FALL 2023, VOL. 37, ISSUE 3 The Scientist

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FALL 2023 | VOL. 37, ISSUE 3 | WWW.THE-SCIENTIST.COM

TO UNDERSTAND HOW MEMORIES ARE FORMED AND MAINTAINED, NEUROSCIENTISTS TRAVEL FAR BEYOND THE CELL BODY IN SEARCH OF ANSWERS.

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Finding Solutions for Neurodegenerative Diseases Extensive Range of High-quality Recombinant Proteins and Antibodies to Support Neuroscience Research

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3JZWFQ8YJR(JQQX A Panel of Differentiation Markers and Growth Factors Find out More Therapeutic Targets for Neurodegenerative Diseases

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Contents MODIFIED FROM © ISTOCK.COM, WILDPIXEL; © ISTOCK.COM, RUNGRUDEE; MODIFIED FROM © ISTOCK.COM, KOTO_FEJA; MODIFIED FROM © ISTOCK.COM, ARTEMISDIANA

FALL 2023 | WWW.THE-SCIENTIST.COM | VOL. 37, ISSUE 3

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ON THE COVER: MODIFIED FROM © ISTOCK.COM, KOTO_FEJA

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Natural High: Endogenous Psychedelics in the Gut and Brain

A Chronic Itch: Burrowing Beneath the Skin

Defying Dogma: Decentralized Translation in Neurons

AI Has the Last Word

We have barely scratched the surface of itch science and what it indicates about our health.

To understand how memories are formed and maintained, neuroscientists travel far beyond the cell body in search of answers.

Psychedelics are evolutionarily ancient compounds produced by fungi, plants, and microbes. Humans also synthesize psychedelics. Researchers want to know how and why.

BY BRIAN KIM, PhD

A renaissance in natural language modeling may help researchers explore how the brain extracts and organizes meaning. BY NATALIA MESA, PhD

BY DANIELLE GERHARD, PhD

BY IRIS KULBATSKI, PhD

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Department Contents FROM THE EDITOR

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The CRISPR Family Just Got Bigger

Two groups independently discovered that Fanzor proteins in eukaryotic organisms are CRISPR’s genomeediting cousins.

Onward and Upward!

At The Scientist, we are strengthening our roots while reaching for the sky. BY KRISTIE NYBO, PhD

BY IDA EMILIE STEINMARK, PhD

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CROSSWORD

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Biosensors Illuminate Talk Between Neurons

First developed in 2013, a fluorescent indicator has evolved to enable precise glutamate tracking. BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

20 What Makes Hair Turn Gray? Hair-coloring stem cells must swing 67 The Expansion of Volume back and forth between their maturity Electron Microscopy states to give hair its color. A series of technological advanceBY MARIELLA BODEMEIER LOAYZA ments for automation and parallel Microscopes were inaccessible to most CAREAGA, PhD imaging made volume electron of the world until Manu Prakash and microscopy more user friendly Jim Cybulski put their engineering while increasing throughput. prowess to the test. 21 Molecular Signatures of a Broken Heart BY DANIELLE GERHARD, PhD The transcriptional profiles in the brains of BY DANIELLE GERHARD, PhD prairie voles changed after a long breakup, revealing a molecular shift that might help 12 Making Sense of Nonsense them cope with the loss of a partner. A discovery that goes back to the first studies of translation has become BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD the topic of biotech buzz. FOUNDATIONS

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Magnifying Curiosity with a Pocket Microscope

BY IDA EMILIE STEINMARK, PhD PROFILE

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Matthew Disney’s idea of small molecules that target RNA once seemed fanciful. Now, even the pharma industry is pursuing it.

By coloring different organelles simultaneously, cell painting allows scientists to pick up subtle changes in cell function in response to drugs and other perturbations.

BY IDA EMILIE STEINMARK, PhD

58 Lighting Up Diagnostics Brought together by a shared interest in synthetic biology and diagnostics, two researchers are transforming how we label biomolecules.

LITERATURE

Mice Heal Themselves in Response to a Common Signaling Molecule

BY DANIELLE GERHARD, PhD

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A B S S 13 14 15 M I C A T 17 18 8 O L A V R 20 21 S E R O T O 23 C O P 26 D R A W 25

BY IDA EMILIE STEINMARK, PhD

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Since the technique was first published in 2019, prime editing has grown with lightning speed, alongside hopes for what it can achieve.

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Prime Editing Comes of Age

B O O A R 38 B I N O I D 41 S O A R S 44 4 A L T A 47 U L I 3 5 53 54 55 T A M A T E 58 U I D E A

BY IDA EMILIE STEINMARK, PhD

METHODS

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How will a new version of epigenetic clocks aimed at validating the age of people older than 100 years of age balance accuracy and anonymity?

S A I D C O U G 36 37 E N D O C A N N A 39 40 A C E A C T O R 3 42 43 L E S O T H O 45 46 6 B E E P 8 50 1 48 49 51 52 G L U E T H E R 57 Y O U R N A U R

BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

New Epigenetic Clocks May Confirm Extreme Age

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Timothy Hand leads a research team that explores how maternal immune signals shape the infant intestinal microbiota.

BY IDA EMILIE STEINMARK, PhD

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When Microbes Meet the Immune System

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A newly discovered way to induce scarless healing in mice depends on a highly conserved signaling pathway that is also present in humans.

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BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

A Quest to Drug RNA

E R G O S E E N

Cell Painting: Exploring the Richness of Biological Images

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FROM THE EDITOR

Onward and Upward! At The Scientist, we are strengthening our roots while reaching for the sky. BY KRISTIE NYBO, PhD

A

s SARS-CoV-2 burst onto the scene in late 2019, the world clamored for information as basic, translational, and clinical research suddenly became the topics of household conversations. Misinformation spread like wildfire, and everyone from middle school pupils to government leaders began questioning if what they heard and read was trustworthy. The Scientist stepped up to the challenge. We leaned heavily into the public health space to provide accurate, up-to-date information, and we widened our reach to ensure that scientists and nonscientists alike had the details they needed to choose their own paths through the pandemic. Now, as the dust settles following these turbulent years, like many of you, we at The Scientist are learning from our experiences and refocusing our priorities for the future. The Scientist originally launched with scientist readers in mind. With the hunger for pandemic-related information fading from public view, we are returning to our roots and rededicating ourselves to serving the needs of life science researchers at the bench. To meet this goal, we have searched among our readers to assemble our team. Each of our editors joins us with a PhD degree in the life sciences, years of experience conducting research , and extensive science communications expertise, which you will see as you read their enthralling stories. As a team, we are tuned in to your scientific interests, entertainment needs, bubbling curiosities, and networking wishes because we are scientists ourselves. I’m excited to introduce you to your content creation team today! In building our refocused editorial strategy, we have relied heavily on your recommendations and guidance. We are extremely grateful for the messages you have sent via email, social media, and forms on our website letting us know what topics you are eager to read about and how you like to see those stories presented. With this

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feedback in mind, we have exciting plans to increase the number of feature stories we run in each print issue and online, expand our conference coverage, introduce new columns, and present the topics of utmost interest to our readers in a relatable, relevant, and engaging way. We also have several exciting new developments for our online programs. In support of your desires to stay up to date on the latest advances and discoveries in the life sciences while also productively working your way through long to-do lists, we introduced the TS Digest in July 2023 and will publish new issues monthly going forward. This new publication focuses on presenting fun, bite-sized content such as flash nonfiction, miniature podcasts, videos, infographics, puzzles, interviews, and surprising columns such as “Just Curious” and “Epic Fail” that come directly from our readers’ questions and experiences. You can peruse the mobile-friendly TS Digest while waiting for a gel to run, a meeting to start, a quick centrifuge spin to finish, or any other time when you have only a few free minutes. In addition, we proudly launched our new journal club this summer, which presents research published by you, our readers, in an online interactive webinar format. We would love to have you join us for these world-wide journal club discussions, either as a listener or as a presenter. In January 2024, we will launch a new newsletter program that is uniquely tailored to your interests. We’ll present the latest breaking science news, long- and short-form stories, podcasts, webinars, and resources to propel your research forward. We will focus in on your preferences to ensure that each piece of content we send is relevant to you. You can tailor your own reading experience by updating your newsletter preferences ahead of that change. We are confident that these changes will better meet your needs and create a pub-

lication that you can’t put down or scroll past. Of course, to achieve this goal and our overarching goal of “exploring life, inspiring innovation,” we need your help! Keep an eye on your inboxes; we plan to conduct a reader survey in the coming weeks to find out exactly what you think of these changes, and we eagerly await your feedback, story topic requests, and research experiences to highlight in our magazine. J

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MEET THE NEW TS TEAM

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KRISTIE NYBO, PhD Group Content Director Kristie joined The Scientist in 2019 with 17 years of experience in life science publishing. Prior to joining, she directed news content and managed peer review for the journal BioTechniques for more than a decade. She has also served in public relations communications and medical writing and editorial roles. Kristie earned her PhD at the University of California, Los Angeles (UCLA) in neuroscience and conducted research at UCLA and at the National Institute of Environmental Health Sciences on brain development and signal transduction in mouse models. She currently also serves as content director for Drug Discovery News.

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MEENAKSHI PRABHUNE, PhD Editor in Chief Meenakshi joined The Scientist in 2023. She is passionate about disseminating science and brings several years of experience in diverse communication roles including journalism, podcasting, and corporate content strategy. Meenakshi earned her PhD in biophysics at the University of Goettingen, which sparked a life-long love for interdisciplinary biological sciences.

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MELISSA KAY MSc | Engagement Specialist Melissa joined The Scientist in August 2023 as the engagement specialist. She holds a master’s of science degree in cellular and molecular biology and a graduate diploma in science communication from Laurentian University. Melissa leads the social media strategy and audience engagement efforts for The Scientist and Drug Discovery News.

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NIKI SPAHICH, PhD Senior Science Editor, Team Lead Niki earned her PhD in genetics and genomics from Duke University, where she studied membrane proteins responsible for Haemophilus influenzae infectivity. She shifted her research focus to mechanisms of Staphylococcus aureus virulence during her postdoctoral fellowship at the University of North Carolina Chapel Hill. After various teaching and science communication experiences, Niki joined The Scientist’s creative services team in 2019. She is currently a senior science editor who leads content creation for the team.

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DANIELLE GERHARD, PhD Assistant Editor Danielle earned her PhD in psychology and behavioral neuroscience from Yale University and completed postdoctoral research in neuroscience and psychiatry at Weill Cornell Medicine. During her graduate and postgraduate training, she examined cellular and molecular mechanisms underlying stress and depression. In April 2023, Danielle joined The Scientist’s editorial team as an assistant editor. Her writing has also appeared in Drug Discovery News and BioTechniques. She is based in the UK.

NATHAN NI, PhD Senior Science Editor Nathan earned his PhD in physiology from Queen’s University in 2013, where he investigated the role of inflammatory leukotriene pathways in exacerbating cardiac injury during myocardial infarction. He then completed a two-year postdoctoral training stint in Toronto’s University Health Network, where he explored the effects of aging on stem cell effectiveness. Nathan joined The Scientist in 2016. He now serves as a senior science editor and leads the scientific services program, which offers editorial and design services directly to scientists.

MARIELLA BODEMEIER LOAYZA CAREAGA, PhD | Assistant Editor Mariella joined The Scientist in 2023 after completing postdoctoral research at the Uniformed Services University of the Health Sciences studying sex differences on the effects of chronic stress and traumatic brain injury. Mariella holds a PhD in neuroscience from the Universidade Federal de Sao Paulo, and a certificate in science communication from the University of California, San Diego.

IRIS KULBATSKI, PhD Assistant Science Editor Iris, a neuroscientist by training and word surgeon by trade, is an assistant science editor with The Scientist’s creative services team. Her work has appeared in various online and print publications, including Discover Magazine, Medgadget, National Post, The Toronto Star, and others. She holds a PhD in medical science and a certificate in creative writing from the University of Toronto.

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LAURA TRAN, PhD Assistant Editor Laura joined The Scientist as an assistant editor in 2023. She has a background in microbiology and earned her PhD in biomedical sciences from Rush University. Her research focused on how circadian rhythms and alcohol affect the gut.

DEANNA MACNEILL, PhD Assistant Science Editor Deanna earned their PhD from McGill University in 2020, studying the cellular biology of aging and cancer. They have an endless curiosity about telomeres. Deanna has a multidisciplinary academic background ranging from chemistry to metacognition to microbiology. They joined The Scientist’s creative services team in 2022.

SHELBY BRADFORD, PhD | Assistant Editor Shelby earned her PhD in immunology and microbial pathogenesis from West Virginia University, where she studied neonatal responses to vaccination. She completed an AAAS Mass Media Fellowship at StateImpact Pennsylvania, and her writing has also appeared in Massive Science. She participated in the 2023 flagship ComSciCon and volunteered with science outreach programs and the Carnegie Science Center during graduate school. Shelby joined The Scientist as an assistant editor in August 2023.

CHARLENE LANCASTER, PhD | Assistant Science Editor Charlene earned her PhD in cell biology from the University of Toronto, where she studied how macrophages resolve phagosomes. She has also conducted research on how vitamins can increase bone formation in osteoblast cell culture. She joined The Scientist’s creative services team in 2023.

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BD CELLVIEW™ IMAGE TECHNOLOGY:

Bringing New Images to Flow Fluorescence-activated cell sorting (FACS) and microscopy have long been trusted methods for scientists to study single cells within a population. Previous attempts to combine the two technologies have been met with the challenge of speed, or rather the lack thereof. Current camera-based image technologies are not fast enough to support high-throughput cell sorting at speeds ranging from thousands to tens of thousands of events per second. T e novel o e BD CellView™ Image Technology overcomes this challenge by using fluorescence real-time imaging The with w t radiofrequency-multiplexed ency-multiplexed excitation. Combining BD CellView™ Image Technology and high-throughput cell c ll sorting g unlocks l k new and diverse applications beyond immunology, including oncology, cell biology, plant b biology, microbiology, p l gy, and genomics.1,2

BD D CELLVIEW™ C IMAGE G FEATURES3 BD CellView™ Ce e Image ge Technology y enables e bles imaging using scatter and fluorescent detectors to reveal the internal and characteristics a d external e e l spatial p c ce c of a cell, without the use of a traditional camera. In flow w cytometry, y y, fluorochrome e stained ained cells pass through laser beams one at a time, producing signals that are collected electronic With co ec e as e ec o c pulses. p th BD CellView™ Image Technology, the blue laser beam is directed through an acousto-optic a p deflector e ec or and a d split p t into 100 separate beams. The unique optics cause each beam to blink at its own cell p passes through ffrequency. q y Ass the e ce hrough the array of beams, each beam generates a signal that is collected onto a PMT. Frequencies q i s are e mathematically lly distinguishable, making it possible to separate each beam from the complex combined signal g l generated g d by y a passing g cell. The frequencies are constructed to produce a complete 2D image of the cell. With W t BD CellView™ Ce e ™ Image ge Technology, traditional flow parameters like scatter and fluorescence can be combined with w th iimage ge features e e such ch as eccentricity, total intensity, size, and many more. In addition, a light loss detector collects co ec s the e incident c e light ght as it passes through a pinhole to produce images similar to brightfield images collected on a standard a da d microscope. cope. ECCENTRICITY is the ratio of the shortest and longest axis of a particle. Low eccentricity indicates that the axes are close to the same length, while high eccentricity represents a signal with variable axes lengths. MAX INTENSITY is measured as the brightest pixel in an image.

SIZE is the number of pixels that are brighter than the defined background brightness.

RADIAL MOMENT is the average distance of the signal from the center of the region of analysis. When measuring the location of two fluorescent signals relative to one another, CORRELATION is the degree of signal overlap.

DELTA CENTER OF MASS is the distance between the two fluorescent signals.

DIFFUSIVITY is a measure of how concentrated or spread out a signal is within the cell.

Spatial Sorting: Phagocytosing Cells Antigen presenting cells (APCs) engulf pathogens such as bacteria by phagocytosis, which is crucial for immune surveillance and activation. After phagocytosis, APCs present pieces of the pathogen on their cell surface to activate T cells for a strong immune response. Detecting phagocytosis by flow cytometry has been technically challenging because a signal on the cell surface is indistinguishable from a signal within the cell by traditional technologies. BD CellView™ provides confirmation of signal location to accurately visualize phagocytosis, distinguishing between bound and internalized particles.

Scientists studied the phagocytosis of pHrodo™ Green E. coli BioParticles™ with BD CellView™ Image Technology. After incubating these green particles with fibroblasts (NIH3T3), the researchers evaluated cells based on features including LIGHT LOSS, RADIAL MOMENT, and ECCENTRICITY. Phagocytosis of pHrodo Green E. coli BioParticles by fibroblasts was confirmed with real-time imaging features to identify fibroblasts containing green fluorescence.

THE SCIENTISTS BEHIND THE RESEARCH4 This work was carried out at VIB Flow Core by Dr. Tania Løve Aaes (Postdoctoral Researcher, VIB-UGent Center for Inflammation Research), Julie Van Duyse (Flow Cytometry Expert, VIB Core), and Gert Van Isterdael (Head of Flow Core, VIB).

This approach has important implications for future infectious disease research. BD CellView™ Image Technology provides the tools to detect and enumerate phagocytosis in both classical (e.g. macrophages) as well as nonclassical (e.g. fibroblasts) phagocytes, and also sort these cells for downstream applications including single cell RNA sequencing and cytokine/chemokine secretion studies of function and plasticity. plast .

Achieving Cleaner Resolution of Solid Samples From the discovery of new cell types to unraveling complex signaling networks that shape root development, single cell genomics is redefining plant biology research. However, scientists have not widely adopted single cell genomics in plants due to sample preparation challenges. Clearing plant cell wall debris is essential for high quality single cell experiments. Historically, researchers have relied on cell sorters that use light scattering to distinguish single cells from debris, but larger plant cells can be similar in size to cell doublets and debris, making it difficult to cleanly isolate plant cells of different sizes.4,5

THE SCIENTISTS BEHIND THE RESEARCH4 This work was carried out at VIB Flow Core by Dr. Moritz Nowack (Professor, VIB-UGent Center for Plant Systems Biology), Dr. Rafael Buono (Postdoctoral Researcher, VIB-UGent Center for Plant Systems Biology), and Gert Van Isterdael (Head of Flow Core, VIB).

Sorting Cells by All Major Mitotic Phases

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Cell division is essential for growth and development, and defects in mitotic regulation are commonly associated with cancer. One challenge scientists face when studying mitosis with traditional methods is identifying and isolating cells in each stage of division, without chemically blocking the cell cycle. Researchers at EMBL developed an approach using BD CellView™ Image Technology for sorting large numbers of cells throughout the cell cycle. This novel assay bypasses the need for chemical enrichment of cell cycle phases and provides a tool for sorting cells in all major mitotic stages, including anaphase and telophase, both of which were previously inaccessible by any method.1

H2b-mNG

MI (H2b- mNG)

BD CellView™ Image Technology can assist plant researchers with sample cleanup challenges in their single cell genomics experiments. By combining traditional fluorescent parameters with image-based features such as ECCENTRICITY and SIZE, scientists were able to identify and sort both large and small Arabidopsis root protoplasts while excluding cell doublets and debris. Low ECCENTRICITY protoplasts were further enriched for eGFP expression to isolate cells of a specific lineage, developmental stage, or gene editing status. With BD CellView™ Image Technology, these researchers were able to make rapid and confident sorting decisions, confirmed by real-time images of each gated population during sorting.4

The highly enriched protoplasts obtained with BD CellView™ Image Technology are ideal for downstream single cell and bulk genomic applications, such as transcriptomics and functional genomic screening. BD CellView™ Image Technology has opened the gates to isolating cleaner and less biased samples from plant tissues than traditional cell sorters. 4

MI (H2b- mNG)

Eccentricity (SSC)

pS10 AF647

Eccentricity (SSC)

Researchers engineered cells expressing the histone H2B tagged with a fluorophore called mNeonGreen (H2B-mNG). This tagged histone enables visualization of changes to the chromatin as a cell progresses through mitosis. The scientists identified G2 and mitotic cells (G2/M) based on total DNA content, which has historically been the extent of cell cycle resolution using flow cytometry. Based on FSC and SSC images, the researchers derived RADIAL MOMENT and ECCENTRICITY features that provided information about cell morphology to further differentiate cells in anaphase and telophase. The image-enabled MAXIMUM INTENSITY of H2B-mNG fluorescence helped distinguish cells in different mitotic phases based on chromatin compaction and distribution within the cell.1

Eccentricity (FSC)

Eccentricity (FSC)

Image-enabled visualization of cell morphology and chromosomes, combined with high-speed sorting, allowed researchers to purify populations of cells at each step of mitosis without the need for chemical cell cycle blockers. Cells were sorted with BD CellView™ Image Technology by visualizing stage-specific subcellular molecular changes, such as chromosome segregation, which traditional flow cytometry methods cannot capture.1 Sorted cells can be analyzed for downstream applications, for example evaluation of the transcriptome, proteome, or epigenome. High-speed enrichment of cells based on imaging features provides a powerful new tool for basic research, cell-based diagnostics, cell atlas efforts and high-content image screening.

THE SCIENTISTS BEHIND THE RESEARCH1 This work was carried out at EMBL by Dr. Daniel Schraivogel (Research Staff Scientist, EMBL) and Dr. Terra Kuhn (Postdoctoral Researcher, EMBL).

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Eccentricity (Light Loss)

Once sorted using BD CellView™ Image Technology, cells can be analyzed for specific molecular changes such as transcriptomic, proteomic, or epigenetic profiling. BD CellView™ Image Technology enhances FACS methods with live visual inspection of target cells and novel gating strategies based on real-time images and the spatial distribution of fluorescence signals, a unique capability among cytometers. Combining the spatial information of images with the speed of flow cytometric cell sorting has broad implications for dreaming up new sorting-dependent experimental strategies.1-3

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ABOUT BD As pioneers of the fluorescence activated cell sorting (FACS) technology, BD Biosciences has provided flow cytometry expertise, world class support and continuous innovation over more than 45 years and enabled the routine or complex FACS analysis needed to propel the goals of laboratories around the globe. Our work and our technology are currently changing how the world looks at an individual cell. Every day we partner with our customers to help reveal the identity and function of billions of cells, one at a time. True to BD’s mission of advancing the world of health™, we continue to be at the forefront of discovery to shape the future with new innovations in informatics, genomics, proteomics and image-based sorting, and we proudly offer a large and extensive portfolio of products to address the needs of our customers.

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REFERENCES 1. D. Schraivogel et al., “High-speed fluorescence image-enabled cell sorting,” Science, 375(6578):315-20, 2022. doi:10:1126/science.abj3013 2.

D. Schraivogel, L.M. Steinmetz, “Cell sorters see things more clearly now,” Mol Syst Biol, e11254, 2023. doi:10.15252/msb.202211254

3.

“Take a Behind-the-scenes Look at BD CellView™ Image Technology,” BD Biosciences, https:// www.bdbiosciences.com/en-us/learn/campaigns/cell-view-image-technology#Technology, accessed August 11, 2023.

4.

J. Van Duyse et al. “Improving Plant protoplast cell sorting outcomes with high-speed fluorescence image-enabled cell sorting.” Poster presented at: CYTO 2022, June 5, 2022. Philadelphia, PA

5.

J.R. Wendrich et al., “Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions,” Science, 370(6518):eaay4970, 2020. doi:10.1126/science.aay4970

CROSSWORD

What’s the Transmission? 1. External obliques et al., for short 4. Triangular bones of the lower back 9. Taken by mouth 13. Silicate with a layered structure 15. Test of a drug’s effectiveness, for example 16. 1-Across location 17. Name of five Norwegian kings 18. Fix a loose shoelace, perhaps 19. Polydactyl cats have extra ones 20. A neurotransmitter that affects mood and emotions 22. Unconscious periods 23. Certain Egyptian Christian 24. Drink slowly 25. Use the BioRender program, perhaps 27. Parts of hearts 31. Spoke aloud 32. Big cat, also known as a mountain lion 35. React to a ref’s bad call 36. One of a class of lipid-based neurotransmitters 39. Target of some hypertension drugs: Abbr. 40. Members of a cast 41. Propellers of canoes 42. Country enclosed by South Africa 44. High, in Spanish 45. Member of the family Apidae 46. Dog breed known for its corded coat 48. Old anesthetic with the formula (C2H5)2O 51. Most abundant excitatory neurotransmitter in vertebrates 56. Second-person possessive 57. Country where the Micronesian imperial pigeon can be found 58. Evolution by natural selection is a big one 59. Therefore 60. Open sore in the stomach’s lining 61. Large amounts 62. Observed with the eyes 63. Not available, as a seat 64. Sault ___ Marie, Michigan

DOWN 1. 2. 3. 4.

Cognitive psychologist Tversky Substance stored by the gallbladder Evidence of an old surgery Device for sharpening a razor

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ACROSS

© 2023

5. “___ we all?” 6. ___ Field (home stadium of the New York Mets) 7. Liquid precipitation 8. Liquid in a yard 9. Eight-armed cephalopods 10. Laboratory or office, for example 11. Cell surface ___ 12. Not as much 14. Fruit that matures on a tree but ripens off the tree 21. Drag behind 22. Wispy clouds 24. Impales 25. Certain mating ritual 26. Goes by car, bike, or horse 27. Common growth medium in a microbiology lab 28. WWII menace 29. “Schitt’s Creek” role played by Catherine O’Hara 30. Lays down turf 31. It should be airtight to create a vacuum

32. Temporary data storage 33. Not fooled by 34. Colorful card game 37. Provide food and drink 38. Betting feature of some poker games 43. Uranus moon named for a Shakespearean fairy king 44. In the style of 46. Culinary mash-up? 47. Reversal of direction 48. What ommatidia make up 49. Ripped 50 Like blue whales 51. Fancy party 52. Uncontrollable factor in some scientific discoveries 53. Big fusses 54. Structure for a geological researcher, perhaps 55. Lack of difficulty 57. Fruit with a woody wall

Answer key on page 5 FALL 2 02 3 | T H E S C IE N T IST

9

FOUNDATIONS

Magnifying Curiosity with a Pocket Microscope Microscopes were inaccessible to most of the world until Manu Prakash and Jim Cybulski put their engineering prowess to the test. BY DANIELLE GERHARD, PhD

I

n 2011, while waiting for his new lab to be built at Stanford University, bioengineer Manu Prakash traveled to labs around the world, including in his home country of India. When he came across limited or broken microscopes in several labs, Prakash was disappointed. “Almost two-thirds of all biological and life science insights and research work utilize microscopes, while very few people have access to microscopy as an infrastructure,” said Prakash. With a new purpose for his bioengineering knowledge, he set out to build an affordable multipurpose microscope. “Science has a crisis associated with access and affordability,” said Prakash. A basic light microscope that costs at least $100 could be a big amount for small labs in developing countries or impractical for use outside of the lab. Inspired by pencils and ballpoint pens, the most accessible and utilized tools he could think of, Prakash set the price point for his microscope to be produced at a mere $1. The challenge that remained was to actually build it.

Kirigami microscopes take shape

To address cost, the team looked to flat manufacturing or printing microscopes. After throwing out what seemed like hundreds of failed ideas, they landed on the Foldscope, an optical microscope based on kirigami, a variation of origami that involves cutting along with folding. The engineering duo had to consider several features and functions in their thrifty design, including the lens material, aperFOLDSCOPE INSTRUMENTS, INC; © ASHLEIGH CAMPSALL

Around this time Jim Cybulski, a mechanical engineer who is now president of Foldscope Instruments, Inc., joined Prakash’s lab as a graduate student. He teamed up with Prakash to design the $1 microscopes. “The first challenge was trying to get the same level of performance as a conventional microscope but at a very low cost,” said Cybulski.

The Foldscope is a portable, water-proof microscope that is small enough to fit in a pocket.

1 0 T H E SC I EN T I ST | the-scientist.com

ture, focus, and panning. Buried in the design is a hefty mathematical calculation on how to make ultrasmall lenses.1 “What is not obvious to people when they play with this is that there are optics the size of a grain of salt in it that enable all of this to be possible,” said Prakash. The resulting Foldscope’s microscopic ball lens is made from borosilicate glass, a cheap yet effective material that fits into a small piece of plastic designed to provide a single optimal aperture. After placing a sample on a glass slide, a three-dimensional kirigami design feature allows users to toggle the lens to pan and zoom in on the sample using their thumbs. Light from the sun, a lamp, or light-emitting diode (LED), passes through the sample, aperture, and lens to reach the eye and magnify the sample. This pocket-sized inexpensive design achieves approximately two-micron resolution and 140X magnification. Using a magnetic coupler, users can attach the kirigami microscope to a phone camera to attain even greater magnification. “Frugal science has the connotation in many people’s minds that somehow frugal also means low tech,” said Krishnaswamy VijayRaghavan, who was a developmental biologist at the National Center for Biological Sciences and former principal scientific advisor to the government of India. He was not involved with the development of the Foldscope. “What is admirable about [Prakash’s] approach is it might be frugal, but it’s extraordinarily complex science.” After unveiling their technology in a publication in 2014, the team soon realized that they had another problem to solve.1 Prakash recalled an air of skepticism and confusion around what they were trying to do. To convince the scientific community of the Foldscope’s merit, they needed to get the kirigami microscope into people’s hands.

FOLDSCOPE INSTRUMENTS, INC

A microcosm of curiosity Prakash wanted to do more than build a tool. “Communities are far more powerful because tools will come and go,” said Prakash. His team planned to assemble 10,000 Foldscopes, but after a flood of applications poured in from around the globe, they overshot their goal. They shipped off 70,000 Foldscopes for free, no rules attached. “We were not asking anything of anyone. We were just sharing the tool and wanted people to be curious and explore,” said Prakash. To provide a space for this growing community to flourish, the team created Microcosmos. “It’s a huge database that’s collected by our users almost like Wikipedia for the microscopic world,” said Cybulski. By magnifying the world around them, Foldscope users have achieved a lot, including discovering a new strain of cyanobacteria from a drinking water pipeline in Pune, India and screening soil samples for microorganisms that produce the agricultural and medicinal enzyme naringinase.2,3 The Foldscope also serves as a platform for a harmful algal bloom monitoring program run by the Open Field Collective.

Manu Prakash (left) and Jim Cybulski (right) won a Golden Goose Award in 2022 for the Foldscope.

In the spirit of using Foldscope for curiosity, Prakash has made his own amazing discoveries. While on a vacation in Lake Tahoe with his kids, he peered through a Foldscope at a water sample from the lake when a cell he was examining seemingly disappeared right before his eyes. He took the sample back to the lab, cultured the cells, and made the discovery that cells can talk to each other using pressure waves.4 Although there are potential applications in diagnostics, animal health, agriculture, and public health, the Foldscope is primarily an educational tool. “When you get kids together, it’s stunning how much enthusiasm there is, and it just transforms the kids into questioning,” said VijayRaghavan. “It is a magnet for curiosity.” Cybulski and Prakash officially launched the company Foldscope Instruments, Inc. in 2016, and they continue their quest to provide affordable, user friendly microscopes. The team recently launched a new and improved Foldscope 2.0. “Although we are very proud of where we are, we also know we have a long way to go,” said Prakash. “That is why this is the type of a project that is a lifetime goal, not just something that we do and put aside.” J

References 1. Cybulski JS, et al. Foldscope: Origami-based paper microscope. PLoS One. 2014;9(6):e98781. 2. Nitnaware KM, et al. Whole-genome characterization and comparative genomics of a novel freshwater cyanobacteria species: Pseudanabaena punensis. Mol Phylogenet Evol. 2021;164:107272. 3. Patil SV, et al. A novel screening method for potential naringinase-producing microorganisms. Biotechnol Appl Biochem. 2019;66(3):323-3277. 4. Mathijssen AJTM, et al. Collective intercellular communication through ultrafast hydrodynamic trigger waves. Nature. 2019;571:560-564.

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FOUNDATIONS

Making Sense of Nonsense A discovery that goes back to the first studies of translation has become the topic of biotech buzz. BY IDA EMILIE STEINMARK, PhD

B

20 years ago, I was literally dreaming about how [tRNAs] could be applied. —Caroline Köhrer, Alltrna

“In other words, you have a mutation in a gene that you put in E. coli A and it doesn’t produce any protein,” said RajBhandary. “But if you put it into E. coli B, it will now produce the protein. So, E. coli B has something that suppresses the effect of the original nonsense mutation.” 1 2 T H E SC I EN T I ST | the-scientist.com

Seymour Benzer, shown here holding a large Drosophila model, was one of the pioneers of the tRNA field.

Benzer had previously documented tRNAs as “adaptors” of mRNA to amino acids,4 so he suggested that they might play a role. That suspicion was confirmed three years later when Mario Capecchi and Gary Gussin, then at Harvard University, identified a nonsense codon suppressing tRNA in a bacteriophage.5 “These bacteria and phages actually use them regularly, often as part of a stress response,” said Caroline Köhrer, an RNA scientist who worked alongside RajBhandary for 20 years at MIT. She is now at Alltrna, a biotechnology company working on suptRNAs. “In the 1960s and 1970s, there was a quite a bit of work on naturally occurring tRNAs, but of course, that is quite different from something you can use as a therapy.” In 1982, researchers finally attempted to use sup-tRNAs against a disease.6 At the University of California, San Francisco, geneticist and hematologist Yuet Wai Kan wanted to treat a version of the blood disorder `-thalassemia caused by a nonsense

WILLIAM A HARRIS

etween 10 to 15% of all genetic diseases have the same underlying cause: a nonsense mutation.1 This type of mutation results from a stop codon in the middle of a gene sequence, which terminates translation prematurely and leads to half-finished proteins. Instead of trying to develop cures for these diseases one by one, some scientists and start-up companies now believe that a single drug could target and fix the nonsense mutations in many diseases at once. That drug is a nonsense suppressor tRNA. Nonsense suppressor tRNAs, or sup-tRNAs, read through premature stop codons in the mRNA caused by nonsense mutations, allowing protein synthesis to continue. It’s easy to see how that might make a handy therapy, and although it has only recently appeared on the biotech radar, it’s an idea that stretches back to the very beginnings of the RNA biology field. “This research has been going on forever, really since understanding translation,” said physiologist John Lueck who researches suptRNAs at the University of Rochester. Retired RNA biologist Uttam RajBhandary, formerly of Massachusetts Institute of Technology (MIT), remembers the beginning of that research. “It all happened in the early to mid1960s,” he said, recalling the three geneticists then driving the field: Sydney Brenner at the University of Cambridge, Alan Garen at Yale University, and Seymour Benzer at the University of Purdue. Two of their experiments in 1962 were particularly important: one revealed that nonsense codons of bacteriophage mutants were suppressed in some bacterial hosts but not in others,2 and the other showed that a nonsense mutation in one E. coli strain could be overruled by a “suppressor mutation” in a different strain.3

© ISTOCK.COM, SELVANEGRA

mutation. To try and fix the mutation, he and his team mutated a human tRNA gene in a phage/E. coli system to produce a suptRNA and injected it into a frog ovary cell carrying a `-thalassemia patient’s mRNA. “They saw that the tRNA could actually correct the `-thalassemia,” RajBhandary said. Despite this early success, sup-tRNA therapy did not take off. “[It] was this concern that natural termination codons would be suppressed, and that would be toxic,” Lueck said. “Does [sup-tRNA] really do that? That answer is no.” He and his colleagues demonstrated as much in 2019,1 showing that sup-tRNA readthrough of natural stop codons was minimal compared to that of premature stop codons, likely due to extra termination context in the mRNA sequence around natural stops. That wasn’t the only hurdle, however. RNA synthesis is tricky, and the products degrade easily. Additionally, Köhrer said, fifteen years ago, researchers were still asking fundamental questions about the details of tRNA nonsense suppression. With advances in genome sequencing and mass spectrometry, those questions became easier to answer, catapulting the field forwards. To someone like Köhrer, seeing Alltrna’s preclinical mouse work and finally appearing to stand on the cusp of viable tRNA therapeutics gives immense satisfaction. “20 years ago, I was

During normal translation, tRNAs (shown here in dark violet) bring the amino acids (red) corresponding to the codons on the mRNA (multicolored) inside the ribosome (blue/purple), but nonsense mutations stop that process prematurely.

literally dreaming about how [tRNAs] could be applied,” she said. “Imagine my absolute joy and excitement when I think about today.” J

References 1. Lueck JD, et al. Engineered transfer RNAs for suppression of premature termination codons. Nat Commun. 2019;10(1):822. 2. Benzer S, Champe SP. A change from nonsense to sense in the genetic code. Proc Natl Acad Sci USA. 1962;48(7):1114-1121. 3. Garen A, Siddiqi O. Suppression of mutations in the alkaline phosphatase structural cistron of E. coli. Proc Natl Acad Sci USA. 1962;48(7):1121-1127. 4. Chapeville F, et al. On the role of soluble ribonucleic acid in coding for amino acids. Proc Natl Acad Sci USA. 1962;48(6):1086-1092. 5. Capecchi MR, Gussin GN. Suppression in vitro: Identification of a SerinesRNA as a “Nonsense” Suppressor. Science. 1965;149(3682):417-422. 6. Temple GF, et al. Construction of a functional human suppressor tRNA gene: an approach to gene therapy for `-thalassaemia. Nature. 1982;296(5857):537-540.

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FOUNDATIONS

Cell Painting: Exploring the Richness of Biological Images By coloring different organelles simultaneously, cell painting allows scientists to pick up subtle changes in cell function in response to drugs and other perturbations.

Actin cytoskeleton/Golgi

Endoplasmic reticulum

Mitochondria

Nucleoli

Cell Nuclei

Combined

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cientists have been peeking through microscopes and taking pictures of cells for decades. The methods available for visualizing highly complex cells have changed over time, moving from simpler morphology-focused imaging to single-cell specific fluorescence microscopy. Despite these improving approaches, there is a vast amount of information left unexplored in cells, like hidden treasures at the ends of breadcrumb trails. Anne Carpenter, a cell and computational biologist at the Broad Institute, wanted to explore this vastly untouched world. “As a cell biologist, I felt that labeling the major organelles would

1 4 T H E SC I EN T I ST | the-scientist.com

In the cell painting assay, researchers use fluorescent dyes to label different organelles.

It was a phrase we only used informally in the lab. At some point, we thought about adding 30 dyes at once to the cells and calling it “cell graffiti.” —Anne Carpenter, Broad Institute

BROAD INSTITUTE CENTER FOR THE DEVELOPMENT OF THERAPEUTICS

BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

probably give us the broadest readout of how the cells were experiencing their environments,” she said. Changes in the physical characteristics of major organelles can provide great insight into the cells’ states. For instance, in myopathy conditions such as Duchenne muscular dystrophy, mitochondria in muscle and skin cells swell and show reduced matrix density.1 In collaboration with fellow scientists, Carpenter started developing an assay to paint cellular organelles and allow researchers to uncover subtle changes in them. The result was the cell painting assay, which the team introduced in 2013.2

back from this more complex approach to keep the assay easy to use and interpret. “Now that you have a high dimension representation of the state of the cell, any problem that needs to group things together based on biological states is available for being solved,” said Shantanu Singh, a computer scientist who leads a research team with Carpenter at the Broad Institute. One of these applications is predicting the mechanisms of action of compounds, a process that can be time consuming, especially when evaluating the activity of new molecules, said Slava Ziegler, a biochemist at the Max Planck Institute of Molec-

If cells with the disease look differently, you can potentially do a drug screening to identify drugs that make the diseased state cells look healthier. —Anne Carpenter, Broad Institute

Since Carpenter and her colleagues wanted to paint the organelles simultaneously, the first key step was to choose the right combination of organelle-specific dyes to highlight as many features of the organelles as possible. They came up with a mix of six stains that are distinct from each other as they target specific parts of the cellular structures. As an example, the mitochondria dye binds to the organelle’s membrane, whereas the nuclei stain binds to adenine-thymine pairs in DNA. As opposed to antibodies that label specific molecules inside the cells, cell painting dyes provide a less biased way of looking at cellular changes since they stain the organelles as a whole, revealing hundreds to thousands of different morphological features such as textural patterns, sizes, and shapes. To create a profile of the cells, scientists then combine all of the features from the different organelles that are imaged and analyzed using cell imaging software such as CellProfiler, another of Carpenter’s creations. The term profiling, Carpenter explained, describes the process of extracting a large set of morphological features from cell images and letting the patterns that arise lead researchers to discoveries. Early on, the researchers viewed this approach as painting, but they didn’t officially name the assay “cell painting” until later on.3 “It was a phrase we only used informally in the lab. At some point, we thought about adding 30 dyes at once to the cells and calling it ‘cell graffiti,’” she recalled. Eventually, the team pulled

ular Physiology who uses the cell painting assay in her research. “The cell painting assay is more unbiased because it still focuses on a certain feature, which is morphology of the cells, but not on a certain process, like a signaling pathway,” she explained. By using it, “we thought we would get broad coverage of the bioactivity of all of these compounds synthesized in house.” As diseases might alter the morphology of organelles, as in the case Duchenne muscular dystrophy, researchers can also use the cell painting assay to identify profiles associated with a disease and compare those to the profiles of healthy cells. “If cells with the disease look differently, you can potentially do a drug screening to identify drugs that make the diseased state cells look healthier,” Carpenter said. Observing cells through microscopes is a powerful way to look at life on a small scale, Singh explained, and the cell painting assay reveals cellular features that might not have been obvious with other methods. From a qualitative description to the measurement of a few and now thousands of features, Carpenter believes that cell painting is changing the way researchers look at these highly complex biological snapshots. “It is pretty exciting for researchers to use images as a data type as opposed to just confirming what they see by eye,” she said.

References

It is pretty exciting for researchers to use images as a data type as opposed to just confirming what they see by eye. —Anne Carpenter, Broad Institute

1. Pellegrini C, et al. Melanocytes--a novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy. J Cell Physiol. 2013;228(6):1323-1331. 2. Gustafsdottir SM, et al. Multiplex cytological profiling assay to measure diverse cellular states. PLoS One. 2013;8(12):e80999. 3. Bray MA, et al. Cell painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes. Nat Protoc. 2016;11(9):1757-1774.

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The Literature Mice Heal Themselves in Response to a Common Signaling Molecule A newly discovered way to induce scarless healing in mice depends on a highly conserved signaling pathway that is also present in humans. BY IDA EMILIE STEINMARK, PhD

The researchers at the University of Kentucky and the Hubrecht Institute embedded beads soaked in ERK-activation growth factor mix (shown in white in this fluorescence microscopy image of a mouse ear) into the ears of common mice, and the tissue regenerated in response (shown in green).

1 6 T H E S C I E N T I ST | the-scientist.com

Now, researchers at the University of Kentucky and the Hubrecht Institute have shown that boosting the activation of extracellular signal-regulated kinase (ERK), a common signaling molecule, can help common mice heal themselves in the same way as spiny mice do,2 suggesting that the ability to regenerate could lie dormant in other mammals, including humans.

The regenerative capacity in mammals is still there. —Monica Sousa, University of Porto

The team suspected that ERK was important for the spiny mouse’s self-healing because ERK plays an important role in tissue healing. They began by exploring whether injury-related activation of ERK

ANTONIO TOMASSO

T

he seemingly magical regrowth of salamander tails and starfish arms after injury has always captured the attention of scientists. In recent years, they have also been excited about the spiny mouse, a small rodent discovered in 2012 to shed and regrow its skin1 because the existence of a regenerating mammal hints at the possibility of scarless healing in mammals more broadly.

Cryo-EM: Building on a history of invention and innovation

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Cryo-transmission electron microscopes Thermo Fisher Scientific offers a range of cryo-electron microscopy instruments suited to a variety of analytical needs. With the Thermo Scientific Tundra™ Cryo-TEM, you can expand the possibilities of your biochemical research without prior microscopy experience and at a more affordable price point. The Thermo Scientific Glacios™ 2 and Krios™ G4 Cryo-TEMs are capable of producing higher resolution results with increased capabilities. capabilities

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