you don’t dominate the sea, you feel part of it.”  —Shawna Williams

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soPlexis’ CodePlex Secretome is a fully-automated, costeffective solution for running an entire multiplexed proteomics workflow. The technology provides researchers a faster, optimized approach for generating multiplexed bulk cytokine data, while minimizing variability from user input. The CodePlex Secretome solution uses just 11 µL per sample replicate, an improvement over status quo multiplexed bulk analysis. With the modular design, users load eight to 64 samples per run. Only five minutes of hands-on time is required. All enzyme-linked immunosorbent assay (ELISA) procedures are performed automatically, and there is no need for additional washing and incubation stations, centrifuges, vortexers, or plate readers. The CodePlex family of chips can generate data on the same day, and the state-of-the-art IsoSpeak bioinformatics software

Researchers use CodePlex to shed light on mechanisms behind severe COVID-19 Many individuals infected with severe COVID-19 develop selflimiting disease and recover. However, some individuals develop severe respiratory disease marked by lung infiltrates and reduced oxygen saturation, which can progress to acute respiratory distress syndrome (ARDS), multi-organ failure, and death. The exact role of immune responses in severe COVID-19 has been unclear, because they have largely been studied in circulation. A better understanding of how airway and blood immune cells function together is urgently required to dissect disease mechanisms and to develop new treatment and prevention strategies.

features advanced visualizations. One IsoLight or IsoSpark system serves as a hub for single-cell and population proteomic analysis, allowing up to 32+ cytokines to be measured at a time. By contrast, typical multiplexed bulk analysis requires multiple steps and user interaction points, up to ten hours of hands-on sample preparation time, multiple systems to generate and analyze data, and manual user input for data analysis and visualization. The unique advantages of CodePlex Secretome were demonstrated in a study published in the journal Immunity. The results provide a dynamic view of ongoing respiratory and systemic immunity in severe COVID-19, revealing key roles for airway immune cells in both protective and inflammatory immune responses. These findings could have important implications for patient monitoring and treatment for COVID-19, as well as future infectious challenges to the respiratory tract.

IsoPlexis’ highly multiplexed functional proteomics characterized the cytokine and chemokine profile of severe COVID-19 airway cells and plasma.

To address this need, researchers leveraged IsoPlexis’ low volume bulk highly multiplexed solution and IsoSpeak software to investigate dynamic immune processes involved in severe COVID19. They examined the relationship between immune processes in the respiratory tract and in circulation using phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples obtained at multiple time points from COVID-19 patients. All 15 patients, who ranged in age from 14 to 84, were hospitalized in intensive care units, exhibited clinical features of ARDS, and required mechanical ventilator support and intubation. The samples were collected starting 24 to 36 hours after intubation and continued daily for up to 10 days. The researchers used IsoPlexis’ Human Adaptive Immune Panel for the CodePlex Secretome chip to characterize the cytokine and chemokine profile of these samples and compare them to samples from healthy subjects.

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Paving the way to clinical translation Based on the results, airway T cell measurements could be a useful biomarker to monitor patients and stratify their risk. In addition, promoting lung-localized immune responses could be beneficial in vaccines. Respiratory targeting could be considered for the immunocompromised or elderly, or for individuals who are otherwise unable to develop effective antibody responses to currently available SARS-CoV-2 vaccines. This strategy could also be used as a booster for those at risk of infection due to frequent interactions with others through their living or work situations. Moreover, the findings suggest that targeting airway-derived cytokines such as CCL2 through CCR2 antagonists or other airwayspecific mediators may be more effective than treatments targeting systemic inflammation, either globally with steroids, or specifically with cytokine blockade, in reducing lung damage or even promoting recovery from ARDS in severe COVID-19. Overall, the study elucidates the roles of airway and blood immune cells in both protective and inflammatory responses to COVID19, providing novel insights that may enable the development of improved methods for monitoring and treating COVID-19. IsoPlexis’ highly multiplexed functional proteomics characterized the cytokine and chemokine profile of severe COVID-19 airway cells and plasma, providing functional insights. Obtaining these insights was possible with IsoPlexis’ platform, which highly multiplexes cytokines with walk-away automation, drastically reducing hands-on time. IsoPlexis provides key functional insights in a fraction of the time, on one integrated proteomics hub.

Reference  1. P. Szabo et al., “Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19,” Immunity, 54(4):797-814.e6, 2021.

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IsoPlexis’ highly multiplexed CodePlex Secretome provides measurements in paired airway and plasma samples from COVID-19 patients and demonstrates MCP-1, MIP-1α, and MIP-1β, granzyme B, IL-7, and TNF-β were significantly increased in airways compared to blood.1

This analysis revealed that pro-inflammatory chemokines and cytokines were prominent in the airways of patients with severe COVID-19, with only some of these proteins present in the blood. Moreover, the results suggest that airway T cells and myeloid cells, primarily monocytes and macrophages, may influence disease outcomes. Higher T cell frequencies in airways, but not in blood, correlated with younger age and survival from severe COVID-19, providing evidence that T cells in the respiratory tract may have a protective

role. By contrast, higher frequencies of myeloid cells in COVID-19 airways correlated with mortality and older age. IsoPlexis’ CodePlex Secretome revealed that these cells featured hyperinflammatory signatures, producing high levels of chemokines such as C–C motif chemokine ligand 2 (CCL2). In blood samples from the COVID-19 patients, CCL2 was not detected, but circulating monocytes expressed its cognate receptor: C-C chemokine receptor type 2 (CCR2). In addition, aberrant CD163+ monocytes predominated over conventional monocytes in COVID19 blood samples and were found in corresponding airway samples and in damaged alveoli. Taken together, these results suggest that airway myeloid cells recruit aberrant monocytes from circulation into the respiratory tract via a CCL2-CCR2 chemokine axis, thereby perpetuating inflammatory responses and driving lung damage in severe COVID-19.

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Sound from cars, aircraft, trains, and other man-made machines is more than just annoying. It increases the risk of cardiovascular disease. BY THOMAS MÜNZEL AND OMAR HAHAD



06.2021 | THE SCIEN TIST 27

Indirect harm In 1950, Karl Kryter, then the director of the Operational Applications Laboratory at the Air Force’s Air Research and Devel28 T H E SC I EN T I ST | the-scientist.com

living in the vicinity of Düsseldorf airport in Germany with a window tilted open. Using questionnaires, blood analyses, and physiological tests of endothelial function, we established that one night of simulated aircraft noise exposure reduced self-reported sleep quality, elevated circulating levels of stress hormones such as adrenaline, stiffened blood vessels, and caused vascular endothelial dysfunction, the latter two reflecting early subclinical signs of atherosclerosis and being independent predictors of future cardiovascular events and disorders.8 Importantly, comparing participants exposed to 30 versus 60 aircraft noise events per night revealed a dose-dependent worsening of endothelial function.9 Moreover, previous exposure to 30 aircraft noise events caused 60 events to have larger adverse effects on endothelial function. Thus, rather than any sort of habituation to the noise, there appeared to be a priming effect: prior exposure amplified the negative effect of noise on endothelial function.

opment Command in Washington, DC, emphasized the potential health effects of the so-called nonauditory effects of noise.5 He proposed that such effects are the result of the stimulation of the body’s neural systems that are not exclusively linked to audition, including the autonomic nervous system, which controls systemic responses and arousal reactions of an organism, and the cortical and subcortical brain centers responsible for cognitive performance. In 1968, Gerd Jansen from the Max Planck Institute of Molecular Physiology in Dortmund, Germany, provided evidence linking noise to cardiovascular problems. Examining 1,005 German industrial workers, Jansen reported the occurrence of physiological changes such as peripheral circulation issues, heart problems, and equilibrium disturbances, which were more pronounced in very noisy industries compared with less noisy industries.6 These early observations hinted that chronic noise exposure may cause cardiovascular disease, but it was unclear how. In 2003, Wolfgang Babisch, a senior research officer at the German Federal Environmental Agency, developed the noise reaction model, which describes two pathways for determining the adverse health effects induced by noise. In the first, known as the auditory/direct pathway, exposure to noise louder than 90–100 decibels (such as a jackhammer) causes inner-ear damage that can lead to hearing loss and tinnitus. In the second, the nonauditory or indirect pathway, low-level noise exposure of 50–60 decibels (such as a conversation) interferes with communication, concentration, daily activities, and sleep, resulting in annoyance, mental stress, and subsequent sympathetic and endocrine activation.7 It was the latter pathway that Babisch suspected was the central player for noise-induced cardiovascular effects. Specifically, he hypothesized that if the exposure is persistent and chronic, noise contributes to a pathophysiological phenotype that is characterized by increased stress hormone levels, high blood pressure, and accelerated heart rate. As a consequence, the body generates its own cardiovascular risk factors, including high cholesterol and glucose levels, increased blood viscosity, and activation of blood coagulation. If stress persists for years, cardiovascular diseases such as hypertension, coronary heart disease, heart failure, arrhythmia, and stroke can begin to manifest, along with mental stress or related disorders such as depression and anxiety, which are themselves known to negatively affect cardiovascular health.

Translational aircraft studies in people In 2013, to take a more controlled look at the effects of traffic noise, we and our colleagues conducted our first field study involving the exposure of healthy subjects to simulated aircraft noise overnight in their homes. On control nights, we simply had participants play a recording of normal background noise in their home on a standard portable audio system placed on their nightstands. On other nights, we had them play a looped recording of aircraft noise taken in the bedroom of a resident

noise itself that’s a problem, but one’s emotional reaction to it. In 2019, Michael Osborne of Massachusetts General Hospital and colleagues demonstrated that, after five years of exposure to transportation noise such as that caused by road and aircraft traffic, higher activity in the amygdala, a brain region involved in emotional processing, stress perception, and emotional reactions, is linked with an increased risk of heart attack, stroke, heart failure, and death through mechanisms involving heightened arterial inflammation. 13 Noise annoyance, it seems, is a so-called effect modifier, meaning that the cardiovascular side effects of noise are greater in people who are getting annoyed and therefore experiencing increased stress responses compared with those who are not. Whatever the cause, evidence is now accumulating to demonstrate that noise pollution is resulting in endothelial dysfunction, ultimately leading to high blood pressure, arrhythmia, heart attack, heart failure, and stroke.14

Molecular mechanisms

Exposure to transportation-related noise is related to the annual loss of up to 1.6 million cumulative years of healthy life among people in Western Europe.  —World Health Organization, data from 2011

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M

ore than 100 years ago, the German physician and Nobel Prize winner Robert Koch predicted that “one day mankind will have to fight the burden of noise as fiercely as plague and cholera.” He was right. While many sounds in our environments are quite pleasant, noise, defined as unwanted sound, has the potential to cause real damage to our bodies and minds. The principal sources of environmental noise are transportation and industrial operations. Since Koch’s time, researchers have come to recognize that such noise can cause sleep disturbances, elicit anger, and trigger conditions such as tinnitus and coronary heart disease caused by reduced blood flow to the organ. Noise can also lead to memory and learning impairments in children. In 2011, the World Health Organization (WHO) concluded that exposure to transportation-related noise—specifically from aircraft, vehicles, and trains—is responsible for the annual loss of up to 1.6 million cumulative years of healthy life among people in Western Europe. 1 The cardiovascular burden of traffic noise is particularly insidious, with annoyance reactions and sleep disturbances leading to an increased risk of heart disease. A 2015 report from the European Environment Agency linked exposure to car, truck, plane, and train sounds with nearly 1.7 million additional cases of hypertension, 80,000 additional hospital admissions, and 18,000 premature deaths due to coronary heart disease and stroke in Europe each year.2 A few years later, a metaanalysis conducted on behalf of the WHO supported these conclusions, with seven high-quality longitudinal studies collectively establishing that road traffic noise exposure was associated with an 8 percent increased risk of coronary heart disease.3 In addition to being associated with an increased incidence of coronary heart disease, noise may serve as an acute trigger of cardiovascular problems. For example, a study published earlier this year established that for nighttime deaths, noise exposure levels two hours preceding death were significantly associated with heart-related mortality.4 Despite these indications of the hazards of noise, research concerning adverse health effects of noise pollution is not well supported financially or politically, and the underlying mechanisms by which noise increases the risk of cardiovascular disease are not well understood. Our research group in the Department of Cardiology at the University Medical Center of the Johannes Gutenberg University of Mainz in Germany and others aim to uncover these pathophysiological processes. This will not only provide a method for quantifying the degree of physiological stress triggered by noise, but also help to identify novel pharmacological agents or noise mitigation measures that could be used to prevent, manage, or treat noise-induced disease.

A surprising result to come out of our first field study was that the adverse effects of nighttime noise on endothelial function were ameliorated by the administration of vitamin C, which we gave to some participants after noise exposure. Vitamin C is an antioxidant, a scavenger of oxygen-derived free radicals. Thus, this finding hinted that increased oxidative stress within the vasculature may be responsible for noise-induced endothelial dysfunction.

More recently, we exposed healthy subjects to simulated nighttime train noise and similarly found that one night of exposure greatly impaired sleep quality and endothelial function. In addition, proteomic analysis of participant blood samples revealed substantial changes in circulating proteins that pointed to a higher susceptibility to inflammation and blood clotting. Only a few other studies have provided mechanistic insight into the relationship between traffic noise exposure and cardiovascular disease. In 2017, Maria Foraster and her colleagues at the Swiss Tropical and Public Health Institute found, much as we did, that a decade of exposure to nighttime noise events, mainly related to road traffic noise, was associated with increased arterial stiffness in a cohort of 2,775 Swiss participants.10 That same year, a pooled analysis of more than 144,000 people in two large European cohorts from Norway and the Netherlands indicated that long-term exposure to road traffic noise was associated with higher levels of inflammation, blood lipids, and fasting glucose.11 Babisch proposed that annoyance reactions to noise may play an important role in the extent to which noise-exposed subjects develop cardiovascular disease.12 That is, it’s not the

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A

NOISE STRESS REACTIONS

NOISE-TRIGGERED PLAQUE RUPTURE

Fibrin Circulating cortisone Blood clot

Nighttime noise can disrupt sleep and cause cognitive and emotional responses via activation of the amygdala.

Red blood cell

Disrupted sleep can also activate the autonomic nervous system and the endocrine system, leading to increases in circulating levels of stress hormones such as cortisone.

Acute noise stress can cause a physical disruption of the plaque, leading to cardiovascular disease, including acute and chronic coronary syndrome, stroke, arrhythmia, arterial hypertension, and heart attack, plus mental health disorders such as depression and anxiety.

PLAQUE BUILDUP

ENDOTHELIAL DYSFUNCTION

NOISE DAMAGE PATHWAYS

Cholesterol Lipids

© LAURIE O’KEEFE

Epidemiological data have long linked exposure to noise such as aircraft, railway, or traffic sounds to increased risks of cardiovascular disease. And in recent years, experimental work has been revealing the biological mechanisms underlying that link. Specifically, researchers are finding that noise activates the brain’s limbic system, which plays a role in emotional regulation, the release of stress hormones into the blood, and controlling of the sympathetic nervous system. These stress responses can lead to cerebral and vascular inflammation, oxidative stress, and altered gene expression, sometimes culminating in endothelial dysfunction and cardiovascular disease.

Macrophage

Such chronic stress can cause high cholesterol, high blood glucose, high blood pressure, increased blood viscosity, and the activation of blood coagulation—all cardiovascular risk factors. Stress can also increase the permeability of the endothelium to inflammatory cells such as macrophages, leading to endothelial dysfunction.

Smooth muscle cells

If stress persists, a buildup of cholesterol and immune cells below the endothelium leads to plaque formation and eventually smooth muscle cells and lipids accumulate.



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Planning for a less noisy future

Unlike other major cardiovascular risk factors, noise pollution cannot be treated by doctors and patients but rather by politicians.

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

Thomas Münzel is the chief of the Department of Cardiology at the University Medical Center of the Johannes Gutenberg University Mainz in Germany. Omar Hahad is a postdoctoral researcher in the same department. Both are members of the German Center for Cardiovascular Research partner site in RhineMain, Mainz, Germany. They can be reached at [email protected] and [email protected].

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References

© SHUTTERSTOCK.COM, IVECTOR

To further elucidate the molecular mechanisms responsible for nonauditory noise-induced cardiovascular side effects, we established a novel mouse model and employed various noise pollution protocols. In the first study, we exposed mice to simulated aircraft noise around the clock for four days and observed increased blood pressure and elevated concentrations of stress hormones such as cortisol, noradrenaline, angiotensin II, and dopamine, along with raised blood pressure, suggesting the animals were stressed.15 This was accompanied by endothelial dysfunction and increased production of reactive oxygen species (ROS) within the vascular wall. Blood vessels are lined with endothelial cells that produce powerful vasoconstricting and vasodilating substances such as the radical nitric oxide (NO.). But ROS—which are produced in cases of hypertension, high cholesterol, diabetes, chronic smoking, and other conditions that are risk factors for cardiovascular disease—attack and degrade NO., thus limiting its bioavailability. This leads to stiffer vessels, higher blood pressure, and the initiation of plaque buildup in arteries. It appeared that this might be the initial pathway by which noise causes cardiovascular damage. In addition to the increased production of ROS in the vasculature, our mouse study revealed that noise could trigger oxidative stress in the brain. In a subsequent study, which followed the same noise exposure protocols as the first mouse study, we

confirmed high ROS levels in the frontal brain region of mice and documented significant neuroinflammation in that area.16 This observation is particularly interesting because these cerebral effects may explain, at least in part, the impaired cognitive development seen in children exposed to noise. We identified two radical-forming enzymes, the phagocytic nicotinamide adenine dinucleotide phosphate oxidase isoform 2 (Nox2) and endothelial nitric oxide synthase (eNOS), as sources for increased ROS production in our noise-exposed mice. Nox2 is mainly found in inflammatory cells such as macrophages and monocytes, and the eNOS we detected tended not to be coupled with its cofactor or substrate. Under normal conditions, eNOS produces NO, which has important vasodilating and antiatherosclerotic effects, but after noise, the enzyme becomes “uncoupled”—it switches to a pro-atherosclerotic state, producing the reactive oxygen species superoxide (O2.-) instead of NO. Indeed, mice lacking the Nox2 gene suffered almost no ill effects from noise exposure, confirming oxidative stress as a key player in noise-induced cerebral and cardiovascular damage. We also detected a downregulation of genes encoding antioxidant pathways as well as evidence for more inflammation of the vasculature, which may further increase oxidative stress and thus may aggravate endothelial dysfunction and arterial hypertension. Importantly, we did not observe these outcomes in a control group of mice, which was exposed to white noise at the same volume as the animals in the aircraft-exposure group. This indicates that the sound pressure level per se is not causing the damage. Moreover, the effects were only seen when the mice were exposed to noise during the day, when the animals are normally sleeping, suggesting that impaired sleep quality, including frequent fragmentation of sleep and/or too-short sleep, is a driver of noise-induced adverse health effects. In all, the findings in humans and mice indicate that noise activates inflammatory and oxidative stress pathways in the vasculature and the brain, leading to endothelial and cerebral dysfunction. (See illustration on page 30.) This aligns with the pathophysiological pathways at play in traditional cardiovascular risk factors such as smoking, obesity, diabetes mellitus, and hypertension. These and the novel risk factor of noise appear to work similarly to increase cardiovascular risk.

While the mechanisms underlying the cardiovascular side effects of environmental noise remain an active area of investigation, experimental and epidemiological studies from the last several years clearly demonstrate that exposure increases the risk of disease. Unlike other major cardiovascular risk factors, however, noise pollution cannot be treated by doctors and patients but rather by politicians. Policies should work to bring noise exposure levels in line with the new guidelines developed by the WHO,17 which lowered the recommendations for mean daily noise sound pressure levels to 45 decibels for aircraft noise, 53 decibels for road traffic noise, and 54 decibels for railway noise, with even stricter limits for nighttime hours, in order to reduce the burden of disease from noise. Importantly, noise and air pollution have many of the same sources—aircraft, trains, and road vehicles. Research suggests that the direct and indirect social costs of noise and air pollution in the European Union could be nearly €1 trillion, accounting for premature death and disease. That far exceeds the costs caused by alcohol and smoking, which have been estimated to cost €50 billion– €120 billion and €540 billion, respectively. We must better understand the response to coexposure to noise and air pollution, as well as the synergistic effects of both exposures on surrogate measures such as blood pressure and diabetes. Other open questions include the effects of cardiovascular therapy on noise- and air pollution–related cardiovascular risks and the influence of noise on circadian rhythms. Finally, we will need to address the combined effects of noise and lifestyle factors such as diet, stress, and exercise to fully tackle the noise problem. g

7. W. Babisch, “Stress hormones in the research on cardiovascular effects of noise,” Noise Health, 5:1–11, 2003. 8. F.P. Schmidt et al., “Effect of nighttime aircraft noise exposure on endothelial function and stress hormone release in healthy adults,” Eur Heart J, 34:3508–14a, 2013. 9. J. Herzog et al., “Acute exposure to nocturnal train noise induces endothelial dysfunction and pro-thromboinflammatory changes of the plasma proteome in healthy subjects,” Basic Res Cardiol, 114:46, 2019. 10. M. Foraster et al., “Exposure to road, railway, and aircraft noise and arterial stiffness in the SAPALDIA study: Annual average noise levels and temporal noise characteristics,” Environ Health Perspect, 125:097004, 2017. 11. Y. Cai et al., “Long-term exposure to road traffic noise, ambient air pollution, and cardiovascular risk factors in the HUNT and lifelines cohorts,” Eur Heart J, 38:2290–96, 2017. 12. W. Babisch et al., “Noise annoyance—a modifier of the association between noise level and cardiovascular health?” Sci Total Environ, 452–53:50–57, 2013. 13. M.T. Osborne et al., “A neurobiological mechanism linking transportation noise to cardiovascular disease in humans,” Eur Heart J, 41:772–82, 2020. 14. T. Münzel et al., “Adverse cardiovascular effects of traffic noise with a focus on nighttime noise and the new WHO noise guidelines,” Annu Rev Public Health, 41:309–28, 2020. 15. T. Münzel et al., “Effects of noise on vascular function, oxidative stress, and inflammation: mechanistic insight from studies in mice,” Eur Heart J, 38:2838–49, 2017. 16. S. Kröller-Schön et al., “Crucial role for Nox2 and sleep deprivation in aircraft noise-induced vascular and cerebral oxidative stress, inflammation, and gene regulation,” Eur Heart J, 39:3528–39, 2018. 17. World Health Organization, “Environmental noise guidelines for the European Region,” 2018.

1. World Health Organization, “Burden of disease from environmental noise: Quantification of healthy life years lost in Europe,” 2011. 2. Danny Houthuijs et al., “Health impact assessment for noise in Europe,” ETC/ACM Technical Paper 2014/9 (Bilthoven, The Netherlands: European Topic Centre on Air Pollution and Climate Change Mitigation, 2015). 3. E. Van Kempen et al., “WHO environmental noise guidelines for the European region: A systematic review on environmental noise and cardiovascular and metabolic effects: A summary,” Int J Environ Res Public Health, 15:379, 2018. 4. A. Saucy et al., “Does night-time aircraft noise trigger mortality? A case-crossover study on 24 886 cardiovascular deaths,” Eur Heart J, 42:835–43, 2021. 5. K.D. Kryter, “The effects of noise on man,” J Speech Hear Disord Monogr Suppl, 1:1–95, 1950. 6. G. Jansen, “Effects of noise on health,” Ger Med Mon, 13:446–48, 1968.



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« MICROBIAL CONNECTIONS: Nanotubes can be seen extending

from E. coli in this scanning electron micrograph of a coculture of E. coli and Acinetobacter baylyi.

Not one to give up, Pospíšil struck up a collaboration with Imrich Barák of the Slovak Academy of Sciences. Barák had access to more sophisticated imaging technology, and Pospíšil says he hoped that he could use it to capture the elusive structures. Together, the researchers prepared Bacillus subtilis for microscopy using glass slides and coverslips, standard tools of the trade. One day, they saw that the cells were moving on the slide. To immobilize them, “we just decided to push the glass coverslip down,” says Barák. When they did, lo and behold, nanotubes began bursting forth from nearly every cell. Looking closer, Pospíšil and Barák found that the cells were dying. This observation, published last October, ran contrary to all the previously published work on nanotubes, which posited these structures as conduits between living, healthy cells.4 If nanotubes were indeed features of dying cells, then the suggestion that the structures were central to material transfers that seemed to play a role in microbial growth and survival was unlikely to hold true, says Barák. “We realized that probably this is the end of this nice story of different functional nanotubes.”

What’s the Deal with Bacterial Nanotubes?

What are bacterial nanotubes?

Several labs have reported the formation of bacterial nanotubes under different, often contrasting conditions. What are these structures and why are they so hard to reproduce?

I

n 2011, the microbiology community learned of a brand-new feature of bacteria: nanotubes.1 Scientists later showed that these membranous, hollow connections between bacteria allow the transfer of materials such as amino acids2 and toxins that inhibit growth.3 These tubes were unlike anything the researchers had seen before: in contrast to the conjugative pili that transfer genetic material during bacterial “sex,” the nanotubes were made of lipids, not proteins. They were also more promiscuous than pili, often linking microbes of different species, and even connecting bacteria with mammalian cells. It was starting to look like bacterial nanotubes were long-overlooked features of microbiology.

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

Jiří Pospíšil, a graduate student at the Czech Academy of Sciences, was enamored with these novel bacterial structures—so much so that in 2016, two years into his PhD studying RNA polymerases, he started working on nanotubes on the side. But as he set out to replicate some of the studies that first visualized the nanotubes, he quickly ran up against a wall. Despite performing a host of different experiments, he could not reproduce the earlier results. Although he did occasionally spot nanotubes forming between cells, this happened very rarely and only under specific conditions, making Pospíšil question the ubiquity and importance of nanotubes in the microbial world.

CHRISTIAN KOST, OSNABRÜCK UNIVERSITY

BY SRUTHI S. BALAKRISHNAN

As with many scientific advances, bacterial nanotubes were discovered by accident. In 2008, Sigal Ben-Yehuda, a microbiologist at the Hebrew University of Jerusalem in Israel, and her then–graduate student Gyanendra Dubey were studying sporulation in B. subtilis. One of their experiments involved mixing two types of B. subtilis, one that expressed green fluorescent protein (GFP) and one that did not, and letting them grow together. When they observed the cells under a light microscope, they noticed weak fluorescence coming from some of the cells that originally did not have GFP but were lying close to cells that did. “I thought that [it was] a bleaching artifact,” says Dubey, who until recently worked as a researcher at the Institut Pasteur. Ben-Yehuda and Dubey performed a battery of tests to figure out what was going on. They did experiments using other fluorescent small molecules, such as calcein, and found that these also seemed to jump between cells, as did genetic material such as plasmids or RNA. Bacteria can exchange material using contactindependent systems, such as simple diffusion and vesicles, or contact-dependent systems, such as conjugative pili and secretion systems. But the scientists observed material-sharing even in strains of B. subtilis that were incapable of these well-known modes of transfer. “That made us think that there was some cytoplasmic exchange happening that we were [previously] not aware of,” says Ben-Yehuda. When she and Dubey examined B. subtilis with highresolution scanning electron microscopy (HR-SEM), they saw tubes extending from the bacteria to adjacent cells. “We could see them again and again and again,” says Ben-Yehuda. Surprised that these structures had not been reported before, they

dug through older papers for electron microscope (EM) images of bacteria. To their delight, they “could see them all over the place all the time,” says Ben-Yehuda. She surmised that bacterial nanotubes had been overlooked simply because no one really knew what they were. In 2011, the team published its first paper reporting the existence of bacterial nanotubes that acted as channels connecting bacterial cells.1 Until the discovery of bacterial nanotubes, the only known contact-dependent systems that could transfer material between bacterial cells were made of proteins, and researchers had shown these conduits to be very picky about the cells they connect to; conjugative pili, for instance, will only form between bacteria that carry specific plasmids and others that carry certain receptors. Ben-Yehuda’s group found that, unlike these protein-based systems, nanotubes are made almost entirely of lipids and do not seem to care about who they partner up with. Often looking like a string of connected vesicles, nanotubes vary in width and facilitate cytoplasmic connections between cells.5 The cytoplasmic link distinguishes nanotubes from outer membrane vesicles that package and secrete material from the space between the two membranes of Gram-negative bacteria. Curious about how widespread bacterial nanotubes are, BenYehuda’s team tested various other species of bacteria. The lab next door, led by Ilan Rosenshine, worked on enteropathogenic E. coli (EPEC), which attaches to its host cells to extract nutrition through hitherto unknown molecular machinery. Ben-Yehuda thought that it might be nanotubes. She approached Rosenshine, and asked him to share some EPEC samples with her. In 2019, they published two papers together, one reporting that EPEC did indeed produce nanotubes when attaching to host cells,6 and the other showing that nanotubes can connect Gram-positive and Gram-negative bacteria, and that the microbes require a set of five proteins, called the CORE complex, to do so.7 Neither EPEC nor B. subtilis cells that lacked this complex could produce nanotubes. “We couldn’t believe that all these different systems, from EPEC and from Bacillus, were the same,” says Ben-Yehuda. Meanwhile, several other labs were also hard at work documenting the presence of nanotubes in their own bacterial systems. In 2015, Christian Kost of the University of Osnabrück in Germany and his colleagues engineered two species of bacteria, E. coli and Acinetobacter baylyi, to each lack a specific amino acid. Paired populations of the microbes, with each of the pair lacking a different amino acid, would only survive if they grew together and exchanged amino acids. When the researchers tagged amino acids with fluorescent markers and observed them under a microscope, they saw that the amino acids were exchanged between the two cell types. They tracked the movement of these fluorescent markers over time and found that the transfer was happening through lipid-based tubes. Using EM, the scientists then confirmed that these were bacterial nanotubes.3 That same year, a paper from Marie-Thérèse Giudici-Orticoni’s group at the French National Centre for Scientific Research and Aix-Marseille University reported seeing nanotube-like struc

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We realized that probably this is the end of this nice story of different functional nanotubes. 

—Imrich Barák, Slovak Academy of Sciences

were indeed nanotubes. They concluded that the nanotubes might be making it easier for the bacteria to communicate, possibly promoting the cells’ ability to form biofilms in hostile environments. With so many labs having observed bacterial nanotubes under different conditions and in actively growing cells, Pospíšil was not anticipating that he would have any trouble doing the same.

Elusive structures When Pospíšil first set out to visualize bacterial nanotubes, he immediately noticed that the structures occurred at a much lower frequency than had been reported by Ben-Yehuda’s lab. “The greatest challenge to study nanotubes was actually to be able to detect nanotubes,” says Libor Krásný, Pospíšil’s advisor at the Czech Academy of Sciences in Prague. In fact, the researchers only found one nanotube for every 500 or so cells they scanned. “We [saw] something which looks like nanotubes, but in very, very low frequencies,” says Oldř ich Benada, head of the EM group in the Institute of Microbiology of the Czech Academy. “And we had to scan a lot of frames to find some of those structures.” Benada says he grew skeptical that nanotubes existed at all. A couple of years into their exploration of nanotubes, Krásný met Ben-Yehuda at a conference and discussed his group’s difficulties observing the structures. Ben-Yehuda later provided them with the same bacterial strains and protocols her lab had used,

but the Czech group remained unable to see the nanotubes. The researchers also tried and failed to reproduce some of the material transfer experiments that Ben-Yehuda’s lab had performed. That was when Pospíšil and his Slovak Academy of Sciences collaborator Barák started applying pressure to their coverslips and inadvertently discovered that this made the bacteria produce lots of nanotubes.4 They immediately started testing varying amounts of pressure to see what could most reliably make the cells produce nanotubes. They found that applying about 80 kilopascals, which they achieved by placing a 2.5-kilogram weight on the coverslip for 10 seconds, almost immediately resulted in several nanotubes extending from each cell. When Pospíšil used a marker called SYTOX Green, which only stains dead or dying cells, he saw that nanotubes were exclusively produced by green cells. This told him that the pressure was killing the cells, which produced nanotubes as a result. Even though pressing down on the coverslip is a common practice, Barák says he thinks that pressure-induced nanotubes have never been reported before because people tend to ignore dead cells. He adds that in decades of using dyes that stain cell membranes and pressing on coverslips, “I didn’t ever see nanotubes . . . because I didn’t concentrate on them.” Now, he knows that if he carefully fixes the cells and uses a good microscope, he will likely see nanotubes all over the place. When the group published its work in December 2020, Barák says, the response from the scientific community was largely positive. The paper was shared widely on Twitter, drawing comments from a few scientists who questioned whether the nanotubes were indeed real structures. Alex Merz, a biochemist at the University of Washington, has been a vocal critic of some of the papers from Ben-Yehuda’s lab, especially the recent one detailing the involvement of the CORE complex and showing that nanotubes can connect Gram-positive and Gram-negative bacteria.7 These two sets of bacteria have very different cell envelopes—Gram-positive bacteria have a single lipid membrane covered by a tough peptidoglycan cell wall, whereas Gram-negative bacteria have an additional lipid membrane outside of a thinner cell wall. When a nanotube extends from a Gram-positive bacterium toward a Gram-negative bacterium, does it interact only with the outer membrane of the Gram-negative recipient, or does it actively engage with the inner membrane of the recipient cell? Merz says he believes that answering these questions is vital to figuring out whether bacterial nanotubes are indeed functional structures or artifacts. He calls for the use of higher-resolution microscopy techniques, which have not been used in the recent papers, that would allow one to closely observe how the nanotubes interface with the two types of bacterial cell envelopes. “I need to see more to be persuaded,” he says. Erin Goley, a biological chemist at Johns Hopkins University School of Medicine, echoes Merz’s concerns. “I am skeptical of them being real, functional structures,” she says. Aside from how the nanotube membrane is organized, she is puzzled by the seemingly generalist nature of nanotubes. Moreover, as nearly

every contact-dependent system seen so far has its own tightly regulated machinery, Goley finds it strange that nanotubes seem to lack a master regulator. Like Merz, she is interested in seeing high-quality cross-sectional images of the nanotubes, particularly at the points where they contact the bacterial cells. The kinds of techniques both Goley and Merz recommend will let scientists eliminate traditional sample preparation artifacts and preserve

cells in their native states, which would help “show you actually what’s going on with the membranes . . . who’s contacting what and which membranes are contiguous with which,” says Goley. Kost says he thinks that the pressure-induced nanotubes seen by the Czech group might reflect the self-organization of lipids once they burst out of dead cells, similar to an older study showing that protocells, self-organizing lipid vesicles capable of interacting

WHAT ARE BACTERIAL NANOTUBES? Unlike cellular appendages such as the pili used for mating, injectisomes that transfer virulence proteins, and flagella that power swimming in many microbes, bacterial nanotubes are made solely of lipids and can connect the cytoplasm of different microbial species.

© NICOLLE FULLER

tures connecting Clostridium acetobutylicum, a Gram-positive bacterium, and Desulfovibrio vulgaris, which is Gram-negative.8 The contact between cells, which they observed using scanning electron microscopy (SEM) and other techniques, appeared to allow the cells to exchange molecules in order to survive harsh growth conditions, such as a shortage of nutrients. More recently, in mid-2020, Jinju Chen’s group at Newcastle University documented the presence of nanotubes in Pseudomonas aeruginosa, a common, biofilm-forming pathogen found in hospitals. Faced with nanopillars—tiny spikes that are often used to make medical devices with surfaces that can deter the formation of harmful biofilms—the bacteria aligned themselves between the pillars and used nanotubes to connect different cells.9 By using strains that were incapable of forming other contact-enabling structures such as pili, the group confirmed that the connecting structures they saw

Bacterial Nanotubes

Conjugative Pili

Type 3 Secretion Systems, e.g., Injectisomes and Flagella

Composition; structure

Lipids; segmented

Proteins; helical

Multiprotein complex; tubular

Length

1–40 μm

1–2 μm

0.8–2 μm

Width

30–130 nm; commonly 40–70 nm

6–11 nm; lumen diameter ~3 nm

8–10 nm; lumen diameter ~2.5 nm

Materials transferred

Antibiotic resistance factors, metabolites, toxins

Plasmids

Injectisomes for the transfer of virulence proteins; flagella for motility

Proteins involved in formation

CORE complex (same proteins as the flagellar base) and hydrolases that help make a hole in the cell wall

“Transfer” (Tra) class of proteins such as pilin, TraL, and TraF

The injectisome complex has various proteins such as secretin, stalk protein, and needle filament; the flagellar apparatus has its own set of dedicated proteins that form the base, stalk, and tip.



VO L. 35 ISSUE 2 | THE SCIEN TIST

A

SOURCES OF VARIATION IN NANOTUBE STUDIES

STEP 3: PREPARE THE BACTERIA FOR IMAGING For fluorescence microscopy, bacteria are either made to express fluorescent proteins or are treated with membrane-staining dyes. Glass slides and coverslips can be coated with different compounds to allow the bacteria to stick better. For electron microscopy, cells are dried and treated with heavy metals before imaging.

To interrogate bacterial nanotubes, researchers first grow the bacteria in culture, then use sophisticated microscopy techniques and different readouts such as fluorescent tags to check whether cells are exchanging materials. The behavior and morphology of bacterial cells can differ depending on the specifics of each step, giving rise to varied observations and, sometimes, conflicting results.

STEP 1: GROW THE BACTERIA Bacteria can be grown either on solid media or in liquid broth. Depending on the species, growth conditions can influence bacterial behavior and appearance.

Without pressure

With pressure

Differences in sample preparation can affect the reliability with which bacterial nanotubes are observed. One lab, for example, could only see nanotubes if they pressed down on the coverslip before imaging the cells, whereas other labs never needed to do so in order to observe the structures.

STEP 4: IMAGE THE BACTERIAL NANOTUBES Fluorescence and electron microscopy, or sometimes a combination of both, are most commonly used to observe nanotubes. The structures are hard to see using fluorescence microscopy (at right), a lower-resolution form of imaging, because there are no specific markers for nanotubes. Using higher magnification electron microscopy (below), nanotubes can be easily resolved and distinguished from structures such as pili and flagella.

Various methods have been used to observe nanotubes, but some labs have been unable to observe certain types of material transfer.

11:4963, 2020; CHRISTIAN KOST, OSNABRÜCK UNIVERSITY

STEP 2: TEST FOR THE TRANSFER OF MATERIALS BETWEEN TWO BACTERIA Observing whole cells or colonies can reveal the transfer of fluorescent proteins, plasmids, metabolites, and more.

ILLUSTRATIONS BY © NICOLLE FULLER; NAT COMM,

Bacteria grown in both types of media have been seen to produce nanotubes, but some labs have been unable to observe them in either one type or the other.

Nanotubes can only be identified with training and experience; scientists need to be able to distinguish them from the background noise of fluorescence microscopy and artifacts of electron microscopy.

with their environments, can form long lipid tubes under certain conditions.10 “I think, yes, under some conditions, cells show this phenomenon. But this certainly does not rule out nanotubes” playing a role in living cells, he adds. Ben-Yehuda’s group stands by its work showing the commonplace formation of nanotubes among living microbes. “We have never had to place the cells under any sort of pressure,” says Amit Baidya, a postdoc in the lab. Ben-Yehuda also notes that bacterial colonies that they imaged were still capable of growing and dividing after having made nanotubes. Even in her lab, however, the researchers sometimes struggle to observe nanotubes. A major challenge is the lack of tags for nanotubes, forcing scientists to rely on membrane staining and resulting in very noisy images. “It’s like looking at the sun and trying to see something very small,” says Ben-Yehuda. Baidya agrees that the process of identifying nanotubes is finicky. “You have to be really, really careful while making the samples,” he says. “If you are not focused that day . . . if you are in a hurry, you will not see nanotubes.”

A replication crisis? The studies on bacterial nanotubes differ in many aspects: the bacterial species or strains, growth conditions, assays to detect material transfer, and microscopy techniques to image the structures themselves. And, as Pospíšil’s study highlighted, not all labs have success with all the conditions. The growth conditions of bacteria, including temperature, acidity, and aeration, are known to affect the physiology and appearance of cells, says Paula Montero Llopis, who directs the

light microscopy facility at Harvard Medical School. For example, B. subtilis comes in two forms: swarmers, which can freely swim around, and chains, which are made of cells that are connected to one another and therefore cannot move, explains Montero Llopis, whose work has revealed that the latter are more likely to form when the bacteria are grown at a low temperature in media containing casein hydrolysate, a source of amino acids. “I found that the percentage of swarmers vs. chains in the population is highly dependent on how you grow the cells,” she tells The Scientist in an email. When it comes to nanotubes, Pospíšil and Kost both used liquid media, whereas Ben-Yehuda’s lab grew the bacteria on solid agar plates. Neither Pospíšil nor Kost was able to observe nanotubes from bacteria grown on solid media, while Ben-Yehuda’s lab failed to see them on cells in liquid media. Kost attributes different experimental outcomes to the bacterial species each lab handles: Ben-Yehuda’s lab works on B. subtilis, which is a soil bacterium and prefers solid surfaces, whereas Kost’s lab works on E. coli, which likes liquid cultures. Even when labs are trying to replicate one another’s studies, comparing results will be tricky, Kost notes. “We try to replicate all the materials that we use as good as possible, but you never know,” he says, having faced similar problems when shifting his own lab from one university to another. Not only is there variation among manufacturers of various reagents, Kost explains, but batch differences among chemicals can foil replication attempts even when using otherwise identical products for growing or imaging bacteria.

PUTATIVE FUNCTIONS OF NANOTUBES Numerous studies have identified various possible roles for bacterial nanotubes, which researchers have observed under different growth conditions. Function

Species found

Culture conditions

Citations

Transfer of materials (RNA, proteins, amino acids, toxins)

Bacillus megaterium, B. subtilis, Clostridium acetobutylicum*, Desulfovibrio vulgaris*, E. coli, enteropathogenic E. coli, Staphylococcus aureus

Solid or liquid media, depending on the study

Cell, 144:590–600, 2011; Nat Comm, 11:4963, 2020; Nat Comm, 6:6283, 2015; Cell, 177:683–96.e18, 2019

Adhesion to mammalian cells

Enteropathogenic E. coli

Liquid media

Cell, 177:683–96.e18, 2019

Adhesion to nanopillars and the formation of biofilms

Pseudomonas aeruginosa

Liquid media

Soft Matter, 16:7613–23, 2020

Stress response in dying cells placed under pressure

B. subtilis

Liquid media

Nat Comm, 11:4963, 2020

*Indirect evidence 4 0 T H E SC I EN T I ST | the-scientist.com

I am skeptical of them being real, functional structures. 

—Erin Goley, Johns Hopkins University School of Medicine

These differences can also spill over into how the scientists prepare the bacteria for imaging and the kind of imaging technique used. When preparing bacteria for EM, two of the most common steps are drying the cells out and then coating them with metals. “These techniques . . . can be really robust, but they do carry a significant hazard of artifacts,” says Merz. He also cautions against chemical fixation, a technique that uses chemicals such as paraformaldehyde to “freeze” cells in time, as it is more likely to give rise to artifacts. Among the labs that have observed nanotubes using EM, the imaging modalities and sample preparation techniques used have varied widely. The different and sometimes contrasting conditions required to observe nanotubes are not lost on nanotube skeptics. Ariane Briegel, a professor of ultrastructural biology at Leiden University, says she believes that the structures seen in EMs might in fact be drying artifacts, which is why they cannot be observed consistently. “I do believe that there is exchange [of materials] between the cells, but I am not sure how far I trust the nanotubes to be real,” she tells The Scientist in an email. Goley concurs, adding that the lack of replicability has only added to her skepticism. But to Kost, the fact that a variety of conditions have been used to produce nanotubes is evidence that they do indeed exist. “What I found very encouraging is that . . . there’s so many labs that report very, very similar phenomena.” In fact, the apparent ubiquity of nanotubes has spurred him to push forward. In 2019, his group found that the transport of amino acids through bacterial nanotubes was unidirectional—nanotubes that originated from bacterium A would shuttle materials from bacterium A to bacterium B, but not the other way around. This implies that the nanotubes provide an element of control and are not just tubes connecting the cytoplasms of two cells.11 Kost says that ongoing studies in his lab indicate that the nanotubes might not just be mediators, but key instigators of bacterial cooperation. In unpublished work, the lab has observed that the mere presence of nanotubes makes cells choose cooperative strategies over competition, he says. “This completely changed the way that I look at ecological interactions. This seems to suggest that these nanotubes are the key for cooperation to evolve.” Ben-Yehuda’s group, meanwhile, has continued to unearth details of nanotube formation. The researchers recently discovered, for example, that nanotube-producing bacteria first send to recipient cells hydrolase activators, which trigger cell wall

remodeling to allow the nanotubes to penetrate and send other material through.12 Somehow, cells also appear to direct where the nanotubes go, Ben-Yehuda says. “They can grow everywhere in the space, no? [But] they always grow directly towards each other. . . . That makes me suggest that it’s not random.” Pospíšil does not see this sort of directionality in the nanotubes extending from his dying bacteria, but he has observed a preference in the regions from which the nanotubes emerge. In vertically elongated B. subtilis cells, Pospíšil has observed that the nanotubes are produced mostly from the poles of the cells (the top and bottom), rather than from along the sides. 4 Tim Errington, the director of research at the Center for Open Science (COS), point outs that contrasting results are not always bad. “Different points of view [are] exactly what you want in science, because you shouldn’t [ just] accept something you’re saying. . . . You have to keep revisiting it.” One of the ways Errington thinks that the differences regarding bacterial nanotubes can be resolved is through a method known as adversarial collaboration, where two labs with opposing hypotheses “precommit” to a set of experiments designed to test replicability, do the experiments, and then compare results. Ben-Yehuda remains unperturbed by Pospíšil’s findings and is excited to explore the myriad unanswered questions about bacterial nanotubes. “Over the years, we established that they are real and they are there, although some people think they are not. Science is a marathon, and we are running a marathon, not a sprint.” Sruthi S. Balakrishnan is a freelance science journalist based in Bangalore, India. You can follow her on Twitter @sruthisanjeev.

References 1. G.P. Dubey, S. Ben-Yehuda, “Intercellular nanotubes mediate bacterial communication,” Cell, 144:590–600, 2011. 2. S. Pande et al., “Metabolic cross-feeding via intercellular nanotubes among bacteria,” Nat Comm, 6:6238, 2015. 3. O. Stempler et al., “Interspecies nutrient extraction and toxin delivery between bacteria,” Nat Comm, 8:315, 2017. 4. J. Pospíšil et al., “Bacterial nanotubes as a manifestation of cell death,” Nat Comm, 11:4963, 2020. 5. G.P. Dubey et al., “Architecture and characteristics of bacterial nanotubes,” Dev Cell, 36:453–61, 2016. 6. R.R. Pal et al., “Pathogenic E. coli extracts nutrients from infected host cells utilizing injectisome components,” Cell, 177:683–96.e18, 2019. 7. S. Bhattacharya et al., “A ubiquitous platform for bacterial nanotube biogenesis,” Cell Rep, 27:334–42, 2019. 8. S. Benomar et al., “Nutritional stress induces exchange of cell material and energetic coupling between bacterial species,” Nat Comm, 6:6283, 2015. 9. Y. Cao et al., “Bacterial nanotubes mediate bacterial growth on periodic nanopillars,” Soft Matter, 16:7613–23, 2020. 10. T.F. Zhu, J.W. Szostak, “Coupled growth and division of model protocell membranes,” J Am Chem Soc, 131:5705–13, 2009. 11. S. Shitut et al., “Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells,” Environ Microbiol, 21:1306–20, 2019. 12. A.K. Baidya et al., “Donor-delivered cell wall hydrolases facilitate nanotube penetration into recipient bacteria,” Nat Comm, 11:1938, 2020.



06.2021 | THE SCIEN TIST 41

EDITOR’S CHOICE PAPERS

The Literature CELL & MOLECULAR BIOLOGY

Fungal Squeeze

Slow-growing fungus

Fast-growing fungus

THE PAPER

S. Fukuda et al., “Trade-off between plasticity and velocity in mycelial growth,” mBio, 12:e03196–20, 2021.

4 2 T H E SC I EN T I ST | the-scientist.com

SLOW AND STEADY: When slow-growing fungi arrive at a gap smaller than the width of their hyphae, they can squeeze through and keep growing once they reach the other side. Fast-growing fungi tend to stall, either before their hyphae make it through the gap (top right), or because the hypha becomes depolarized—that is, it loses its internal organization—by the time it emerges from the channel (bottom right). Researchers used live-cell imaging to help explain the difference: faster growers have more vesicles carrying material needed for hyphal elongation and a bigger buildup of pressure at the tip, causing swelling that disrupts normal growth.

was fast-growing fungi’s downfall: the rush of vesicles and pressure buildup at the tip blocked growth through the channel or disrupted the hypha’s internal organization by the time it reached the other side. Slower-growing species had time for internal rearrangement without such a buildup. The results indicate a tradeoff between speed and plasticity, Takeshita writes. “Fast-growing fungi possess the advantage of covering quickly new nutrient-rich substrates or free open spaces. . . . On the other hand, the slow growth of fungi may help them to adapt better to different niches or to grow on more complex substrates.” Roger Lew, a biophysicist at York University in Toronto who was not involved in the work, says the team’s findings will

advance the study of cell growth more generally. “We’re looking at something that’s relevant to a whole range of different kinds of developmental patterns in different kingdoms,” he says. Pollen tubes in plants grow in a similar way to hyphae, he notes, and both are of interest in biotechnology, with applications to crop breeding and disease management, respectively. Further work could incorporate sensors to measure the pressure experienced by fungi growing in channels and provide more information about how the organisms make growth decisions, Lew says. Setups such as the one used in the study allow researchers to do “a lot of imaginative stuff. . . . There are possibilities out there, for sure.”  —Catherine Offord

© JULIA MOORE, WWW.MOOREILLUSTRATIONS.COM

Much of a filamentous fungus’s life involves infiltrating organic tissue: weaving its hyphae between cells in decaying animals, for example, or, in the case of some pathogenic species, invading plants through tiny pores in their leaves. The tips of these fungi grow by synthesizing new cell wall on the extending side, but scientists have puzzled over how they control growth through such tight spaces. Norio Takeshita of the University of Tsukuba in Japan approached the question by growing seven species of fungi in a microfluidic device with tiny channels, the narrowest just 1 micrometer across— smaller than the diameter of typical hyphae. His team used live imaging techniques, some involving labeling intracellular components with green fluorescent protein, to see how each species handled the challenge. The species turned out to respond in one of two ways: one group of fungi grew through the channels just fine, Takeshita notes in an email to The Scientist. “Surprisingly, however, the other group . . . stopped growing in the channels, or stopped after the channels.” Fungi in that second group had much faster hyphal elongation rates than those in the first did. Scouring the images for explanations, Takeshita and colleagues discovered a link between growing speed and the movement of intracellular vesicles, which shuttle proteins and other molecules within cells. “To elongate the membranes and cell walls quickly, a large number of vesicles containing [the necessary] material are supplied to the mycelial tip,” Takeshita says. This

OVERACTIVE: Patients with Takotsubo syndrome (left) had elevated

BUSTED: Vaccines against poxvirus and melanoma induced more memory

DISEASE & MEDICINE

IMMUNOLOGY

Unbreak My Heart

Barrier Boost

THE PAPER

THE PAPER

A. Radfar et al., “Stress-associated neurobiological activity associates with the risk for and timing of subsequent Takotsubo syndrome,” Eur Heart J, ehab029, 2021

K. Rakhra et al., “Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells,” Sci Immunol, 6:eabd8003, 2021.

Takotsubo syndrome, also known as broken heart syndrome, is a rare, reversible condition with symptoms mimicking a mild heart attack. A disease that disproportionately affects women, TTS is triggered by stressful events such as bankruptcy, the death of a loved one, or divorce, and results in a weakening of the heart’s left ventricle such that it becomes temporarily misshapen. Previous work has shown that TTS patients have elevated activity in their amygdala, a brain region involved in stress response. What has never been clear, however, is whether “this activity in the brain happens as a result of the syndrome or whether it began many years before,” says Shady Abohashem, a nuclear cardiologist at Harvard Medical School. Abohashem and his colleagues retrospectively analyzed full-body PET/CT scans from 104 patients, most of whom had cancer and 41 of whom had developed TTS since first being scanned, and 63 individually matched controls. The team calculated ratios of the activity in each person’s amygdala to that of two brain regions that attenuate the stress response, the temporal lobe and the prefrontal cortex. Higher amygdala activity was associated with an increased risk for TTS, and among those with the condition, patients with higher ratios had developed TTS roughly two years earlier following the imaging than those with lower ratios. “We can now show that this syndrome happens as a result of chronic stress over years that makes you vulnerable to developing the syndrome more easily and sooner than [less stressed] people,” Abohashem says. “This study confirms our suspicion that there’s a relationship between amygdala activity and future risk of Takotsubo,” says Janet Wei, a cardiologist at Cedars-Sinai Medical Center who was not involved in the work. The results, she adds, “necessitate further study to see why these patients have higher amygdala activity and how it actually regulates the acute response.”  —Amanda Heidt

Most vaccines are injected into muscle, where they induce systemic immunity. A goal of many vaccine developers is to engineer inhalable formulations that would build up powerful immunity localized in the mucous membranes that line organs such as the lungs. But to do so, vaccines need to breach that mucous membrane and head to the lymph nodes within the lungs, where they can instruct the immune system to generate memory T cells, key players in long-term immunity. To accomplish that, Darrell Irvine, a biological engineer at MIT, and his team looked to albumin, a protein that naturally crosses mucous membranes. The researchers engineered a peptide vaccine against vaccinia (an animal poxvirus) with a lipid tail that binds to albumin and gave it to mice. As hoped, albumin shepherded the vaccine across the mucous membrane, transporting it into the lungs and lymph nodes, where it persisted for two weeks. When exposed to the virus, all of the mice that received the new vaccine intratracheally (to imitate an aerosolized vaccine for humans) survived, while those vaccinated with a standard vaccine or subcutaneously with the new vaccine all died. The researchers also tested an albumin-binding vaccine against melanoma. When mice given this vaccine were later injected with melanoma cells to mimic metastases in the bloodstream, 8 out of 10 survived for 100 days, compared to 4 out of 10 that received a subcutaneous vaccine. The key to the enhanced immunity was ensuring that the antigen stayed in the animals’ systems for at least two weeks, Irvine says. Standard peptide vaccines that lack a lipid tail typically degrade rapidly in vivo. “This suggests that any vaccine that promotes antigen/adjuvant persistence over such a timescale may be more effective for promoting protective T cell responses.” “It will be particularly exciting” if the strategy works in humans, says Alistair Ramsay, a microbiologist at Louisiana State University who was not involved in the study.  —Emma Yasinski

EUR HEART J, DOI:10.1093/EURHEARTJ/EHAB029, 2021; FLICKR, NIAID

activity in their amygdalas (arrows) before their diagnosis compared with patients who never developed the condition (right).

T cells (shown) in mice when they entered through the lungs than when administered via another route.



06 . 2021 | T H E S C IE N T IST 4 3

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SCIENTIST TO WATCH

Adriana L. Romero-Olivares: Fungus Tracker Assistant Professor, Department of Biology, New Mexico State University BY AMANDA HEIDT

LESLIE COUSE

G

rowing up in Hermosillo, Mexico, Adriana L. RomeroOlivares spent many weekends among the stately saguaros of the Sonoran Desert. Her father, drawing on his knowledge of local plants and animals, taught her the basics of biology, a path she followed after a high school teacher suggested she study science. Toward the end of her undergraduate career at the Autonomous University of Baja California, she says, she fell in love with molecular biology. “I had no idea what this invisible world looked like, but once I did, it blew my mind.” Romero-Olivares stayed at the University of Baja for her master’s degree, using genetic tools to characterize assemblages of fungi across the Baja Peninsula’s many microclimates. Her unpublished results confirmed that fungi display the same biogeographical tendencies as plants and animals—namely, organisms evolve to thrive in their environments. Fungal communities from similar ecosystems were more alike, regardless of the geographical distance that separated them. With climate change warping ecosystems worldwide, RomeroOlivares wondered how fungi might adapt, a pressing question given their importance in global nutrient cycling. Fungi grow by breaking down detritus, unlocking nitrogen for other species and releasing carbon into the atmosphere. To study whether warming prompts physiological changes in fungi, Romero-Olivares joined fungal biogeochemist Kathleen Treseder at the University of California, Irvine, in 2012 for her PhD work. Romero-Olivares started her doctoral research by growing strains of the mold Neurospora discreta at 16 ℃ or 28 ℃ for 1,500 generations. She then exposed these “adapted” molds and their parent strains in the lab to warming while monitoring their growth rate, biomass, spore production, and CO2 respiration. As temperatures climbed, adapted strains produced more spores—perhaps to ensure their survival under higher temperatures—and devoted less energy to growth, therefore decomposing less organic material and respiring less carbon (BMC Evol Biol, 15:198, 2015). These early experiments with Neurospora were “the driver of many ideas that I still have” about how fungi adapt to changing conditions, Romero-Olivares says.

To see if these findings held true in the field, she traveled to Treseder’s long-term research site in the boreal forests of Alaska, manipulating conditions inside greenhouses to assess how warmer conditions changed the fungal community. Again, Romero-Olivares saw a tradeoff. As temperatures increased, fungi became stressed, shifting their energy toward basic metabolism and away from decomposition of organic material, thus weakening the flow of nitrogen to other species (Front Microbiol, 10:1914, 2019). The nature of their decomposing changed, too. When stressed by rising temperatures, fungi broke down more recalcitrant carbon, a type that normally resists decomposition and remains locked in the ground (PLOS ONE, 12:e0179674, 2017). Even though decomposition decreased overall, which should theoretically reduce carbon emissions, warmer fungi’s swap to recalcitrant carbon could ultimately weaken soil’s ability to store carbon, Romero-Olivares says. Her dissertation research, Treseder tells The Scientist, is “one of the first studies to explicitly test how microbes evolve in response to climate change. It’s almost a whole new field.” While previous research has focused on fungi’s immediate responses, Romero-Olivares takes an evolutionary perspective, assessing how such changes affect species over the long term. “She basically started that for fungi,” Treseder says. After getting her PhD in 2017, Romero-Olivares started a postdoc at the University of New Hampshire, studying the genetics underlying fungal traits. She assessed the frequency of nitrogen uptake and decomposition genes in 879 fungal genomes and categorized the species using traits such as their functional group—whether they were pathogenic or plant symbionts, for example—their growth morphology, or the enzymatic pathways they use to drive decay. Climate change, her results suggest, will increase the abundance of fungal species with more nitrogen-uptake genes, such as yeasts and, significantly, animal pathogens (Microb Ecol, doi:10.1007/s00248-021-01687-x, 2021). Her use of many different approaches, from culturing microbes to mining big data, makes Romero-Olivares’s work particularly strong, says Camille Defrenne, an ecosystem ecologist at Oak Ridge National Laboratory who has not participated in RomeroOlivares’s research. “She’s a very complete scientist with a holistic view of how microbes respond to global change. She really represents the next generation of women in science.” Romero-Olivares launched her lab at New Mexico State University last August. In addition to continuing her climate work, she’s also expanding into new territory in her own backyard. “While we know that deserts work in very different ways than other ecosystems, we don’t know what role fungi play,” she says. Beyond serving as the desert’s decomposers, “it’s an exciting challenge to figure out what else they might be up to.” g

06. 2021 | T H E S C IE N T IST 4 5

BIO BUSINESS

Making Poop Profitable

ally, because FMT is not a Food and Drug Administration (FDA)–approved treatment, UM had to file an investigational new drug (IND) application to offer it. The rise in FMT’s availability really began in 2013, when the FDA announced that it would exercise “enforcement discretion” by no longer requiring INDs for the use of FMT in recurrent or refractory C. diff infections. OpenBiome, a nonprofit stool bank that had started up in 2012 with the aim of supplying local hospitals treating C. diff patients, saw that 2013 decision as an opportunity to expand, says Carolyn Edelstein, the organization’s executive director. “When the FDA issued enforcement discretion, we saw increasing need from the clinical community that was offering FMT, wanting to be able to just make sure that they could directly treat patients,” she says. “And so we began scaling up to meet the interest in using FMT to treat C. diff patients who weren’t responding to standard antibiotic therapies.” Over time, OpenBiome came to supply most clinics in the US that offered FMT—including many that, like UM, had started out by screening donors and preparing stool themselves.

With multiple microbiota therapeutics in the pipeline for recurrent Clostridium difficile infection, clinicians foresee a shift in treatment options for the condition. BY SHAWNA WILLIAMS

A

4 6 T H E SC I EN T I ST | the-scientist.com

Increased screening

The use of FMT to treat C. diff dates back nearly 40 years; one of the first case studies on its successful application was published in 1983. But the procedure didn’t spark widespread attention until the turn of the millennium, when, faced with the rise of increasingly virulent and antibiotic-resistant strains of the bacterium, doctors starting searching for new ways of helping patients who didn’t respond to antibiotics, or whose infections would subside only to recur again and again. In some cases, antibiotics even seemed to promote C. diff infection, with the bacteria taking hold after the commensal microbiota had been depleted by the drugs. Patients often acquired C. diff while in the hospital being treated for other conditions. In the early 2010s, evidence for FMT’s efficacy against recurrent C. diff infec-

POOP PILL: A fecal microbiota transplant capsule filled by staff at OpenBiome

tion accumulated, and many academic medical centers began to offer the procedure. Patients would often be asked to recruit their own donor, such as a family member, and if that person tested negative for infectious diseases, their fresh stool would be homogenized with saline solution or milk in a household blender, filtered, and then administered. But there were hurdles to offering FMT. At the University of Michigan (UM) medical center, which started performing the procedure around 2012, gastroenterologist and researcher John Kao recalls that it was difficult to get permission from hospital administrators. “I have comments like, ‘What are you doing? This is like bloodletting.’” Addition-

COURTESY OF OPENBIOME

Rising popularity

COURTESY OF OPENBIOME

lexander Khoruts wishes we’d all stop using the F-word. In addition to its yucky connotations, the term “fecal transplant” is an inaccurate description of the procedure he helped pioneer, he argues, since “you can’t transplant feces.” Rather, it’s the intestinal microbiome that gets engrafted, says Khoruts, a gastroenterologist at the University of Minnesota who coauthored the first detailed how-to guide for the procedure. Accordingly, he says, he prefers the term “intestinal microbiota transplant.” As director of the university’s Microbiota Therapeutics Program, he regularly works with people who undergo the procedure, and “I’ve seen the patients really have a sigh of relief when you lose the ‘fecal’ word.” Khoruts realizes that the term fecal microbiota transplant, or FMT, is likely here to stay, however. The procedure, which involves transferring carefully screened donor stool via colonoscopy, enema, or a pill, has gone mainstream over the past decade. FMT is now a go-to treatment for recurrent or refractory infections with a bacterium known as Clostridium difficile (C. diff for short), which causes sometimes debilitating gastrointestinal symptoms and can be fatal if not successfully treated. Researchers are also investigating the efficacy of FMT for a range of other conditions. Khoruts and other observers say the use of FMT is poised for major changes with the expected entry onto the market of commercial microbiota therapeutics that consist of processed stool components or consortia of lab-grown intestinal bacteria. While many clinicians who perform FMT expect these changes to be largely positive, some, including Khoruts, have concerns about how they might affect patients’ access to treatments.

An advantage of obtaining FMT material from OpenBiome is that the organization handles the necessary pre-transplant screenings. “As time has gone on, the screening has gotten more and more involved and more in depth,” both for donors and for the stool, explains Stacy A. Kahn, a professor at Harvard Medical School and director of Boston Children’s Hospital’s FMT and Therapeutics Program. “There’s no cost-effective way for an individual provider to adequately screen the stool as comprehensively” as a stool bank such as OpenBiome, Kahn says. The importance of this screening has been highlighted in recent years by highprofile safety incidents linked to FMT, including a patient death at Massachusetts General Hospital in 2019 that was ascribed to antibiotic-resistant E. coli in the donor’s MEETING DEMAND: OpenBiome’s Gina Mendolia

fills fecal microbiota transplant capsules.

stool, and six infections with pathogenic E. coli after FMT with stool from OpenBiome that prompted an FDA warning last year. The FDA noted in its warning that FMT with contaminated stool might also have contributed to the deaths of two other patients, although this could not be definitively determined. OpenBiome announced changes to its screening protocols at the time of the warning, while also arguing that the deaths were unlikely to be due to FMT. From July 2020 until this May, OpenBiome stopped providing FMT materials except in emergency cases as it worked to develop and validate a test for SARS-CoV-2 in stool. For emergency cases, it used stock collected before December 1, 2019. The organization announced this February that, due to financial difficulties and the likely entry onto the market of FDA-approved commercial microbiota therapeutics for C. diff, it will be phasing out its services. OpenBiome isn’t planning on going away, says Edelstein, but will instead focus on supporting microbiome researchers—for example, with its library of more than 100,000 human stool aliquots.

Drugs on the horizon Even before high-profile safety issues cropped up, some companies spied an opportunity to develop an easier-to-use and more consistent FMT product. Around a decade ago, FMT was “forcing doctors to be manufacturers and manufacture a drug,” says Ken Blount, chief scientific officer of microbiota therapy company Rebiotix, which started up in Minnesota in 2011. “And that ultimately means the FMT you get . . . in Connecticut versus San Diego

versus Boston is a different thing.” The lack of an FDA-approved product also means that FMT isn’t available everywhere, he adds, and usually isn’t covered by insurance. To plug this gap, Rebiotix began developing what it calls a Microbiota Restoration Therapy with the goal of obtaining FDA approval. Rebiotix’s product, dubbed RBX2660, now in Phase 3 testing for recurrent C. diff infection, is not only screened for safety but also tested to ensure that it contains a certain amount of viable bacteria per dose, Blount says. He expects the company will present data from that trial later this year. RBX2660 is designed to be delivered via enema; Rebiotix also has a second, oral formulation in development. Another product nearing the end of the development pipeline is Seres Therapeutics’s SER-109, for which the Massachusettsbased company announced positive Phase 3 results last year. That orally administered product consists of a consortium of bacteria purified from stool in which all live organisms have been killed, leaving spores behind. Finch Therapeutics, also headquartered in Massachusetts, has a stool-based product that has completed Phase 2 testing; a spin-off from OpenBiome, Finch is headed by Edelstein’s husband, Mark Smith, who previously served as OpenBiome’s president and research director. Nearby Vedanta Biosciences has developed a non-stool-based consortium of bacteria that has also undergone Phase 2 testing for recurrent C. diff infections. Khoruts predicts that once a commercial microbiota therapy is approved for recurrent C. diff infection, clinics will

BIO BUSINESS

I would love to just have a product that I can prescribe, and that’s passed through all the regulatory hurdles and has FDA approval and is covered by insurance.

MICROBIOTA THERAPEUTICS IN DEVELOPMENT FOR C. DIFFICILE INFECTION Preclinical

Phase 1

Phase 2

Phase 3



Rebiotix: RBX7455 Oral capsule of lyophilized microbes from donor stool Finch: CP101 Oral capsule of lyophilized microbes from donor stool

Rutgers Robert Wood Johnson School of Medicine in New Jersey. In reality, treating other indications with FMT likely won’t be as straightforward as it has been for C. diff, Chen says. “For whatever reason, with C. diff you don’t have to be that selective about the donor stool—clearly there are safety things and screening that we do for infectious agents, but beyond that it seems like most acceptable donor stool will work for any patient,” she says. For other conditions, studies so far indicate that “there seems to be some matching that needs to happen in order to have that effect, and we still don’t fully understand why certain patients do well

Vedanta: VE303 Orally administered consortium of lab-grown bacteria Rebiotix: RBX2660 Liquid suspension of microbes from donor stool, delivered as enema Seres: SER-109 Orally administered consortium of purified spores of Firmicute bacteria from donor stool

Despite this overall optimism, there is a possibility that replacing the current sourcing system with commercial products could 4 8 T H E SC I EN T I ST | the-scientist.com

IN THE PIPELINE: Several companies have

developed microbiome therapeutics for recurrent or refractory C. difficile infection and are vying to gather the data needed to apply for approval from the US Food and Drug Administration.

peutics in children, and while commercial therapies could potentially be used off-label in pediatric patients, Kahn says, insurance is unlikely to cover that. While the move to commercial products could create access issues for some patients who would benefit from FMT, it could also make the therapy available to patients who are unlikely to benefit. FDA’s enforcement discretion notice was specific to the use of FMT for C. diff infection that doesn’t respond to other therapies, meaning that treating other conditions with stool still requires an IND. But preliminary experimental results suggesting FMT could also be an effective therapy for ailments ranging from intestinal disorders such as inflammatory bowel disease to cancer to autism have raised excitement among patients and their families that outstrips what’s currently clinically feasible, providers say. “We sometimes have patients cold calling asking for FMT for a variety of indications beyond C. diff,” says Lea Ann Chen, a gastroenterologist at

and why certain donors seem to be better than others.” Given these circumstances, Khoruts says he’s worried about the potential for FMT to be prescribed inappropriately, because “once something is approved [for one condition], physicians are free to use it for other things off label.” Unless the FDA steps in to restrict off-label uses, “there’s going to be potential for abuse of misappropriating these drugs for something that we don’t know how to use them for,” he adds. Sahil Khanna, who leads the Mayo Clinic’s FMT program for C. diff, agrees: “We need to set up some guardrails to avoid off-label use, to prevent potential harm to people.”

There are some microbiota therapeutics already in the pipeline for disorders other than C. diff infection, although they are not as advanced. Vedanta, for example, has therapeutics in development for inflammatory bowel disease, solid tumors, food allergy, and multidrug resistant organisms, while Rebiotix is developing products for vancomycin-resistant enterococci infection, pediatric ulcerative colitis, drug-resistant urinary tract infections, and a condition involving the buildup of ammonia in the liver. In the long run, Edelstein says, she expects that the field will move further from its fecal roots once researchers pinpoint the types of bacteria that can effectively treat various disorders. “Once we have reliable access to products that are directly sourced from humans, we think the next step is to help make sure that we can isolate and identify those strains that can potentially be a key component of future therapies.” g

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Accessibility concerns

inhibit access to these treatments, some experts note. Khoruts, who helped develop a process for making an oral formulation with freeze-dried bacteria that was licensed by Finch, says he’s concerned companies could charge prohibitively high prices for their microbiota therapies, leading insurance companies to “put up barriers of some kind” that make it difficult for patients to get the therapeutics. Khoruts’s program manufactures its own capsule-based microbiota therapeutic for recurrent C. diff, which he says patients aren’t charged for. He says he’d like to scale that up by starting a nonprofit that could supply clinicians throughout the country whose patients couldn’t otherwise access FMT—but there are legal barriers to doing so, again because of litigation risk. Pediatric patients are a group that’s particularly at risk of losing access to FMT with OpenBiome’s planned phaseout, says Kahn. C. diff infection isn’t as common in kids as in adults, she says, but as with adults, its prevalence has been increasing in recent decades. “I think that people don’t understand how debilitating this is for kids,” who commonly spend so much time in the bathroom that it’s difficult for them to attend school or participate in activities, Kahn elaborates. Companies aren’t currently testing their microbiota thera-

THE SCIENTIST STAFF

move away from providing traditional FMT with either their own preparations or OpenBiome’s material. That’s due to the costly screening process needed today, he says, and to concerns about litigation risk should safety issues crop up with a homemade product that isn’t FDA-approved. Other clinicians who spoke with The Scientist largely concurred with that prediction, while noting it will depend somewhat on as-yet-unknown factors such as how much companies charge for the therapies and whether insurance companies cover them. “I would love to just have a product that I can prescribe, and that’s passed through all the regulatory hurdles and has FDA approval and is covered by insurance,” says Krishna Rao, the medical director of the fecal microbiota transplant program for C. diff infection at the University of Michigan and a consultant for Seres. Reezwana Chowdhury, a gastroenterologist at Johns Hopkins University School of Medicine, likewise says she’s “excited about all the things that will be coming out into the market. And I do think it’s going to give our patients more options.”

—Krishna Rao, University of Michigan

The future of microbiota therapeutics

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06.2021 | THE SCIEN TIST 49 BioprocessingSummit.com

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READING FRAMES

The Brains Behind the Brain Improving the function of inhibitory neurons could be key to developing more-effective treatments for a variety of brain disorders. BY LAUREN AGUIRRE

W

hen we think about how the brain works—or how to fix it—we tend to think of neurotransmitters such as serotonin or dopamine. But the brain is an electric organ, its currency the impulses that fly across thousands of miles of neurons. As I describe in my new book, The Memory Thief and the Secrets Behind How We Remember: A Medical Mystery, more electrical activity is not always better. In fact, hyperactivity in the hippocampus—the brain’s memory center—is an early sign of Alzheimer’s disease that is gaining overdue interest as a therapeutic target. Neurons come in two main “flavors,” excitatory and inhibitory. When an excitatory neuron receives enough input from other excitatory neurons, it fires, passing that signal along its axon to partners downstream. Inhibitory neurons usually tell other neurons not to fire. They are less plentiful than excitatory neurons but more diverse. In some ways, they are the real brains of the system, the machines in the background that pace and coordinate a ceaseless hum of electrical activity. The best-studied inhibitory neuron is called a basket cell, so named because its axon splits into many filaments and wraps like a basket around the cell body of other neurons, the point where it can exert maximum control. Basket cells have a relatively simple job: they act as gatekeepers, allowing excitatory neurons to fire or preventing them from doing so. A single basket cell can control and synchronize the output of hundreds or even thousands of excitatory neurons, switching them on and off with precise timing and setting up a rhythmic tug-of-war that creates brain waves. Brain waves, in turn, allow information to be coordinated and transmitted across long distances. When inhibitory neurons stop working well,

this delicate balance between excitation and inhibition degrades, and brain waves become less coherent. When researchers first identified hippocampal hyperactivity as an early Alzheimer’s symptom, they assumed it was compensatory, a way to turn up the volume on weak communication between neurons. Researchers now understand that this loss of inhibition is like background static that interferes with memory retrieval, and clues point to inhibitory neurons as essential players in the chain of events that occurs as Alzheimer’s progresses. For example, even cognitively normal older adults have hyperactivity in the hippocampus and accumulation of tau protein along with it. In addition to sticky amyloid beta plaques, these toxic tau proteins are a defining feature of the disease. Another clue is that seizures, which occur when excitatory neurons fire uncontrollably, are more common in people with Alzheimer’s than without, are thought to accelerate its progression, and may appear in the early stages—perhaps even before other signs of disease. A third clue is that one type of brain wave, called gamma, is weaker in people with Alzheimer’s. These insights suggest that adjusting the balance between excitation and inhibition could improve memory and slow down the disease’s progression. Researchers are investigating several approaches to recalibrating that balance. The furthest along is a Phase 3 clinical trial of a widely used anti-seizure drug called levetiracetam. The US company behind the trial, AgeneBio, is testing whether an extended-release, very low dose reduces background hyperactivity enough to improve memory in the earliest stages of Alzheimer’s. A second angle of attack is to manipulate the brain waves generated by inhibitory neurons. Researchers at a com-

Pegasus Books, June 2021

pany called Cognito Therapeutics, at MIT, and elsewhere are running several independent trials that use external flickering lights and audio to entrain and strengthen gamma rhythms. A third tack, currently being tested in mice, is to transplant genetically enhanced inhibitory neurons into the brain. Faulty electrical communication is also thought to play a role in other brain disorders and diseases, including epilepsy, schizophrenia, depression, and autism. Our understanding of inhibitory neurons is in its infancy compared to what we know about neurotransmitters. Because neurotransmitters play multiple roles and therefore have many side effects, they can act like a pharmacological blanket laid down over the whole brain’s delicate workings. Perhaps, if researchers figure out how to target the inhibitory neurons involved in each illness, they could develop more sophisticated ways of helping hundreds of millions of people around the world who suffer from these debilitating brain diseases. g Lauren Aguirre is a science journalist whose work has appeared in the PBS series NOVA, The Atlantic, Undark Magazine, and STAT. Read an excerpt from The Memory Thief at the-scientist.com.

06. 2021 | T H E S C IE N T IST 51

FOUNDATIONS

Leader of the Pack, 1903–1994

R

uth Ella Moore, the first Black woman in the United States to get a doctorate in the natural sciences and to join the American Society for Microbiology (then the Society of American Bacteriologists), would also become the first woman to head up a department at Howard University. The mold-breaking scientist had diverse research interests and was a dedicated teacher and mentor.  Moore was born in 1903 in Columbus, Ohio, where she grew up with her parents and two older brothers. She earned her bachelor’s, master’s, and history-making doctoral degree in microbiology at The Ohio State University. Moore’s 1933 dissertation was on the bacterium that causes tuberculosis. At the time Moore was going through school, very few universities in the US were admitting Black students—Ohio State was one of the few—and virtually none hired Black faculty. Historically Black colleges and universities would be the only realistic chance for Moore to work in academia, and she began teaching at Howard University’s medical school in 1933. Moore was the sole author of a 1938 paper in The Dentoscope, a publication from Howard’s School of Dentistry, that discusses the association of Lactobacillus acidophilus (then known as Bacillus acidophilus) with dental caries, or cavities. She noted that the bacteria were abundant in mouths where tooth decay was present and largely absent where it wasn’t. Thinking the bacteria might play a role in the formation of the caries, she hypothesized that the saliva in caries-free mouths might have antibiotic properties, or that high-carb diets might promote growth of the bacteria. (Only in this century would it be understood that the presence of L. acidophilus is a result, not a primary cause, of dental caries: the microbe can only survive in the mouth if decay lesions are already present, and then it contributes to their progression.)

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

POMP AND CIRCUMSTANCE: Ruth Ella Moore graduates with her bachelor’s degree from Ohio State, circa 1926.

In 1948, Moore was appointed acting head of Howard’s Department of Bacteriology, and in 1955 its head, becoming the first woman to lead a department in the school’s history. She held the position until 1958. She was the driving force behind the decision to change the department’s name to the Department of Microbiology, which better reflected the scope of the rapidly evolving field. While at Howard, Moore investigated how sensitive certain bacteria were to antibiotics. She was promoted to associate professor and continued to teach at Howard until 1973, but there is no record of her ever receiving tenure. Moore faced racism and misogyny throughout her career, explains microbiologist Marian Johnson-Thompson, a

professor emerita at the University of the District of Columbia who met Moore at American Society for Microbiology (ASM) meetings. For example, Jim Crow laws made it difficult for Moore to attend ASM meetings, especially when they were held in the South, where Black members weren’t allowed to stay at the same hotels as their white colleagues. Some conference venues were strict about which rooms Black meeting attendees could be in and wouldn’t permit them to eat lunch on the premises.  “She [was] a very proud woman,” recalls Johnson-Thompson. “She did not complain at all about the fact that she was denied participation.” In those days, Johnson-Thompson explains, it was easy to be labeled a troublemaker, and what few opportunities Moore did have could have been taken away. At Howard, Moore focused above all on teaching and mentorship. Her dedication to up-and-coming Black microbiologists was recognized in 1986 when the ASM’s Minority Committee gave her a symbolic lifetime achievement award. In her spare time, she was a talented seamstress who created sleek, modern looks. A collection of her garments is on permanent display at Ohio State. Despite Moore’s multiple firsts, many details of her life and career have been lost to time. The failure of her contemporaries to record such details is “another manifestation of racism and sexism,” says Douglas Haynes, vice chancellor for equity, diversity, and inclusion at the University of California, Irvine, who has written about the gender and racial inequality Moore experienced. “In the eyes of people in authority, Black women did not merit attention.” Moore retired from Howard in 1973. She passed away in 1994 in Rockville, Maryland, at the age of 91. In 2005, the US House of Representatives passed a bill posthumously honoring Moore and other Black female trailblazers in STEM for their contributions. g

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