VOL. 37, ISSUE 4, WINTER 2023 The Scientist

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WINTER 2023 | VOL. 37, ISSUE 4 | WWW.THE-SCIENTIST.COM

RECENT ADVANCES IN MODELING THE HUMAN PLACENTA MAY INFORM PLACENTAL DISORDERS LIKE PREECLAMPSIA.

Revealing Immune Responses with Adaptive Immune Receptor Repertoire Profiling

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cDNA 3’

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mRNA/GSP Hybridization

mRNA

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The DriverMap human and mouse AIR-RNA profiling assay uses targeted multiplex RT-PCR to profile all functional TCR and BCR isoforms (TRA, TRB, TRG, TRD, IGH, IGK, and IGL chains) in a single experiment while excluding non-functional pseudogenes and open reading frames. A single-tube multiplex RT-PCR followed by next-generation sequencing (NGS) enables robust rapid profiling of human or mouse RNA, DNA, or both. The simultaneous profiling of DNA and RNA enables the identification of antigen-activated clonotypes to provide insight into the immune response.

TECHNOLOGY: What Is the DriverMapTM Adaptive Immune Receptor (AIR) Repertoire Profiling Assay?

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3 Transcription & splicing

2 V to DJ recombination

1 D to J recombination

V-segments

T cell and B cell receptors (TCRs and BCRs) are key drivers of adaptive immunity. TCR and BCR coding sequences are arranged via a process known as V(D)J recombination. Within a given cell, variable (V), diversity (D), and joining (J) segments are randomly selected from many variants and combined to generate the full-length receptor. Scientists estimate that there are approximately 200 million T and B cell clones with distinct receptor sequence combinations present in human blood.

BACKGROUND: Receptor Diversity

Revealing Immune Responses with Adaptive Immune Receptor Repertoire Profiling

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constant regions

(D) (D)

V

(D)

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FwdGSP Extension

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NGS

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UMI

UMI

UMI

UMI

Anchor 2

Anchor 2

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3’

3’

1 2 3

Use NGS with to profile clonotypes present in each sample. UMI-labeled primers enable quantitative analysis of clonotype representation.

Amplify AIR clonotypes either with the multiplex PCRtargeting CDR3 variable and conservative regions of the T- and B-cell receptors, or with the full-length assay that captures CDR1, CDR2, and CDR3 regions.

Isolate total RNA or genomic DNA from any type of immune sample such as whole blood, PBMCs, cancer biopsies, tissue samples, FFPE, or dried blood microsamples.

How Does the DriverMap AIR Profiling Assay Work?

AIR repertoire profiling can be done with both genomic DNA (gDNA) and RNA, and each has its own pros and cons.

To Start With DNA or RNA?

Immunosequencing amplifies rearranged CDR sequences, and when combined with high-throughput sequencing technology, has the power to enumerate and quantify thousands of TCR or BCR clonotype sequences simultaneously. Scientists can use this information to characterize the abundance and distribution of lymphocytes, as well as to track clonal migration over time during situations such as disease progression or immunologic responses.2

The Power of Immunosequencing

transmembrane region

Anchor 1

1

PCR from Anchor Primers

3’

P5

The variable region of both BCRs and TCRs contain two variable regions polypeptide chains: light and heavy. These chains each contain three complementarity-determining regions (CDR1, CDR2, and CDR3). The V(D)J recombination junction is located in CDR3, making it the most diverse of the three CDRs and directly involving it in antigen recognition. CDR3 is therefore the primary focus of much scientific investigation.

TCR and BCR Structure

3’

or ch An

4 Translation & assembly

cDNA Synthesis from RevGSP

r2 ho nc

Cannot identify Ig isotype Can assess T/B cell counts per clonotype

Can identify Ig isotype Cannot assess T/B cell counts per clonotype

Coverage

Quantitation

200K

250K

DriverMap AIR

140K

160K

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DriverMap AIR profiling and immunophenotyping assays complement each other. The direct parallel profiling of AIR clonotypes and targeted expression levels of 500 T/B cell-typing markers in sorted T cell fractions facilitates TCR repertoire characterization in specific immune cell subsets, including CD8 naïve, CD8 effector, CD4 naïve, CD4 effector, and regulatory T cell fractions, providing greater insight into immune samples.

DriverMap AIR assay repertoire profiling is sensitive and reproducible. The assay reproducibly identifies and quantitates the most abundant 100500 clonotypes (with specific mRNA copy number exceeding 10) present in RNA samples. It also detects 3,000-5,000 medium-abundancy clonotypes reproducibly if replicates are used. Finally, the assay amplifies 50,000-500,000 low-abundancy clonotypes which are present at single-copy mRNA levels.

Running both AIR-RNA and AIR-DNA assays on the same sample allows for the identification of antigen-activated clonotypes by showing transcriptional activation of TCR and BCR genes in immune responses.

The DriverMap AIR assay facilitates simultaneous profiling of all CDR3 or full-length variable region TCR (TRA, TRB, TRD, TRG) and BCR (IGH, IGK, IGL) clonotypes in a single test-tube reaction. The comprehensive analysis of both TCR and BCR repertoires helps researchers better understand how both arms of the adaptive immune system work synergistically.

Cd4Te

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gDNA-ImmunoSEQ®

mRNA-DriverMap™ AI

Parallel profiling of TRB clonotypes and immune cell phenotypes in FACS-sorted CD4 naïve, CD4 effector, CD8 naïve, CD8 effector, and regulatory T cell fractions.

Cd4Tn

Immunophenotyping of T/B marker genes

Amplifies many non-rearranged and non-functional sequences

Only amplifies functional/expressed genes

Functionality

Adaptive immune repertoire (AIR) Profiling

Relatively low

10-100 fold higher than DNA

RESULTS:

types

gDNA

Sensitivity

RNA

types

PBMC1

PBMC2

PBMC1

PBMC2

SMART-based

120K

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8 9 10 11 12 13 14 15 16 17 18 19 20 21 Sample Number

The DriverMap AIR assay detects 1.5-2x more clonotypes than conventional gDNA-based assays.

0

20K

40K

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Cells (intact or lysed), tissues, fluids

Easy, inexpensive Simpler Cannot provide information on receptor chain pairings; cannot link phenotypes with clonotypes

Sample

Complexity and Cost

Scaling

Data Depth

Provides information on receptor chain pairing for single-cell sorting; links cell phenotypes with clonotypes

Less complex

Modern, less expensive

FACS-sorted cellular fraction or single cells

Medium

T/B cell fractions

Can link clonotype repertoire with paired-chain information and cell phenotype

More complex

Harder, more expensive

Intact live cells

Lower

Single-cell analysis

Immune receptor repertoire profiling is an important analytic tool for disease research in many areas, including cancer,2 cell and organ transplantation,3 autoimmunity,4 and infectious pathologies such as COVID-19.5 The DriverMap AIR repertoire profiling assay offers researchers the ability to characterize their immune cell samples in a comprehensive and sensitive manner. The assays are available as ready-to-use kits or as a service with a rapid one-month turnaround.

High

Throughput

Bulk analysis

The bulk AIR assay is the best strategy for cost-effective, quantitative immune receptor repertoire analysis of a large number of biological samples with the goal of identifying antigen-activated clonotypes or disease/treatment-associated TCR/ BCR biomarkers. Single-cell analysis is the most informative approach, but also the most costly strategy for the phenotypic characterization of cell encoding TCR/BCR clonotypes and receptor chain pairings identified through AIR bulk analysis.

Bulk analysis or single-cell sequencing for AIR repertoire assays?

The DriverMap AIR assay identifies 3x more clonotypes than a conventional SMART®-based 5’-switch oligo TCR assay.

0

50K

100K

250K Number of Clon

Number of Clon

Immune receptort repertoire profiling.n Your new superpower. ™ Adaptive Immune Receptor o Introducing the DriverMapM

(AIR) Profiling Assay Start with total RNA or DNA • Comprehensive profiling ofn all 7 TCR/BCR isoforms u • Accurate detection of functional isoforms only, not pseudogenes or ORFs s • Robust results from blood, tissue, FFPE or any immune sample • No specialized instrument required

Now available as a kit or as a service. Learn more at cellecta.com/drivermap-air

Who we are Cellecta Inc., a trusted provider of genomic products and services, has successfully collaborated with the world’s leading pharma, biotech, government, and academic institutions since 2006. Our recognized expertise in viral vector production, functional screening, cell engineering and multiplex qRT-PCR has given rise to a portfolio of offerings useful for loss-of-function and gain-of-function phenotypic screening, cell barcoding, targeted RNA-Seq and adaptive immune receptor profiling, and more. We help power your discovery efforts. www.cellecta.com [email protected] +1 650-938-3910

© 2023 Cellecta, Inc. 320 Logue Ave. Mountain View, CA 94043 USA

Cellecta Inc. is a trusted provider of genomic products and services for drug target and biomarker discovery. Since 2006, we have collaborated with the world’s leading pharma, biotech, government, and academic institutions. We apply our extensive expertise in viral vector production, functional screening, custom cell engineering and multiplex RT-qPCR to provide a variety of products and services, including: •

CRISPR and RNAi pooled libraries and loss/gain-of-function screening services to identify genetic pathways responsible for phenotypes and biological responses

•

Cell barcode libraries and constructs for cell tracking and analysis of clonal variations within cell populations

•

Transcriptome profiling, TCR /BCR profiling and digital spatial profiling for biomarker discovery

•

Custom cell engineering projects for cell assay development, and more

From our headquarters in Mountain View, California, we work with researchers worldwide to power their discoveries. Learn how to put our expertise to work for you at Cellecta.com

References: 1.

S. Teraguchi et al., “Methods for sequence and structural analysis of B and T cell receptor repertoires,” Comput Struct Biotechnol J, 18:2000-11, 2020.

2.

I. Kirsch et al., “T-cell receptor profiling in cancer,” Mol Oncol, 9(10):2063-70, 2015.

3.

A. Minervina et al., “T-cell receptor and B-cell receptor repertoire profiling in adaptive immunity,” Transpl Int, 32(11):1111-23, 2019.

4.

H. Katoh et al., “Immune repertoire profiling for disease pathobiology,” Pathol Int, 73(1):1-11, 2023.

5.

P.C. Taylor et al., “Neutralizing monoclonal antibodies for treatment of COVID-19,” Nat Rev Immunol, 21:382-93, 2021.

Helpful Resources Comprehensive Adaptive Immune Receptor Profiling for All Immune Cell Types — flyer Get an overview of the DriverMap AIR Profiling Assay for immune repertoire profiling starting from RNA or DNA DriverMap AIR Repertoire (AIRR) Profiling Tech Guide Learn all about the AIRR profiling techniques, strategies for designing experiments and key applications T-cell and B-cell receptor repertoire profiling for biomarker discovery — scientific poster View an outline of the DriverMap AIR workflow and a rheumatoid arthritis case study Synthetic Spike-in Controls for Immune Repertoire Profiling — flyer Ensure accurate and reproducible immune repertoire profiling results with this set of RNA controls Imunophenotyping of T-Cell and B-cell Receptor Clonotypes for Biomarker Discovery — video (18:40)

For more information and to access the links to these resources, visit Cellecta.net/ts-air-resources

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Contents WINTER 2023 | WWW.THE-SCIENTIST.COM | VOL. 37, ISSUE 4

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ON THE COVER: © SCIENCE PHOTO LIBRARY, MICROSCAPE

Features

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38

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38

Downsizing DNA

The Ephemeral Life of the Placenta

Unraveling the Mystery of Zombie Genes

A Story of FIRE and Mice

Recent advances in modeling the human placenta, the least understood organ, may inform placental disorders like preeclampsia.

Digging into how and why some genes are resurrected after death sounds morbid, but it has practical applications.

BY DANIELLE GERHARD, PhD

BY IRIS KULBATSKI, PhD

Some species remove up to 90% of their genomes during development, but why or how this happens is still a mystery. BY APARNA NATHAN, PhD

Studying how microglia control myelin growth and prevent its degeneration helps scientists better understand and address neurodegenerative diseases. BY NIAMH MCNAMARA, PhD AND VERONIQUE MIRON, PhD

2

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

© SHUTTERSTOCK.COM, SCIEPRO; MODIFIED FROM © ISTOCK.COM, ILYA LUKICHEV; © ISTOCK.COM, BAWANCH AND KUDRYAVTSEV PAVEL

24

Department Contents FROM THE EDITOR

5

46 Tracking Down Innate Immune Cells

When Scientists Collaborate, Science Progresses

in Multiple Sclerosis

A novel PET tracer targeting a receptor in myeloid cells can help monitor disease progression in a mouse model of multiple sclerosis.

Behind every successful scientist, there is another scientist. BY MEENAKSHI PRABHUNE, PhD

BY MARIELLA BODEMEIER LOAYZA

7

CAREAGA, PhD

CROSSWORD

PROFILE

FOUNDATIONS

8

Coming Into the Fold: DNA Origami

48 A Microbial Link to Parkinson’s Disease

In 2006, Paul Rothemund transformed the field of DNA nanotechnology when he unveiled an innovative approach for making shapes and patterns from genetic material.

Haydeh Payami helped uncover the genetic basis of Parkinson’s disease. Now, she hopes to find new ways to treat the disease by studying the gut microbiome.

BY DANIELLE GERHARD, PhD

48

BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

11

Serendipity, Happenstance, and Luck: The Making of a Molecular Tool

50 A Journey With Metabolism, Parasites,

The common fluorescent marker GFP traveled a long road to take its popular place in molecular biology today.

and Cancer

Piet Borst led stellar work on cell organelles, trypanosomes, and cancer drug resistance during the golden age of biology.

BY SHELBY BRADFORD, PhD

Cre-loxP: A Genetic Engineer’s Swiss Army Knife

BY LAURA TRAN, PhD

Standing at the cornerstone of genetic 53 Making Standards Exceptional research, Cre-loxP recombination Samantha Maragh has taken on serves as molecular scissors for the difficult challenge of standardizing precisely manipulating the genome. assays, data norms, and terminology in the ever evolving genome editing field. BY LAURA TRAN, PhD MEENAKSHI PRABHUNE, PHD

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LITERATURE

42 Rebranding Mitochondria

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L A M B S 14 15 C I N T O 8 7 17 18 L Y S O S O 20 S T O 2 23 24 M I T O C 27 26 2 H U M V H 29 30 E R 6 36 W A S L 40 0

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F I T O M E L E N I A S O N

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BY SHELBY BRADFORD, PhD

O M E R I A E A R

Phage display revolutionized peptide screening methods and unlocked opportunities in protein discovery and development.

A A 6 44 45 46 O C T 51 52 1 P P A R A E T R I S 57 R I B O S 62 63 I M O A 66 L A N Y

Finding the One in a Million

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A S E 37 3 38 G E S T 41 1 O W E R 3 43 A T E 49 50 9 0 O L G I A 54 R T 6 56 E D E D D S

BY HANNAH THOMASY, PhD

BY HANNAH THOMASY, PhD

A T K E

44 Biosensors for Colorectal Cancer

PUZZLE ON PAGE 7 N I C E

Recombinase polymerase amplification lets researchers rapidly replicate DNA in the clinic, in the field, or even in the International Space Station.

BY DANIELLE GERHARD, PhD

Engineered bacteria sound the alarm on a common oncogenic mutation.

Whenever, Wherever: Taking DNA Amplification Outside the Lab

O D O T

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S 55 E 61 C S

As scientists realize the multifaceted role of mitochondria, some feel that the “powerhouse of the cell” analogy is out of date.

METHODS

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FROM THE EDITOR

When Scientists Collaborate, Science Progresses Behind every successful scientist, there is another scientist. BY MEENAKSHI PRABHUNE, PhD

MODIFIED FROM © ISTOCK.COM, NIALL

W

hen I first learned about the eukaryotic cell structure, I marveled at the sophisticated simplicity of how organelles synergistically work with one another to ensure smooth cellular processes. On the surface, the interplay of thousands of proteins and the symphony of gene modulations underlying a function seem chaotic. Still, as we look deeper, impressive patterns emerge, both within and between cells and across different tissues. A remarkable aspect of living systems is their ability to communicate and collaborate for success. The gut finds a way to contact the brain; the placenta commandeers nutrients for the fetus; and cells selectively remove parts of their DNA for better organism health. The same fundamentals hold true for scientific success. Anyone who has attended a conference knows that the crowds outside the presentation rooms often surpass those inside. Scientific leaders buzz around the venues to catch up with their peers located across the globe and to brainstorm future projects. Beyond conferences, there is no dearth of triumphant stories where academics found unique ways to strike up an alliance. In today’s digital age of emails and social media, global outreach does not seem like a big deal, but collaboration has been the hallmark of science for a long time, even when communication channels were limited.1 For decades, scientists have managed to transcend disciplines, geographical boundaries, or personal beliefs to share their knowledge and contribute pieces towards solving big-picture puzzles. If we dissect the journey of any breakthrough that drove a field forward, we would no doubt find scientific collabora-

If we dissect the journey of any breakthrough that drove a field forward, we would no doubt find scientific collaboration at the helm driving success.

tion at the helm driving success. As you peruse the articles in this issue, I invite you to look out for those timely connections that propelled cell biology and molecular

biology forward. Whether in the story of a federal agency employee who found her scientific ally to launch a genome editing consortium, a US researcher’s invitation WINTER 2023 | T H E S C IE N T IST

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to a Japanese researcher that led to GFP as a molecular tool, a chance meeting during a seminar that led to significant progress in DNA origami technology, or an interlab study that created new transgenic mice, you are bound to find a narrative that resonates with you. These often altruistic endeavors that help other scientists take their research forward for the greater good of humanity truly embody the spirit of science. This cooperative mentality was critical in helping everyone cope with the COVID19 pandemic. When the world was at a standstill with everyone helplessly trapped at home, this special group of lab-coat-

adorning heroes sprang into action and their years of experience working together globally paid off. Whether by sequencing samples globally to track COVID-19 variants, working together on vaccines, or developing scalable diagnostic tests, dedicated researchers kept each other abreast of their progress to help the world collectively get through the pandemic.2,3,4 The formats and forums may change over the years with evolving needs and technologies, but I hope that the altruistic, collaborative spirit of scientists remains unchanged. If we do begin to lose it, we only need to look within our cells for inspiration to bring it back. J

Hsf 1

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LEARN MORE

BEFORE

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1. Vermeulen N, et al. Understanding life together: a brief history of collaboration in biology. Endeavour. 2013 Sep;37(3):162-71. 2. Chen Z, et al. Global landscape of SARS-CoV-2 genomic surveillance and data sharing. Nat Genet. 2022;54:499–507. 3. Druedahl LC, et al. Collaboration in times of crisis: A study on COVID-19 vaccine R&D partnerships. Vaccine. 2021 Oct 8;39(42):6291-6295. 4. Krijger PHL, et al. A public–private partnership model for COVID-19 diagnostics. Nat Biotechnol. 2021;39:1182–1184.

Our PhD-trained scientific writers can sharpen your manuscript's language and look.

Our Scientific Services team can help you shape your message and deliver it to the people who need to see it. Hia 1 BD2

References

EDITORIAL

Scientific Services Figure 3 Fi

Meenakshi Prabhune, PhD | Editor in Chief The Scientist

COMMUNICATION Our team can polish your message and present it to your audience.

AFTER

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CROSSWORD

Cells and Organelles 1. Animal used in some preclinical research 5. Extremely common type of genetic variation, for short 8. Consider it proper 14. Really loving 15. “Runaway Girl” actress Lili St. ___ 16. Foundation grants for a lab, often 17. Organelle that breaks down biological polymers 19. Illegally taken 20. Like slow city traffic 22. Mount Saint ___, fourth-highest peak in North America 23. Organelle that generates cellular energy 26. Sound made by the plainfin midshipman fish to attract mates 27. Old tape format 28. “___ the season to be jolly” 29. Wipe the data from 31. Succumb to gravity 32. Big ___ theory 36. Word that can follow “living” or “low” 37. Assimilation and ammonification in the nitrogen cycle, for two 39. Taken by mouth 40. Like a salamander’s life history 41. Subunit of an eon 42. Water: Spanish 43. Consumed 44. Month in which Daylight Saving Time ends 47. Some Boolean operators 48. Organelle that packages proteins into vesicles 53. Bat’s prey-detecting system 54. Video game with pieces falling from above 55. After CRISPR-Cas9, say 57. Organelle that synthesizes polypeptides and proteins 61. Tilted to one side 62. Texter’s prelude to a hot take 63. Opera singer’s solo 64. Noble horses 65. Office computer setup: Abbr. 66. 88 Earth days, for Mercury

DOWN 1. Genre-bending artist ___ Nas X 2. One or more 3. The Rockies, e.g.

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4. Upward shove 5. “Move over!” 6. Immature dragonfly, for example 7. Response to “grazie” 8. Term of endearment for a sibling 9. Goes in 10. Bacteria with strains that can be symbiotic with humans 11. Large sheet of paper 12. “To explain...” 13. “___ across the board!” 18. Apparatus with burners 21. Coming up next 23. Artwork created by Montana State University’s Bioglyphs project 24. Fully mature insect stage 25. Sites of archaeological interest 26. Cuts with a saw 30. Create suture 31. Habitat for Pelagibacter ubique 32. Snake that kills through constriction 33. Specialized jargon 34. Micronesian microstate known for its coral cliffs

BY STELLA ZAWISTOWSKI

ACROSS

35. Amorphous solid that’s highly useful to humans 37. Group that looks for life outside of Earth 38. Pay for dinner 43. Came to a consensus 44. Best possible conditions 45. Graphene’s makeup 46. Small musical group 48. Titular literary character who never shows up 49. How some samples are shipped 50. Potato pancake served at Hanukkah 51. Extreme danger 52. Process that may require sample purification 53. Short moments? 56. Degree held by a dentist: Abbr. 58. Magnetite, chromite, or cassiterite 59. Nowhere to be found, for short 60. Body part that contains the vestibular nerve

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

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FOUNDATIONS

Coming Into the Fold: DNA Origami In 2006, Paul Rothemund transformed the field of DNA nanotechnology when he unveiled an innovative approach for making shapes and patterns from genetic material. BY DANIELLE GERHARD, PhD

D

NA, the medium of life, is so deeply associated with the biochemical world that considering its nonbiological applications may seem far-fetched. However, for researchers in the 1980s and 1990s working in the fledgling field of DNA nanotechnology, it was more than a flight of fancy. Nadrian “Ned” Seeman, the father of DNA nanotechnology and formerly a biochemist at New York University, first proposed the idea that DNA is not only a genetic material but also a construction material in his seminal 1982 Journal of Theoretical Biology paper.1 A crystallographer by training, Seeman struggled to crystalize proteins. He wanted to build DNA cages that were strong enough to hold the protein in place long enough to take a great picture. En route to tackling this problem, Seeman created the field of DNA nanotechnology. “Ned had such a huge body of literature that he’s sort of everyone’s go-to inspiration source,” said Erik Winfree, a computer scientist and bioengineer at the California Institute of Technology (Caltech). In the decades that followed, grand ideas transformed into even grander demonstrations as scientists repurposed nucleic acids to store nonbiological information, such as all 154 of William Shakespeare’s sonnets, and build microscopic robots for drug delivery.2,3

what I was talking about,” said Rothemund. He hit a dead end, and following graduation in 1994, he joined a geobiology lab as a technician. Later that year, Leonard Adleman, a computer scientist at the University of Southern California (USC), published a seminal paper in Science in which he used DNA to compute an algorithm.5 “I simultaneously felt sort of scooped and validated that there was something to the idea of encoding information in DNA molecules and doing computing,” said Rothemund.

DNA computers

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

Erik Winfree (left), Bernard Yurke (middle), and Paul Rothemund (right) explore ways to expand the use of DNA.

Winfree recalled Rothemund’s class project on a similar topic and hunted him down to see if he wanted to attend the inaugural one day workshop on DNA-based computers that would be held at Princeton University in the spring of 1995. They scrounged up the money to attend the conference. Winfree’s talk touched on Seeman’s work on the self-assembly of DNA structures: the spontaneous organization of molecules due to attractive forces. After returning to his seat, Winfree felt Seeman tug on his arm. Later that evening, Adleman, Seeman, Rothemund, and Winfree huddled over a red and white checkered tablecloth in a pizza parlor and reflected on the day’s events. “That sort of seeded the next 25 years of my life,” said Rothemund. He joined Adle-

ERIK WINFREE

In the early 1990s as an undergraduate student at Caltech, Paul Rothemund, now a computer researcher there, took a computer science class that introduced him to the potential of DNA. The professor discussed an idea from Charles Bennett, then a physicist at IBM, that a computer built from DNA could simulate a Turing machine wherein hypothetical enzymes could read the information stored in DNA and use that information to alter DNA bases.4 “He said, ‘you know, someday, somebody who knows something about computer science and biology or chemistry will come up with a way to compute using DNA.’ At that moment, I said, ‘Well, maybe I can do that,’” recalled Rothemund. He didn’t have to wait long. Rothemund had the opportunity to further explore the idea of building a molecular computer for a project in another class. There, Rothemund met Winfree, then a graduate student, and introduced him to Seeman’s work. As Rothemund neared the end of his studies, he wanted to continue working on the problem of building DNA computers, so he shopped the project around. Computer science professors felt ill-equipped to advise on the life science aspects of the project, so he turned to life scientists. “The biology professors at Caltech and elsewhere told me that I was crazy, and they had no idea

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man’s lab at USC later that year as a graduate student, while Winfree ventured to the east coast to collaborate with Seeman at New York University on the self-assembly of DNA crystals.6

Folding DNA DNA computing captured the imaginations of many entering the nascent field of DNA nanotechnology by demonstrating a nonbiological application for nucleic acids. However, by the turn of the century, many researchers shifted their focus. “Many in the field convinced ourselves that although this was intellectually stimulating, this was not going to compete with electronic computers,” said Winfree. Rothemund focused his efforts on building nanostructures using DNA or programmable approaches for DNA assembly. Specifically, he considered how self-assembly processes could be treated as algorithms and studied using computer science tools. In 1993, Seeman detailed the construction of complex nanostructures via the self-assembly of molecules.7 He was interested in figuring out how to design molecules that self-assemble to form parallel DNA helices where strands cross over and become part of another double helical line, thus stitching together the helices. Over the next decade, this inspired others in the field to innovate, increasing the number of crossovers and helices. Rothemund and Winfree’s paths crossed again in 2001. Winfree returned to Caltech as a professor, and Rothemund joined his lab as a postdoctoral researcher, where they continued their work on algorithmic self-assembly of DNA.8 DNA is a versatile building block, but Rothemund described DNA as optically, biochemically, and electronically dead com-

pared to other molecules and materials like quantum dots, carbon nanotubes, or antibodies. “But what it can do is you can use the information in DNA sequences to build structures, and then you can use that structure to organize those other things,” said Rothemund. Since the available methods for creating shapes out of DNA were laborious and time consuming, Rothemund set out to develop an easier approach. “And really, that was the idea for DNA origami,” he said. Rothemund’s DNA origami consisted of two main components: a long, single-stranded piece of bacteriophage DNA, which serves as the scaffold material, and a bunch of shorter strands of oligonucleotides, or staple strands, that fix the structure in place.9 He fed his design into a computer program that used principles of Watson-Crick base pairing to determine which sequences were needed to instruct the scaffold strand to fold into the desired shape or pattern. In his one-pot method, Rothemund mixed the scaffold strand with the custom-made staple strands and waited patiently as molecular self-assembly took shape. Then, to confirm the structures, he used atomic force microscopy. At the time, Winfree granted his lab members the freedom to independently explore their interests. “There was a period in 2005 when I didn’t see [Rothemund] around very much,” said Winfree. Eventually, Rothemund re-emerged with something to share. “He showed me his results on the DNA origami, and to tell you the truth, my first reaction was ‘Blech! Where’s the algorithm? Where’s computer science?’” Uninterested in collaborating on the project, Winfree suggested that Rothemund publish it himself, and so he did. Winfree eventually came around to DNA origami. “It’s fantastic. It’s revolutionized the field,” said Winfree. “[Rothemund’s] demonstration was so elegant and thorough that it really opened people’s eyes to how powerful the idea was.”

PAUL ROTHEMUND

DNA origami’s spiritual successors

For the first DNA origami experiments, Rothemund created one-third of a square. By adding only one-third of the required staple strands, only onethird of the square folded, resulting in rectangular shapes captured using atomic force microscopy. The “ss” label denotes unfolded single-stranded scaffold; “s,m” denotes a stable monomer; and “u,m” denotes an unstable monomer. The scale bar is 100 nm.

DNA origami isn’t the only approach for DNA assembly, but it’s robust and relatively easy.10 “It’s the ability to create a geometrically structured testbed for your experiment with each molecule in the right place, and now, hundreds to thousands of molecules,” said Winfree. “That was unprecedented. That opened up an ability to do experiments in all sorts of fields that people previously couldn’t do.” For Winfree, that was putting short DNA sequences in the correct order to trigger self-assembly. For others, it was putting enzymes in the right order to produce a cascade of enzyme reactions or quantum dots in a particular organization to control optics. “One of the things that DNA origami has been able to do since that paper was published is not something that’s widely used in everybody’s cell phone or something like that, but it’s a research tool for other things,” said Rothemund. He noted that these custom instruments for biology allow researchers to begin asking questions about proteins or other biomolecules and even translate these ideas into therapeutics and molecular diagnostics.11,12 WINTER 2 02 3 | T H E S C IE N T IST

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tures, with the idea that they could potentially enter bacteria and replicate. Shih noted that without a master scaffold strand, the construction is harder to control. “It’s kind of like herding cats,” said Shih. Recently, Shih’s research group has been busy developing what he referred to as the spiritual successors of DNA origami. The scale of DNA origami structures is limited by the length of the scaffold strands, which is typically on the order of 10,000 nucleotides. Therefore, building anything bigger than that requires forgoing the leading scaffold strand. To address this, Shih and his team recently developed crisscross DNA origami whereby a controller molecule on the scale of a single DNA origami directs the construction of a larger 1,000 DNA origami structure.14 “They’re more like well-trained dogs than cats,” said Shih. Shih hopes that these advances will facilitate the construction of bigger nanorobots with the size and complexity of a bacterial or mammalian cell. He views this as a complementary technology advancement to the broader field of synthetic biology where scientists modify the genomes of living cells. “But they’re still living cells,” said Shih. They’re still surrounded by a membrane; they still have metabolism; and they still do DNA replication. “It’s important, technologically, to have alternate schemes that maybe don’t have a membrane, that are not beholden to the normal process of DNA replication and translation, that maybe can be deployed in environments that are hostile to living cells,” said Shih. J

William Shih, a biochemist at Harvard Medical School, employs principles of DNA origami in his quest to build nanoscale objects, including molecular robots. In 2005, at a conference in Albany, Shih learned what Rothemund had been up to. “I saw his talk, and my jaw dropped because these images that he produced—nobody had seen anything like this,” said Shih. He recalled a particularly memorable image of DNA origami with “Ned” patterned on top in homage to Seeman. Still mesmerized by Rothemund’s creations, Shih returned to his lab, scrapped what he was doing, and pivoted to Rothemund-style DNA origami. “To me, what’s most special about DNA origami is that you have an excess of building blocks that do absolutely nothing except when they see a copy of this master controller scaffold strand,” said Shih. Ten copies of the scaffold strand produce 10 DNA origami structures if there are sufficient staple strands. Around the same time that Rothemund tinkered with DNA at Caltech, Shih worked as a postdoctoral researcher down the road at the Scripps Research Institute. In 2004, he published a paper in Nature demonstrating the construction of a nanoscale octahedron.13 However, Shih’s DNA folding approach required the assembly of a substantial number of short, cloneable DNA struc1 0 T H E SC I EN T I ST | the-scientist.com

References 1. Seeman NC. Nucleic acid junctions and lattices. J Theor Biol. 1982;99(2):237-247. 2. Goldman N, et al. Towards practical, high-capacity, low-maintenance information storage in synthesized DNA. Nature. 2013;494:77-80. 3. Douglas SM, et al. A logic-gated nanorobot for targeted transport of molecular payloads. Science. 2012;335(6070):831-834. 4. Bennett CH. Logical reversibility of computation. IBM J Res Develop. 1973;17(6):525-532. 5. Adleman LM. Molecular computation of solutions to combinatorial problems. Science. 1994;266(5187):1021-1024. 6. Winfree E, et al. Design and self-assembly of two-dimensional DNA crystals. Nature. 1998;394(6693):539-544. 7. Fu TJ, Seeman NC. DNA double-crossover molecules. Biochemistry. 1993;32(13):3211-3220. 8. Rothemund PWK, et al. Algorithmic self-assembly of DNA Sierpinski triangles. PLoS Biol. 2004;2(12):e424. 9. Rothemund PWK. Folding DNA to create nanoscale shapes and patterns. Nature. 2006;440:297-302. 10. LaBean TH. Reminiscences from the trenches: The early years of DNA nanotech. In: Jonoska N, Winfree E, eds. Visions of DNA Nanotechnology at 40 for the Next 40. Natural Computing Series. Springer, Singapore; 2023:55-67. 11. Andersen ES, et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature. 2009;459:73-76. 12. Ochmann SE, et al. Optical nanoantenna for single molecule-based detection of Zika virus nucleic acids without molecular multiplication. Anal Chem. 2017;89(23):13000-13007. 13. Shih WM, et al. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature. 2004;427:618-621. 14. Wintersinger CM, et al. Multi-micron crisscross structures grown from DNAorigami slats. Nat Nanotechnol. 2023;18(3):281-289.

PAUL ROTHEMUND

Paul Rothemund created shapes, like smiley faces, and patterns, such as DNA lettering, using his DNA origami method.

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Serendipity, Happenstance, and Luck: The Making of a Molecular Tool The common fluorescent marker GFP traveled a long road to take its popular place in molecular biology today. BY SHELBY BRADFORD, PhD

B. WEINSTEIN, NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT, NATIONAL INSTITUTES OF HEALTH VIA FLICKR.COM

F

ollowing chemist and marine biologist Osamu Shimomura’s success in determining the structure of luciferin from a crustacean at the Nagoya University, Frank Johnson, then a biologist at Princeton University, invited him to study luminescence in his lab. When Shimomura arrived at Princeton University in 1960, he had no ambitions for novel proteins and their applications. Johnson was interested in in the blue light produced by a species of jellyfish called Aequorea aequorea (now known as Aequorea victoria), Shimomura began looking into the phenomenon. At the time, scientists believed that all luminescence involved luciferin and luciferase. However, the duo did not purify any luciferinrelated proteins from the jellyfish. This led Shimomura to believe that the blue light must come from another protein, but he struggled to isolate it.

tein must be in the sink, and that it certainly wasn’t luciferase. The aquarium in the lab drained into the sink, so Shimomura got to work testing the components in it to see if they elicited blue light from his samples. Calcium turned out to be the culprit. By removing calcium from the samples, Shimomura and Johnson isolated the protein responsible for the blue light, which they named aequorin.1 This was the first example of a light-activated protein, which they called a photoprotein.

A new way to luminesce

Today, we call them fluorescent proteins. Alongside aequorin, Shimomura and Johnson also purified a small amount of another protein that glowed green instead of blue. They called it “green protein,” but at the time, they could not purify sufficient amounts to study it further. While collecting and saving these samples for future analysis, they continued exploring aequorin to characterize its structure and chemistry.

Shimomura attempted to isolate this mystery protein under various conditions. He finally found that he could inactivate the luminescence with acid and obtain cell-free extract from the jellyfish tissues. Satisfied with this finding, he dumped the used samples down the sink. To his surprise, they glowed bright blue. He reasoned that the substrate for his mystery luminescent pro-

I wanted to have a tool like this. I thought this would be wonderful. —Martin Chalfie, Columbia University

Fluorescent proteins enable researchers to study biological processes in living organisms, as shown in this zebrafish.

WINTER 2 02 3 | T H E S C IE N T IST 1 1

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Across the country in the mid-1960s, James Morin, a field biologist who is now a professor emeritus at Cornell University, became fascinated by bioluminescence. “It was amazing to me that so many organisms that didn’t really have the capacities for detecting luminescence would be luminescent,” he said. This curiosity led him to the hydrozoan Obelia geniculata when he began his graduate studies at Harvard University. He noticed that the luminescence he saw from these animals in the ocean was green, whereas in his prepared samples, it was blue. “That indicated there must be some kind of coupling,” Morin said. It turned out that O. geniculata, like Shimomura’s and Johnson’s A. aequorea, used a calcium-activated protein that emitted blue light. This light activated a second protein that glowed green. Morin published his findings from O. geniculata and coined the term “green fluorescent protein” (GFP) for the first time in 1971.2 Shimomura confirmed that the same events happened in his jellyfish in 1974.3 Shimomura continued to study GFP and identified its chromophore in 1979.4 Ultimately, though, he returned to studying bioluminescence. Morin also was more interested in the function of luminescence and the nervous system of the simple marine species that produced it. “We were just interested in the mechanisms of how that whole system worked,” Morin said. “I’ve never been an applications biologist. I’ve been a discovery kind of biologist.” For the next two decades, GFP remained just an interesting luminescent protein found in some species of marine animals.

ture would need the help of other enzymes in the jellyfish to fold correctly. Chalfie’s phone calls eventually led him to Douglas Prasher, a molecular biologist who worked at the Woods Hole Oceanographic Institute. Prasher cloned the aequorin genes in 1985 and began working on cloning the gene for GFP.5 The two struck up a collaboration. In 1992, Prasher published his paper describing the GFP cDNA clone, which he sent to Chalfie and his graduate student, Ghia Euskirchen.6 With the clone in hand, the two had a decision to make. Since Prasher cut the GFP gene out of the jellyfish genome with restriction digestion enzymes, the clone had extra DNA segments that bookended the GFP gene. If Chalfie and Euskirchen wanted only the GFP DNA, they could amplify just the GFP gene by PCR, but that risked introducing mutations since PCR was error-prone at the time. Alternatively, they could transform the whole clone into E. coli, which would generate highfidelity DNA but retain the extra jellyfish sequence segments. Because of this risk, most groups opted for transforming the whole clone. “I know at least three groups that did that, and it did not work,” Chalfie said. “We did something different.” Taking a risk on error-prone PCR, Euskirchen amplified only the GFP coding sequence and inserted it into an expression vector to transform back into E. coli. When Euskirchen imaged her bacteria using a fluorescent microscope, she saw green glowing back at her. “That immediately said there is nothing else needed from the jellyfish,” Chalfie said.

Not just a jellyfish thing In the 1970s, Morin, then at the University of California, Los Angeles, took Paul Brehm on as a graduate student and introduced him to Obelia and the field of bioluminescence. As a biologist at Oregon Health and Science University, Brehm continued studying bioluminescence in Obelia as an associate professor. In 1989, he presented his work at a seminar at Columbia University; Martin Chalfie, a geneticist at Columbia University who had never heard of GFP before, happened to attend. After Brehm’s talk, Chalfie started tracking down GFP scientists on the phone. “I was looking for where genes were expressed,” Chalfie said. “I wanted to have a tool like this. I thought this would be wonderful.” In 1989, scientists studied genes of interest in cells using newly developed technologies. “But in all of these instances, whether it’s antibodies, or in situ hybridization, or using a reporter like beta galactosidase, one needed to really prepare the samples. That meant first they were fixed,” Chalfie said. “As a result, one had really a snapshot in time.” There was no way to visualize what genes were active in a living organism. Chalfie thought that if GFP could be taken out of these marine organisms and inserted into other cells, it could act as a reporter in a living animal. However, nobody knew if this would be possible. GFP had a cyclized portion in its structure, and many people thought that such a complex chromophore struc1 2 T H E SC I EN T I ST | the-scientist.com

The jellyfish version of GFP just crashed out in E. coli. —Raphael Valdivia, Duke University

While this was a huge milestone in molecular biology, Chalfie believes that he owes some of his success to other contributing factors. “It’s not that I was the first person in the world who ever thought about this,” Chalfie said. “It was a combination of being at the right place at the right time, having the right people in the lab and the right expertise to be able to try it, and then a smidgen of luck.” With this success, Chalfie and his team tested the initial goal of inserting the isolated GFP into the touch-sensing cells of Caenorhabditis elegans. With positive data in hand, the group published the first manuscript with GFP as a reporter in a nonmarine animal.7

An elegant measurement Once the word was out that GFP could be put into other model organisms, the protein found itself visiting labs across the coun-

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try, quite literally in the case of Raphael Valdivia, who is currently a molecular geneticist at Duke University and who was a graduate student in Stanley Falkow’s lab at Stanford University in the mid-1990s. “This was when I had to take my qualifying exams to get into PhD candidacy, and so I had to propose a project outside of my thesis project,” said Valdivia. “I proposed to use GFP as a reporter for gene expression in bacteria.” Valdivia got the clone from Prasher and amplified the GFP coding sequence to reinsert it into an expression vector, similar to Chalfie’s experiments, and then transformed it into E. coli. “But when we expressed it, it did not work very well,” Valdivia explained. Although the bacteria were green, the GFP took a long time to fold and become fluorescent. “The jellyfish version of GFP just crashed out in E. coli,” he said.

Cells express GFP after viral transfection or insertion into the genome.

Valdivia wondered if he could tweak GFP to brighten its fluorescence. Working alongside Brendan Cormack, he created mutants of GFP that targeted the protein’s chromophore.8 They had their brighter mutants within a week. “We didn’t do it with an idea of doing big technological breakthroughs or screens in general. We just needed something that worked better in pathogens so we could screen,” Valdivia said.

How do you make a really elegant measurement that’s biologically relevant and learn things with it? —Geoffrey Baird, University of Washinton

Valdivia eventually gave GFP to the spouse of a postdoctoral fellow in Falkow’s lab who worked at ClonTech, which is now Takara Bio USA, Inc. Through this spurious connection, ClonTech optimized GFP for expression in mammalian cells, licensed, and commercialized as enhanced GFP, or eGFP in 1996. While many groups worked with GFP to change its stability or activity, Roger Tsien, a chemist at the University of California, San Diego made possibly the most striking contributions. With a background in dye chemistry, Tsien saw the value a fluorescent protein could offer biological research. According to Geoffrey Baird, who was a graduate student in Tsien’s lab and is now a clinical pathologist at the University of Washington, his mentor had a fundamental interest that drove his research. “How do you make a really elegant measurement that’s biologically relevant and learn things with it?” Baird asked. Tsien’s goal was to use GFP in fluorescence resonance energy transfer (FRET) to monitor calcium signaling. Although his team successfully generated a few mutants that gave rise to altered colors of GFP, Tsien anticipated that solving the structure of GFP would be important for more modifications. 9-11 “Because it was only really with the structure that one could then sort of say, well, hey, if we made this mutation here, we might be able to make it a much different color,” Baird explained. Tsien’s group solved this structure with help from James Remington’s group at the University of Oregon in 1996.12 Within a week, a second group published their finding of the structure, and the two closely aligned.13 With this information, Tsien’s group got to work exploring what was possible with GFP. “I basically got into the business of making mutations,” Baird said. Baird considers his most important contribution to have come from an accident. He was trying to create brighter GFP clones, and one day, he saw one that didn’t get dimmer after acid exposure. After he sequenced it, Baird realized that he had made a mistake. “This thing shouldn’t be fluorescent at all,” he said. WINTER 2 02 3 | T H E S C IE N T IST 1 3

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“It revolutionized cell biology and molecular biology,” said Baird. Shimomura, Chalfie, and Tsien received the Nobel Prize in 2008 for their work on GFP. “It’s been useful in the way I thought,” Chalfie said, reflecting on GFP’s legacy. “But even more so, it’s been useful in ways that I would have never imagined.” J

Researchers inserted a GFP tag to visualize a transcription factor for a motor neuron in mouse embryonic stem cells and used a red-fluorescing antibody against a marker for neurons to identify these cells.

Instead of a single point mutation, he had accidentally inserted six amino acids into the sequence. But this accident showed that it was possible to distort GFP. Baird informed Tsien of his mistake. “He basically had all of the ideas for what you would do to exploit a serendipitous finding,” Baird said. Ultimately, the team found that breaking GFP and rejoining it in a variety of ways through a process called circular permutation created proteins with applications not only in FRET, but also for studying protein interactions just by observing if a split GFP molecule became active again.14 As Tsien had hoped and anticipated, GFP was a very useful measuring stick.

Revolutionizing biology From an obscure protein that wasn’t even abundant enough to study, scientists have now published more than 40,000 papers that reference green fluorescent protein. Today, GFP tags proteins to view them inside of cells under microscopes and to isolate cells of interest using flow cytometry and fluorescenceactivated cell sorting.15 It has confirmed physical connections between proteins and determined if a protein is made in the first place. GFP has found itself in viruses, yeast, plants, mice, rats, rabbits, pigs, and even nonhuman primates.16-23 1 4 T H E SC I EN T I ST | the-scientist.com

1. Shimomura O, et al. Extraction, purification, and properties of Aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Physiol. 1962;59(3):223-239 2. Morin JG & Hastings JW. Energy transfer in a bioluminescent system. J Cell Physiol. 1971;77(3):313-318 3. Morise H, et al. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry. 1974;13(12):2656-2662 4. Shimomura O. Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett. 1979;104(2):220-222 5. Prasher D, et al. Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein. Biochem Biophys Res Commun. 1985;126(3):1259-1268 6. Prasher DC, et al. Primary structure of the Aequorea Victoria green-fluorescent protein. Gene. 1992;111(2):229-233 7. Chalfie M, et al. Green fluorescent protein as a marker for gene expression. Science. 1994;263(5148):802-805 8. Cormack BP, et al. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1)33-38 9. Heim R, et al. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci. 1994;91(26):12501-12504 10. Heim R & Tsien RY. Engineering green fluorescent protein for improved brightness, longer wavelengths, and fluorescent resonance energy transfer. Curr Biol. 1996;6(2):178-182 11. Sekar RB & Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003; 160(5):629-633 12. Ormö M, et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 1996;273(5280):1392-1395 13. Yang F, et al. The molecular structure of green fluorescent protein. Nat Biotechnol. 1996;14:1246-1251 14. Baird GS, et al. Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci. 1999;96(20):11241-11246 15. Chudakov DM, et al. Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiol Rev. 2010;90(3):1103-1163 16. Oparka KJ, et al. Using GFP to study virus invasion and spread in plant tissues. Nature. 1997;388:401-402 17. Li J, et al. Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter. Biotechnol Bioeng. 2000;70(2):187-196 18. Köhler RH. GFP for in vivo imaging of subcellular structures in plant cells. Trends Plant Sci. 1998;3(8):317-320 19. Yang M, et al. Visualizing gene expression by whole-body fluorescence imaging. Proc Natl Acad Sci. 2000;97(22)12278-12282 20. Hakamata Y, et al. Green fluorescent protein-transgenic rat: a tool for organ transplantation research. Biochem Biophy Res Commun. 2001;286(4):779-785 21. Takahashi R, et al. Establishment and characterization of CAG/EGFP transgenic rabbit line. Transgenic Res. 2007;16:115-120 22. Brunetti D, et al. Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts. Cloning Stem Cells. 2008;10(4):409-420 23. Kurre P, et al. Kinetics of fluorescence expression in nonhuman primates transplanted with GFP retrovirus-modified CD34 cells. Mol Ther. 2002;6(1):83-90

T. MACFARLAN, NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT, NIH VIA FLICKR

References

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Cre-loxP: A Genetic Engineer’s Swiss Army Knife Standing at the cornerstone of genetic research, Cre-loxP recombination serves as molecular scissors for precisely manipulating the genome. BY LAURA TRAN, PhD

W

hen Nat Sternberg, a molecular biologist at the Frederick Cancer Research Center, heard about bacteriophage P1, he was intrigued. Having previously worked on the head proteins of h phage, Sternberg had an appetite for studying phage-host relationships. P1 was largely unexplored, but Sternberg was up for the challenge. Like h phage, P1 infected Escherichia coli and entered the lysogenic cycle. In this stage, the bacteriophage genome integrated itself into the host cell’s genome to achieve replication without destroying the host cell. During lysogeny, the h phage genome incorporated into the host DNA, but the P1 phage displayed a unique feature: It existed as an independent plasmid. P1 seemed to be a suitable model for studying plasmids, so Sternberg set off to construct a P1 library in a h vector to study its genes. He focused on studying site-specific recombination of P1. P1 phage particles were cyclic, and researchers expected recombination to produce a circular map. However, Sternberg noted an unexpected characteristic of P1’s recombination map. It was linear. Sternberg pondered about the underlying mechanism; he suspected that there must be a genetic hotspot for recombination located at the terminal ends of the linear genome to facilitate plasmid cyclization.1

Discovery of the Cre-loxP sandwich Consistent with his hypothesis, Sternberg’s experiments revealed a small fragment of P1 DNA at the end of the genetic map that was likely responsible for site-specific recombinase. He named this locus of crossover in P1, or a loxP recognition site. On further investigation, Sternberg performed deletion mutagenesis studies to identify another necessary component for recombination events, a P1 gene product he named Cre (an anagram for recombination).2 Cre recombined target sequences at two loxP recognition sites. He referred to the DNA sequences flanked by loxP sites as “floxed.” The products of Cre-mediated recombination depend on the orientation of the loxP sites. Two loxP sites oriented in the same direction will excise DNA as a circular loop, while loxP sites aligned in opposite directions will invert the DNA sequence between them. This Cre-loxP sandwich in the P1 bacteriophage laid the foundation for precise genetic manipulation. When Sternberg moved to DuPont in 1984, a member of his lab who followed him to DuPont, Brian Sauer, continued the project. The biochemistry of the system was simple, but Sauer hoped

that Cre’s prokaryotic origins would not hinder its ability to effectively work in eukaryotic cells. On testing loxP sites in yeast chromosomes, Sauer was thrilled to see that Cre recombinase readily recognized loxP sites and actively transported into the eukaryotic nucleus.3 He later showed that Cre-loxP functioned efficiently in mammalian cell lines.4 His next question was whether this system could be implemented to target genes in the germline to obtain strains of genetically modified animals.

The explosive potential of transgenic mice In the late 1980s, Nobel laureates Mario Capecchi at the University of Utah, Oliver Smithies at the University Wisconsin in Madison, and Sir Martin Evans at the University of Cambridge, pioneered methods for gene targeting in mouse embryo-derived stem (ES) cells and homologous recombination as a mechanism for manipulating genes in the mouse genome.5-7 This work demonstrated the feasibility of making specific mutations in ES cells and obtaining genetically modified knockout mice. This series of breakthrough studies catapulted transgenic mice into the spotlight and inspired researchers to follow suit. Klaus Rajewsky, an immunologist from the University of Cologne, took a keen interest in the development of the immune system, especially that of B cells. He closely followed the work on ES cells and applied it to develop one of the earliest knockout models of the immune system by rendering mice deficient of B cells.8 However, the technology had its limitations. Rajewsky wanted to study the function of DNA polymerase ` (pol`), but traditional knockouts were not a viable option. Mice needed the gene early in development, and eliminating this gene was fatal for them. In addition to targeting a gene of interest, geneticists needed to incorporate a selectable marker gene to find correctly recombined cells. Rajewsky used the resistance gene neomycin in his experiments, but he encountered an unexpected roadblock. “Another problem that arose in these early knockout experiments was the selection marker gene neomycin, which was still in the locus and disturbed the phenotypes,” recalled Rajewsky. “I, along with many others, thought about how this could be corrected. Then, we came across Cre recombinase systems.”9 When he read about this system, Rajewsky eagerly sought to introduce this technique into the gene targeting technologies he had already established. WINTER 2 02 3 | T H E S C IE N T IST 1 5

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Across the world, Jamey Marth, a molecular and cellular biologist at the Biomedical Research Center, recognized the method’s potential for modeling gene function. He wanted to study genes that regulated protein glycosylation in animals for closer recapitulation of the human system. Marth recalled early discussions with his team members revolving around Sauer’s previous work with recombinases that cleaved DNA in a very conservative and targeted manner in yeast and mammalian cells. Despite its success in mammalian cell lines, Marth was worried that it would not translate well in an animal model. It was possible that chromatin rearrangement during development might shut down the prokaryotic activity of Cre. However, he was undeterred and designed a Cre-expressing vector with two objec-

tives: obtain Cre expression, and make the mutation cell specific. The results of his experiments surprised him. He and his team demonstrated that Cre-loxP recombination efficiently deleted DNA sequences in specific developing T cells of transgenic animals in 1992.10 “I remember the day we saw the first autoradiograms coming off the machine,” recalled Marth. “We were just overjoyed that this worked so well with high efficiency.” Around the same time, Marth received a call from Rajewsky. Rajewsky wanted to collaborate after reading Marth’s paper on the successful T cell cre transgenic line. Although Rajewsky initially wanted to target the pol` gene in B cells, Marth’s T cell transgenic line was an attractive alternative.

Cre-loxP recombination allows scientists to excise, insert, or invert specific DNA segments with unprecedented accuracy. This works through two key components: a Cre recombinase and loxP sequence recognition sites. The Cre enzyme identifies pairs of loxP sites (arrowheads), which flank (flox) the DNA, and catalyzes reciprocal DNA recombination between the two sites to excise a small piece of DNA.

Q B

Q A

To generate a tissue specific knockout mouse, researchers breed a mouse bearing a Cre transgene under a tissue- or cellA with a homozygous type specific or inducible promoter Q floxed mouse Q B. Target gene

Target gene

Target gene

Target gene

Tissue-specific promoter

Cre

IoxP site

Q C

Q B

The offspring are heterozygous for the floxed target gene Q C and breed with the homozygous floxed mouse Q B. Target gene

Target gene

Target gene

Target gene Cre

Tissue-specific promoter

Q D

The resulting experimental mouse is hemizygous for Cre and homozygous for loxP Q D . This is the necessary genotype required to conditionally knock out the target gene in the specific tissue.

At tissue of interest: Deletion of gene Target gene Target gene Tissue-specific promoter

Cre

Tissue-specific promoter

Cre

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EXPERI Fluorescence microscopy has revolutionized cell and developmental biology research by enabling scientists to visualize cellular processes in real time and dissect molecular events in depth. However, researchers can encounter several problems when imaging their samples through fluorescence microscopy, including bleed-through, photobleaching, high background signal, phototoxicity, and channel misalignment. Fortunately, there are ways to overcome these major challenges.

BLEED-THROUGH

PROBLEM Bleed-through is the phenomenon where the emission from one fluorophore is detected in a different fluorophore’s channel (above middle image).1 This could lead scientists to erroneously conclude colocalization between the fluorophores.

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SOLUTION Researchers avoid bleed-through artifacts by choosing fluorophores with vastly different excitation and emission spectra.1 Additionally, they can employ filters with narrower wavelength ranges or spectral detectors to avoid this problem in most cases.

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EXPERIMEN PHOTOBLEACHING Fluorescence microscopy has revolutionized cell and developmental biology research by enabling scientists to l time and chers can mples thro ugh, phot , and chan ercome th PROBLEM

SOLUTION

Photobleaching is the phenomenon where a cell’s fluorescent signal irreversibly decreases over time because of accumulating damage to the fluorophores.2 This process results from exposure to high intensity excitation light.

Researchers reduce photobleaching by minimizing the light source’s intensity or the exposure time.2 If the scientist is imaging the cells live, they could increase the interval between time points, while mounting media with antifade reagents could help them prevent photobleaching for fixed cell experiments. Moreover, they could also use fluorophores requiring longer excitation wavelengths, such as those excited in the nearinfrared region, to limit photobleaching.3

BLEED-THROUGH

HIGH BACKGROUND SIGNAL OBLEM Bleed-through is the phenomenon where the emission from one fluorophore is detected in a channel (abov cientists to er etween the flu

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PROBLEM

SOLUTION

High background signal obscures the fluorophore’s real signal and reduces the apparent image resolution. This signal is caused by out-of-focus fluorophores detected above or below the focal plane, cell autofluorescence, or incorrect staining.4

Researchers decrease the detection of out-of-focus fluorescence by employing confocal microscopy over wide-field microscopy to image thicker samples.5 They can also reduce autofluorescence by choosing fluorophores that have different spectra than the autofluorescence, changing the fixative, or switching to a less autofluorescent medium when imaging the cells live.4 Moreover, scientists optimize sample staining by altering stain concentrations or changing blocking reagents to diminish the background signal and increase the apparent resolution.

PHOTOTOXICITY

PROBLEM

SOLUTION

Phototoxicity is the phenomenon where repetitive exposure to the light source during live-cell imaging causes damage to the cell that can lead to its death.3 This process leads to the production of reactive oxygen species (ROS), which react with the cell’s biomolecules and organelles to produce the damage. Phototoxicity produces cell morphology changes, including blebbing, apoptotic body formation, and nuclear fragmentation.3,6 This phenomenon often accompanies photobleaching.

Like photobleaching, reducing the light source’s intensity, decreasing the exposure time, or increasing the interval between images can mitigate the phototoxic effects of fluorescence microscopy, while employing fluorophores requiring longer excitation wavelengths also minimizes light-induced damage.3 Additionally, fluorophore choice has a direct effect on the amount of ROS produced in the cell after illumination, indicating that changing the fluorophore could reduce phototoxicity during live-cell imaging.

CHANNEL MISALIGNMENT

PROBLEM

SOLUTION

Channel misalignment is a phenomenon where images acquired for each fluorescent channel do not perfectly align with one another when overlaid (above right image). Scientists encounter this problem when the labeled organelles or biomarkers shift between the acquisition of each channel during live-cell imaging.

Researchers can avoid channel misalignment by employing a beam splitter and dual cameras, which each capture a channel simultaneously.7 They can also acquire the channels concurrently by using a single camera with an emission image splitter, which projects each channel onto a different portion of the camera’s sensors.

Seeing Is Solving Mouse cerebellum captured with a UPLXAPO40X objective on a FLUOVIEW™ FV4000 laser scanning confocal microscope. Sample courtesy of Dr. Katherine Given, Principal Investigator, Neurobiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

At Evident, we understand the value of a vision. We focus on developing advanced life science innovations to meet your laboratory and research needs. Our line of microscopy and imaging solutions is built with quality, clarity, and value in mind. Let’s work together to create an ideal environment for exploring possibilities. For more information, visit EvidentScientific.com

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References 1. 2. 3. 4. 5. 6. 7.

North AJ. Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. J Cell Biol. 2006;172(1):9-18. Ettinger A, Wittmann T. Chapter 5 - Fluorescence live cell imaging. In: Methods in Cell Biology. Vol 123. Academic Press; 2014:77-94. Icha J, et al. Phototoxicity in live fluorescence microscopy, and how to avoid it. BioEssays. 2017;39(8). Waters JC. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol. 2009;185(7):1135-1148. Elliott AD. Confocal microscopy: Principles and modern practices. Curr Protoc Cytom. 2020;92(1):e68. Laissue PP, et al. Assessing phototoxicity in live fluorescence imaging. Nat Methods. 2017;14(7):657-661. Valli J, et al. Seeing beyond the limit: A guide to choosing the right superresolution microscopy technique. J Biol Chem. 2021;297(1):100791.

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Figure 3

BEFORE

Hsf 1

Hia 1 BD2

BD2

BD3

Figure 3. Domain arrangements of Hia and Hsf trimeric autotransporters. The Hia passenger domain is characterized by a repetitive architecture consisting of multiple domain types. Hsf consists of a similar but extended domain arrangement compared to Hia. Domain arrangements were obtained from the daTAA server (http://toolkit.tuebingen.mpg.de/dataa). Signal Peptide

Neck/IsNeck domain

Trp ring domain

GANG domain

KG connector domain

Y1head domain

TTT domain

B-barrel domain

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Signal peptide Neck/IsNeck domain Trp ring domain GANG domain KG connector domain Ylhead domain TTT domain -barrel domain

AFTER

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By crossing Marth’s T cell specific cre transgene with Rajewsky’s conditional pol` allele, Marth and Rajewsky developed mice that lacked DNA polymerase ` in T cells.11 “When we did this work, this kind of technology opened the way to do lots of things beyond conditional targeting,” said Rajewsky.

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Expanding the scope of Cre-loxP recombination The success of Rajewsky’s and Marth’s conditional gene targeting led to an explosion of different Cre transgenic lines with expression profiles in various tissues. Researchers developed additional levels of control over gene expression as a natural extension of this founding technology. Mainly, researchers developed temporal control to circumvent previous challenges that arose from global gene deletion or early developmental Cre recombinase activity. The incorporation of drug- or interferon-responsive promoters allowed researchers to control expression of Cre recombinase. Researchers used tetracycline, type I interferon, or tamoxifen to induce promoter activation.12-14 Andrew McMahon, a developmental biologist from the University of Southern California, and his postdoctoral researcher Paul Danielian, who is now a biomedical editor at General Dynamics Information Technology, refined Cre recombinase control in vivo with mice using tamoxifen. “With a founding technology like Cre-loxP, a lot of resources got built around that,” recalled McMahon. Danielian previously studied steroid hormone regulation, and his ideas guided their subsequent experiments. The duo fused the ligand binding domain of the estrogen receptor to Cre to generate a conditional form of it knowing that it would be sequestered in the cytoplasm in the absence of a ligand. Adding a ligand activated this fusion protein and translocated it to the nucleus to activate Cre activity and induce recombination. This was the first time anybody had conditionally removed gene activity in the developing fetus of the mammalian system.15 Since then, tamoxifen-inducible Cre-loxP has been one of the most widely used inducible systems. “It’s just an illustration of how powerful genetics is. The more you can exquisitely control the process that you’re working with, the more insight you’re going to get into the question that you’re asking,” said McMahon.

Combining cre-ations The Cre-loxP system remains the gold standard method for conditional gene regulation in mice, but it can be costly and time consuming. So, over the last few years, researchers wondered whether to complement this method with another powerful gene editing technique. They found their opportunity with the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated nuclease, Cas9, which together enable highly specific DNA alterations at precise locations within the genome.16 The Cre-loxP-CRISPR combination sparked researchers’ interest in investigating its potential applications. CRISPR could introduce specific mutations or genetic variations and the Cre-loxP recombination system could precisely excise or integrate the genetic elements.

For Stefan Hans, a developmental geneticist at the Dresden University of Technology, this two-step approach offered the best of both technologies for his studies on neuron regeneration in zebrafish.17 The speed of transgenic zebrafish generation and the ability to precisely visualize the target cells were key advantages. “While you can be pretty sure that the cells are mutant, you also need to know where your cells are to understand how they behave. So, this feature is an important aspect because the putative mutant cells are labeled, and we can easily identify and take out these cells for analysis,” said Hans. Although the combination of these techniques was demonstrated in mammalian cells a few years prior, Hans was the first to use it in zebrafish research in 2021.18 The Cre-loxP system left a lasting influence on conditional gene editing, and modern advances have taken gene function and development to new heights. “Techniques come and go with new technology, It’s just like night and day. So, when you have a technique that’s lasted 30 years with no replacement technology, I think that’s kind of remarkable,” said Marth. J References 1. Sternberg N. Demonstration and Analysis of P1 Site-specific Recombination Using h-P1 Hybrid Phages Constructed In Vitro. Cold Spring Harb Symp Quant Biol. 1979;43:1143-1146. 2. Sternberg N, Hamilton D. Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites. J Mol Biol. 1981;150(4):467-486. 3. Saur B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Bio. 1987;7(6):2087-2096. 4. Saur B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988;85(14):5166-5170. 5. Evans MJ, Kaufman MH. Establishment in culture of pluripotent cells from mouse embryos. Nature. 1981;292:154-156. 6. Doetschman T, et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987;330:576-578. 7. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51:503-512. 8. Kitamura D, et al. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene. Nature. 1991;350:423-426. 9. Gu H, et al. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993;73(6):1155-1164. 10. Orban PC, et al. Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci. 1992;89(15):6861-6865. 11. Gu H, et al. Deletion of a DNA Polymerase ` Gene Segment in T Cells Using Cell Type-Specific Gene Targeting. Science. 1994;265(5168):103-106. 12. St-Onge L, et al. Temporal Control of the Cre Recombinase in Transgenic Mice by a Tetracycline Responsive Promoter. Nucleic Acids Research. 1996;24(19):3875-3877. 13. Kühn R, et al. Inducible gene targeting in mice.  Science.1995;269(5229):1427-1429. 14. Metzger D, et al. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A, 1995;92(15):6991-6995. 15. Danielian PS, et al. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Cell. 1998;8(24):1323-1326. 16. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. 17. Hans S, et al. Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish. Nat Commun. 2021;12:1125. 18. Yang F, et al. CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool. Mol Ther Nucleic Acids. 2017;7:378-386.

WINTER 2 02 3 | T H E S C IE N T IST 1 7

Some species remove up to 90 percent of their genomes during development, but why or how this happens is still a mystery. BY APARNA NATHAN, PhD

arie Delattre, a biologist at the École Normale Supérieure de Lyon, has studied the nematode Mesorhabditis belari for nearly a decade now. The microscopic worm first caught her attention for its unconventional approach to reproduction, where only a small fraction of offspring keep their male parent’s DNA.1 But more recently, she was looking at Mesorhabditis embryos under a microscope and noticed something else that was odd: in some embryos, the DNA was shattered into small fragments. As she looked more closely across all stages of embryo development, she noticed a pattern. At the one-cell stage, the DNA looked normal. It stayed intact as the first cell divided into two cells and then three cells. But when the embryo reached the fivecell stage, the DNA fragments suddenly appeared. They started in the nucleus, then moved into the surrounding cytoplasm, and after a few more rounds of cell division, disappeared entirely. Delattre was intrigued. During this brief point in the worms’ development, part of their genomes appeared to vanish. What she observed is a process carried out by dozens of other species. Termed programmed DNA elimination, this process allows organisms to delete specific portions of their genomes dur-

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ing development. “It was a really random discovery,” said Delattre. “Serendipity, I would say.” Her team’s deeper investigation into this phenomenon, recently published in the journal Current Biology, added Mesorhabditis nematodes to the list of species that can carry out this mysterious process.2,3 Even after more than a century of research, there are still many unanswered questions about programmed DNA elimination. “You see that this thing happens very commonly, suggesting it has an important biological role,” said Jianbin Wang, a biologist at the University of Tennessee, Knoxville. “It’s just that we don’t really know what that role is yet.” Researchers around the world are working to answer these questions. Many of them stumbled upon programmed DNA elimination by accident, like Delattre, but now they’re hooked; their research could offer a new understanding of the ever-changing nature of the genome.

New technology takes on an old theory An organism’s DNA starts off in one lonely cell containing the germline DNA passed down from the previous generation. Through many rounds of cell division, new somatic cells emerge T

that contain essentially identical copies of DNA and become the building blocks of the organism. In the 1880s at the Zoological Institute, cell biologist Theodor Boveri studied a species of parasitic worms called Parascaris, which has a relatively large genome compared to other worms— so large that its DNA was visible even through a primitive 19th century microscope. He observed that a large chunk of the germline genome was removed as somatic cells developed.3 More than 100 years later, more sophisticated molecular biology assays revealed that this worm removed an astounding 89 percent of its 2.5-billion-base genome.

As those were still the early days of cell biology, Boveri assumed that this was a normal part of development. But as scientists looked for this process in more organisms, they realized that programmed DNA elimination was not universal. Early work focused on microscopic species, including various species of parasitic worms and single-celled organisms called ciliates. Scientists learned much of what they know about programmed DNA elimination by studying a family of parasitic worms called Ascaris, which removes around one-fifth of its germline genome.3 In the 1980s, researchers finally found a family of vertebrates, the hagfish, that removes between one-

Many species undergo programmed DNA elimination, a process where specific parts of the genome found in the original sperm and egg cells are removed from the cells of the developing body. Different species use varied cellular mechanisms to remove specific parts of their genomes. This process has recently been documented in worms in the Mesorhabditis genus, which eliminate approximately thirty percent of their DNA.

One-cell stage

Cells divide

Kinetochore

Early in the process of Mesorhabditis development, cells still carry the germline genomes from the gametes that produced the first cell. As early as the two or four-cell stage, the DNA begins to fragment. Researchers can see this under a microscope, and it’s one of the first signs that a cell might be preparing for programmed DNA elimination.

As the cells prepare to divide, the DNA assembles into chromosomes. Normally, microtubules latch onto these chromosomes via each chromosome’s kinetochore proteins. However, some DNA fragments lack kinetochores, so the microtubules have nowhere to bind.

Metaphase plate

The chromosomes arrange themselves in pairs along the middle of the dividing cell in a region called the metaphase plate. Without microtubules to guide them there, the unattached DNA fragments do not migrate to the metaphase plate and instead linger in the surrounding areas of the cell. They will likely be targeted for elimination.

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fifth and one-half of its germline genome.4 More recently, studies have shown that nearly all songbirds appear to eliminate parts of their germline genomes.5 “We are exposed to organisms that have programmed DNA elimination every single day,” said Alexander Suh, an evolutionary biologist at the Leibniz Institute for the Analysis of Biodiversity Change and Uppsala University. Recent technologies such as DNA sequencing have bolstered researchers’ efforts to probe this process. By comparing sequences of the genomes of germ cells and somatic cells from the same organism, researchers can look for long

In the last stage of cell division, the microtubules pull the pairs of chromosomes apart, so that each new cell’s nucleus gets one chromosome from each pair. The unattached DNA fragments remain in the center of the dividing cell, where they will be randomly pushed into one of the two new cells.

In a wild-type cell, you would never see this. This would be a red flag. —Marie Delattre, Ecole Normale Superieure de Lyon

stretches in the germline genome that are absent from the somatic genome. These studies have shown that species can eliminate anywhere between 0.5 percent and 90 percent of their genomes.

Each new cell’s nucleus contains one full copy of the somatic genome, while the other DNA fragments remain in the cytoplasm outside the nucleus. Other species such as sea lampreys eliminate whole chromosomes instead of DNA fragments. During cell division, these chromosomes migrate to the metaphase plate along with the other chromosomes, but do not migrate to the poles of the dividing cell. This behavior is known as “lagging” and causes the chromosomes to be excluded from the new cells’ nuclei and eliminated.

After a few more cycles of cell division, the DNA excluded from the nucleus likely degrades in the cytoplasm, as seen in other worms.

Scientists have used sequencing to investigate exactly which parts of the genome are removed and what instructions they encode. Typically, the same regions are removed in every cell of an organism and in every member of a species, although there are differences between species. However, regardless of species, the eliminated regions include large stretches of repeated DNA sequences, which typically do not encode the instructions for proteins.

How to ditch DNA Programmed DNA elimination pops up on almost every branch of the tree of life, but the processes are as diverse as the flora and fauna that use them. The nematodes and unicellular ciliates seem to slice their genomes into small pieces and remove a subset. Vertebrates seem to be more likely to remove full chromosomes.

Understanding the origins of DNA elimination is going to tell us a lot about how our own genome works.

piece of DNA at the tip of the chromosome appeared to drag the chromosome in the opposite direction of the microtubule. As a result, these lagging chromosomes get left behind when the nuclei form and end up degraded in small pockets of the cytoplasm.7 Researchers still don’t know how the lagging chromosomes or discarded DNA fragments are chosen. In worms, Delattre uses detailed maps of the genome and RNA measurements to figure out how the cell knows where to fragment the DNA and what proteins make the cuts. Once she finds a compelling protein candidate, she hopes to delete it from an embryo and observe whether the cells still undergo programmed DNA elimination.

Extreme DNA silencing There’s another major open question: why would species want to eliminate their DNA at all? Removing DNA is an extreme way of keeping genes from being used in cells, according to Jeramiah Smith, a biologist at the University of Kentucky. But cells have other ways of doing this that are less disruptive. For example, they can pack their DNA so tightly that the genes can’t be accessed, or they can use small pieces of RNA that bind to

These different approaches involve the same set of core steps: part of the genome is marked for elimination, and as the cell divides, this DNA is shunted out of the nucleus and ultimately removed from the cell. In ciliates and worms, the cell also needs to slice up the DNA into fragments. How this happens in cells is still largely unknown. But studies from Wang, Delattre, and others are starting to piece together the process. Cell division is a carefully orchestrated dance, where duplicated chromosomes pair up in a double-file line in the metaphase plate at the center of the cell. Long microtubules anchored at opposing ends of the cell latch onto the kinetochore protein at the center of each chromosome. As the cell divides, the microtubules reel in half of the chromosomes to each new cell. In Ascaris, Wang showed that DNA fragments that are ultimately eliminated actually lack kinetochore proteins, so the microtubules can’t bind to them and pull them into the new cells’ nuclei.6 Delattre saw a similar dearth of kinetochore proteins on eliminated DNA in Mesorhabditis.2 Using a fluorescent label, she visualized DNA fragments as tiny dots floating in a ring around the rest of the DNA. The untethered DNA pieces get pushed to the perimeter while the remaining chromosomes segregate. As the cell continues dividing, the fluorescent dots end up outside the nucleus in the cytoplasm, and ultimately fade away. In vertebrate species, where whole chromosomes are eliminated, things go awry when microtubules start pulling the chromosomes apart. Certain chromosomes move slower than the others, a phenomeno≠n called lagging. In a study of sea lampreys, a 22 T H E SC I EN T I ST | the-scientist.com

By looking at Mesorhabditis belaris embryos under a microscope, Marie Delattre noticed that they removed portions of their genomes.

SIMON BIANCHETTI

—Jeramiah Smith, University of Kentucky

MARIE DELATTRE

Scientists observe Mesorhabditis belari worms at various stages of development through a microscope to study programmed DNA elimination.

genes and inhibit protein expression. Species that carry out programmed DNA elimination typically have the machinery to silence genes in these ways too. Another explanation is that eliminated genes might play a role in germ cells during reproduction, where they may need to make an arsenal of proteins that are unnecessary in other cells of the body. Studies in Ascaris and in zebra finches revealed that their eliminated genes have functions in sex organs like the testes, where germ cells originate.8,9 Not all researchers are convinced. In Mesorhabditis, Delattre showed that the eliminated genes seem to follow no pattern and aren’t critical to species survival.2 She thinks that in these worms, the process could mainly serve to remove repeated sequences, and any genes that are removed are just bystanders. It’s also possible that programmed DNA elimination could play diverse r oles in different species. “I would say, at this point, that they’re different processes,” Suh said. Smith agreed and noted that the evolutionary history of DNA elimination is still hazy. He speculated that each major branch of life may have independently developed the ability to eliminate DNA. “They’re doing similar things, but they got there through very different evolutionary trajectories,” he said. Although programmed DNA elimination hasn’t been observed in humans, similar processes can occur in human

cells, but typically only when they are malfunctioning. “In a wild-type cell, you would never see this,” Delattre said. “This would be a red flag.” When human DNA breaks into fragments, a process called chromothripsis, it’s a cellular event so catastrophic that it can cause cancer. When human chromosomes don’t divide into cells properly during embryonic development, we end up with developmental disorders. “Understanding the origins of DNA elimination is going to tell us a lot about how our own genome works,” Smith said. For example, it might help us better understand how these events occur in cancer cells. Knowing how DNA is marked for deletion and being able to do that artificially could even be used as a therapeutic option for disorders caused by extra copies of chromosomes, such as Down syndrome. For now, many scientists agree that the next step is to study programmed DNA elimination in more species. “We probably don’t know the majority of species that actually do this,” Smith said. Suh and his team are doing just that. So far, they have sequenced germline and somatic DNA from around 30 species of songbirds. Every songbird eliminates at least one chromosome, but in some cases it’s the biggest chromosome; in other species it’s the smallest one. The genes on the eliminated chromosomes can be completely different between species but they have one thing in common: Some of the gene sequences are very similar to those found on retained chromosomes, a trend that hasn’t yet been observed in other types of animals.9 “It gets more and more confusing with every species,” Suh said. J

References 1. Grosmaire M, et al. Males as somatic investment in a parthenogenetic nematode. Science. 2019;363(6432):1210-1213. 2. Rey C, et al. Programmed DNA elimination in Mesorhabditis nematodes. Curr Biol. 2023;33(17):3711-3721. 3. Zagoskin MV, Wang J. Programmed DNA elimination: silencing genes and repetitive sequences in somatic cells. Biochem Soc Trans. 2021;49(5):18911903. 4. Smith JJ, et al. Programmed DNA Elimination in Vertebrates. Annu Rev Anim Biosci. 2021;9:173-201. 5. Torgasheva AA, et al. Germline-restricted chromosome (GRC) is widespread among songbirds. Proc Natl Acad Sci USA 2019;116(24):11845–11850. 6. Kang Y, et al. Differential Chromosomal Localization of Centromeric Histone CENP-A Contributes to Nematode Programmed DNA Elimination. Cell Rep. 2016;16(9):2308-2316. 7. Timoshevskiy VA, et al. Germline-Specific Repetitive Elements in Programmatically Eliminated Chromosomes of the Sea Lamprey (Petromyzon marinus). Genes (Basel) 2019;10(10):832. 8. Wang J, et al. Silencing of Germline-Expressed Genes by DNA Elimination in Somatic Cells. Dev Cell. 2012;23(5):1072-80. 9. Kinsella CM, et al. Programmed DNA elimination of germline development genes in songbirds. Nat Commun. 2019;10(1):5468.

WINTER 2023 | T H E S C IE N T IST 2 3

The h al L e of e lacenta in o Y

© SHUTTERSTOCK.COM, SCIEPRO

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e have all had one, and we owe our lives to it. It’s the first organ to develop and it simultaneously serves as the lungs, kidneys, immune system, and digestive tract, to name a few, in a fetus while it develops these systems. Despite being one of the most important organs, the placenta is one of the least understood. “It’s such a fascinating organ,” said Norah Fogarty, a developmental biologist at King’s College London. “We know so little about it, but there’s also this kind of intrigue about the placenta.” This mysterious organ has inspired lore and customs for centuries. Throughout gestation, the fetus depends entirely on the placenta. The discoid-shaped organ serves as a barrier between the parent and child. Although some researchers describe the placenta as an evolutionary battleground due to the mix of maternal and paternal DNA both vying for resources, it is also a space where compromise prevails to ensure the health of both parties. Research has linked abnormal placental development to a number of pregnancy complications, including preeclampsia, fetal growth restriction, placental abruption, and preterm labor, collectively referred to as the great obstetric syndromes.1 Preeclampsia, characterized by high blood pressure and increased protein in the urine after 20 weeks of pregnancy, occurs in 3-5%

of pregnancies.2 Preeclampsia ranges from mild to severe and can be life-threatening for the parent and child. Currently, the only cure is delivery of the baby and placenta. The dearth of treatments for preeclampsia stems from a greater gap in our knowledge. “We also don’t know what’s happening in normal placenta development,” said Fogarty. The lack of physiologically relevant models of placental development stymies efforts to close these knowledge gaps. Although animal models have provided valuable insight into the organ’s development, the placenta is one of the most evolutionarily divergent organs, and considerable differences in the developmental trajectory, morphology, and degree of placental invasion into the uterine wall demand caution when extrapolating data from other species to humans.3 Additionally, several ethical and logistical obstacles hinder the study of early placenta development in humans. These obstacles led researchers to develop in vitro models, but until recently it wasn’t clear how robust these models could be. “The placenta has been difficult to capture in the dish,” said Fogarty. Now, a few key advancements in cell culture techniques over the last decade have breathed new life into the field, and many hope that these models hold the key to unlocking the secrets of the space between. WINTER 2 02 3 | T H E S C IE N T IST 2 5

A black box Following fertilization, one cell becomes two, and those become four and so on, until the zygote transforms into a blastocyst around six days post fertilization (dpf ).4 The blastocyst, or the preimplantation embryo, comprises of an inner cell mass (ICM) swaddled by an outer layer of cells that make up the trophectoderm. The trophectoderm, which is home to nearly 90 percent of the blastocyst cells, develops into the placenta while the ICM gives rise to the fetus. Fogarty wants to understand the molecular processes that orchestrate these early developmental stages. As an undergraduate student at Trinity College Dublin, Fogarty’s interest in fetal health and development began when she enrolled in a course on molecular medicine that focused on treating diseases of adulthood. “It led me to think ‘you know, we’re focusing all this time and research into treating diseases in the adult, but if we can help babies be born as healthy as possible and grow up to be healthy adults, then we would likely eradicate a lot of these diseases,’” said Fogarty. Near the end of her studies, she came across an email that piqued her interest: It was an advertisement about a PhD project on placenta development at the University of Cambridge. She applied and got the position where she studied transcriptional dynamics in the human placenta under the joint supervision of Graham Burton and Anne Ferguson-Smith. Following her doctoral studies, Fogarty joined the lab of stem cell and developmental biologist Kathy Niakan at the Francis Crick Institute to continue her investigations into the molecular drivers of early cell fate. Transcription factors orchestrate trophectoderm development and differentiation. Using comparative analyses, researchers previously demonstrated that two such factors, octamer-binding transcription factor 4 (OCT4) and caudal-type homeobox-2 (CDX2), exhibit temporally and spatially distinct expression patterns in the embryos of mice and humans.5 Considering the divergent expression patterns between the two species, Fogarty was curious about the function of OCT4 during human embryo development. To study this, she turned to CRISPR-Cas9-mediated genome editing. Deletion of the gene encoding OCT4 from early zygotes donated from patients of IVF clinics led to a downregulation of trophectoderm genes, including CDX2, and compromised the development of the blastocyst.6 In contrast, when the researchers manipulated mouse embryos in a similar manner, the blastocyst formed but its maintenance was compromised. Fogarty’s research detailed functional consequences of species-specific gene expression patterns, further illustrating why mouse models may fail to capture key developmental events in humans. Around six to seven dpf, the blastocyst implants into the surface of the uterine wall and begins its expansion.4 This area of the endometrium is transformed early on in pregnancy and acts as a fluffy bed in which the embryo grows. As the blastocyst burrows, the trophectoderm begins to differentiate into subtypes of trophoblast cells, starting with cytotrophoblasts, which are progenitor stem cells in the placenta that give rise to other trophoblasts. 26 T H E SC I EN T I ST | the-scientist.com

This point in development, approximately 14 dpf, corresponds to around the time of the first missed period, and it is typically the earliest point that most people realize that they are pregnant. Early zygote and blastocyst donations from patients of IVF clinics have helped shed light on this black box period of development, but human embryos are a limited resource and ethical concerns restrict their long-term use in the lab. These earliest days of development, although temporally distant from the clinical manifestation of preeclampsia, may lay the foundations for future problems. “In the last few years, there have been more hypotheses being developed that it’s a defect in the cytotrophoblast cell that sets the track for whether preeclampsia will develop or not,” said Fogarty.

“We also don't know what's happening in normal placenta development.” —Norah Fogarty, King’s College London

There are a number of available in vitro models for the study of human placental development with varying degrees of physiological relevance.2,4,7 Choriocarcinoma cells, derived from malignant tumors of trophoblasts, are genetically abnormal and mouse trophoblast stem cells, while a valuable tool, do not fully recapitulate the genetic and molecular milieu orchestrating human placental development. However, 2018 saw a major breakthrough: For the first time, researchers successfully generated bona fide human trophoblast stem cells (hTSC).8 The researchers demonstrated that either trophectoderm from the blastocyst or first-trimester placentas could be used to generate bipotent trophoblast stem cells. Now, researchers finally have access to pure trophoblast cells with the capacity for self-renewal. By tweaking what they feed the hTSC, researchers can transform the cells into different trophoblast subtypes. The hTSC are useful models for studying trophoblasts, while the embryo provides unrivaled access to studying trophectoderm development. Fogarty uses the hTSC alongside human embryos to study the signaling pathways that regulate trophectoderm and early trophoblast development and differentiation. Furthermore, hTSC are a useful platform for optimizing tools and developing hypotheses before testing them in valuable human embryos. “These experiments will further our understanding of hTSC and how they differ from the trophectoderm, but will also give us insights into trophoblast biology,” said Fogarty. She hopes that these tools can one day help reveal how defects emerging early in development set the stage for placental diseases like preeclampsia.

Miniplacentas in a dish As the invasion into the uterine wall wages on, cytotrophoblasts differentiate into syncytiotrophoblasts (SCT), which carve out villi, or

PAULA BALESTRINI

frond-like structures that soon house the fetal capillary system.7 As SCT build larger and larger villi, cytotrophoblasts march forward to conquer a new frontier in search of nutrients to fuel the continued expansion. These rogue cytotrophoblasts go deeper into the uterine wall and differentiate into incredibly invasive extravillous trophoblasts (EVT). Once there, EVT hunt down uterine arteries, enlarge them, and hook them up to the placenta. Finally, around 10 weeks into the pregnancy, the parental circulation reaches the intervillous space.9 By 12 weeks, the placental blueprint is in place. The uterine wall is home to glands, vessels, stromal cells, and immune cells that interact with the invading fetal cells to create a boundary between the parent and fetus.4 The relationship between the parent and the growing fetus is often portrayed as parasitic or antagonistic, a 9-month war waged from within. This is due in part to the highly invasive nature of EVT leaching nutrients, but also the presence of the fetus’ foreign DNA. But Ashley Moffett, a reproductive immunologist at the University of Cambridge, said that the relationship between the parent and the placenta isn’t simply friend or foe. “It’s a compromise, actually.” Moffett, a doctor and pathologist by training, didn’t set out to study the placenta. In the 1980s, she was looking for a job at the hospital in Cambridge. The only available job at the time was in the maternity ward. “I was sort of banished to the maternity hospital without any interest at all in this, but I then, of course, realized that there were these major disorders and that they were completely understudied,” recalled Moffett. “Nobody knew anything about them really.”

While working in the maternity ward, Moffett recalled influential papers published in the early 1980s that suggested that preeclampsia results from a failure of EVT to properly invade the uterine wall.1 She also remembered some peculiar looking cells that she came across in pathology training. “I looked at every single organ in the body under the microscope,” said Moffett. “I realized that there were some cells in the uterus that I’ve never seen anywhere else.” She thought that they might be a kind of natural killer (NK) cell, so Moffett contacted Charlie Loke, one of her undergraduate professors from the University of Cambridge and an expert in reproductive immunology, to study these cells in the lab. After only a month in the lab they figured out that these cells were, in fact, a type of NK cell. “And [Loke] said, ‘you know, you’ll never go back to clinical medicine,’ and I didn’t ever go back to clinical medicine,” said Moffett. “That was the end of my medical career.” Uterine NK cells, which differ substantially from blood NK cells, dominate the immune cell landscape of the uterine wall bordering first trimester placentas.10 Moffett and others went on to characterize this unique immune cell and demonstrate its importance as a mediator between the needs of the mother to retain resources and the needs of the baby to grow. To explore the boundary between the parent and fetus, Moffett and her team used single-cell RNA sequencing on placental and endometrial samples donated by patients who underwent elective pregnancy termination in the first trimester.11 They identified transcription factors that orchestrate cytotrophoblast differentiation into SCT or EVT but also uncovered three subtypes of NK cells with distinct immune regulation and cell-cell communication profiles. Their findings further highlighted the compromise between parent and fetus, suggesting that NK cells keep a check on EVT expansion while these cells protect the fetus from parental immune responses. Around the same time in 2018, Moffett and her team and Martin Knofler’s research team at the Medical University of Vienna separately published the first organoid models for trophoblasts.12,13 These three-dimensional organoid models offered another step towards a physiologically relevant model that recapitulates certain aspects of the in vivo environment. To build a mini-placenta, the researchers isolated proliferative cells from first trimester placenta tissue and cultured them in a special cocktail chock full of growth factors that coax trophoblast development and assembly into a three dimensional blob of cells. Not only did the trophoblast organoids retain transcriptomic and methylation patterns characteristic of in vivo first trimester trophoblasts, but they also developed hormone-secreting SCT with intricate structures akin to villi as well as migratory EVT. These self-replicating mini-placentas even produced enough of the hormone chorionic gonadotropin to test positive on an at-home pregnancy test.12

In her lab at King’s College London, Norah Fogarty studies transcriptional events that orchestrate early placenta development. WINTER 2 02 3 | T H E S C IE N T IST 27

EARLY PLACENTA DEVELOPMENT SETS THE STAGE During early pregnancy, the placenta remodels the uterine environment to support fetal growth.

DAY 0 DAYS 10-12 By 12 dpf, cytotrophoblast cells begin to penetrate the primitive syncytium to form primary villi, which later form the villous placenta.

DAY 1

Primary villi

DAY 2 Primitive syncytium

DAYS 7-9 After implantation, the trophectoderm starts reshaping the endometrium. A layer of cytotrophoblasts—trophoblast progenitor cells—emerges around the same time as the invading primitive syncytium.

DAY 3

DAY 4

DAYS 6-7 Inner cell mass

Blastocyst

DAYS 5-6

Trophectoderm

Approximately five days post fertilization (dpf), the blastocyst develops. The inner cell mass gives rise to the fetus, while the surrounding trophectoderm transforms into the placenta. -scientist.com

Six to seven dpf, the blastocyst attaches to the uterine wall and begins its invasion.

Cytotrophoblasts

Decidua

From weeks three to 10, cytotrophoblast cells escape into the decidua, a specialized layer of endometrium, and differentiate into extravillous trophoblasts. These invading cells remodel spiral arteries to reroute parental blood to the intervillous space.

EARLY FIRST TRIMESTER PLACENTA

Villus

Syncytiotrophoblast

Cytotrophoblast

© JULIA MOORE, WWW.MOOREILLUSTRATIONS.COM

By the beginning of the second trimester, the cytotrophoblast plug breaks down and parental blood begins to enter the intervillous space.

Spiral artery Extravillous trophoblast

FULL TERM PLACENTA

BEYOND WEEK 10 PLACENTA

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In the 1980s, Moffett gained access to rare first trimester pregnancy hysterectomies, which included the entire uterus. She safely tucked these samples away with the hope that one day new tools would emerge to explore their cellular intricacies. Earlier this year, Moffett returned to her historical hysterectomy samples and published a spatially resolved multiomics single-cell atlas that captures the trajectory of trophoblast differentiation as the cells invade and transform the arteries in the uterine wall.14 This rich resource identified transcription factors and key cell-cell interactions, including uterine NK cells in close proximity with EVT. Furthermore, they found many of the same factors expressed on EVT derived from hTSC and primary trophoblast organoids. “We now have a trajectory of the whole invading trophoblast in humans for the first time, and I think the organoids do recapitulate that quite well,” said Moffett. After a long career, Moffett recently handed over the keys to her lab, but she hopes these organoids will go on to provide a relevant platform for studying important placental biology questions that have relevance to placental disorders like preeclampsia.

I realized that there were some cells in the uterus that I’ve never seen anywhere else. —Ashley Moffett, University of Cambridge

Shallow roots As the pregnancy progresses, cytotrophoblast cells keep dividing, and the placenta keeps getting bigger and bigger to keep up with the needs of the growing fetus. Shallow implantation of EVT early in development might be one cause of preeclampsia.1 This results in an insufficient or poor transformation of the arteries and paves the way for a sparsely branched villous tree and weak perfusion network for blood and waste products to travel between the parent and fetus.7 This can cause serious problems later in pregnancy. Mariko Horii trained as an obstetrician in Japan before coming to the University of California, San Diego in 2013 to work with Mana Parast, a placental pathologist. Horii arrived searching for answers to why the placenta grows so poorly as has devastating consequences for some of her patients. At the time, Parast was gearing up to develop in vitro models for studying preeclampsia. A lot of what researchers in the field know about human placental development comes from morphological, immunohistochemical, and transcriptomic analyses of primary first-trimester placental tissue.4 While an incredibly rich source of information, access to these tissues is limited, and isolated cells did not survive for long in a dish, making it difficult to run experiments in the lab. This left scientists with a choice between genetically abnormal cancer cell lines or mouse trophoblast stem cells for their research. A major advance came in 1998 when James Thomson, a stem cell biologist at the University of Wisconsin-Madison, successfully iso30 T H E SC I EN T I ST | the-scientist.com

lated stem cells from human blastocysts.15 Shortly after, his research team demonstrated that they could differentiate human embryonic stem cells (hESC) into hormone-secreting cells akin to SCT by feeding the cells a special media spiked with bone morphogenetic protein-4 (BMP4).16 The subsequent development of induced pluripotent stem cells (iPSC) via the reprogramming of somatic cells provided scientists with a less controversial, more accessible source of human pluripotent stem cells.17 Since then, Horii and others have demonstrated that both hESC and iPSC can transform into trophoblasts and subtypes of trophoblasts by altering the environmental conditions and feeding the cells different molecular cocktails.7,18 While the advent of hTSC and trophoblast organoids in 2018 are major stepping stones, they come with limitations. “Since we have primary cells, then why not use the primary cells for a model system instead?” said Horii. Scientists are still struggling to produce hTSC and trophoblast organoids from full term placentas and currently derive them from either blastocysts or first trimester placentas. Both sources are limited and, in some countries, laws restrict their use. Horii raised another limitation of using cells sourced from early pregnancy. “We don’t have the scientific knowledge to predict from the early first trimester pregnancy materials whether the patient would have developed pregnancy complications or not.” To build this knowledge, Horii and her team turned to full term placentas for iPSC. Either mesenchymal stem cells derived from the umbilical cord or cytotrophoblasts can transform into iPSC when fed a special cocktail.17 Using iPSC derived from placental cells, Horii and others have worked doggedly to refine culture protocols over the years to generate trophoblasts.7,18 Eventually, researchers working with these cells demonstrated that their putative trophoblasts secreted key hormones and expressed the EVT marker HLA-G alongside other key genes expressed by trophoblasts.19,20 Recently, Horii and her team modified their protocol to include a WNT-inhibitor alongside BMP4 to ensure the exclusion of mesoderm cells and differentiation in trophoblast cells resembling cytotrophoblasts.21 However, they struggled to maintain primed stem cells, or iPSC-derived trophoblasts, in a state of self-renewal. To fix this, Horii and her team fed their iPSC the usual fare of BMP4 plus a WNT-inhibitor but swapped the main culture media for one they whipped up for the newfangled hTSC.22 “We were able to finally derive the self-renewing trophoblast stem cells,” said Horii. They further differentiated their new and improved cytotrophoblasts into EVT or SCT using cell type specific differentiation protocols. Horii thinks that the iPSC-derived trophoblast models will be particularly useful for disease modeling because scientists can use iPSC to produce cell types beyond trophoblasts, like blood vessels or stromal cells. Currently, in her own lab, she uses this revamped protocol on cells isolated from term placentas of patients with preeclampsia. Her prior work using an earlier version of the culture protocol suggested that this will be a fruitful avenue for modeling preeclampsia in a dish. iPSC derived from placentas of pregnancies with preeclampsia recapitulate several defects observed in primary placenta tissues, including failure to respond to changes in surrounding oxygen levels and abnormalities in EVT differentiation.23

riage, intrauterine growth restriction, fetal growth restriction, and preeclampsia. “If we can make these insights, there’s going to be massive numbers of patients who can potentially be helped in the future. There’s potential to make a big impact.” J

References

Six days after fertilization, the human embryo holds epiblast cells (red) and the trophectoderm (green). Epiblast cells go on to form the fetus, while the trophectoderm gives rise to the placenta.

NORAH FOGARTY

Fleeting but indelible Following the birth of the baby comes the birth of the placenta as it sheds away from the lining of the uterus. By the end of gestation, the SCT region is incredibly invaginated and convoluted to provide a large surface area for diffusion to the baby. “If you were to spread it out it would be 13 square meters in size,” said Fogarty. That’s about the size of a parking space. Just like that, this transient organ that helped the fetus survive in the womb for the last nine months is gone. Scientists are increasingly appreciating the link between the in utero environment, including placental health, and susceptibility to chronic diseases later in life.24 For example, babies that are born too big or too small relative to their growth potential are at a higher risk for developing cardiovascular disease, diabetes, and obesity in adulthood. These long-lasting effects further emphasize the need for improved screening, prevention, treatment options, and of course, physiologically robust and relevant models. “We now have the tools,” said Fogarty. Trophoblast stem cells, trophoblast organoids, iPSC-derived trophoblasts, extended embryo culture, CRISPR Cas9-mediated genome editing, advanced imaging technology, scRNAseq, and spatial transcriptomics all have a role to play in the study of the placenta. While no model perfectly captures the complexities of this mysterious organ, recent advances, including refined culture protocols and new in vitro systems, will facilitate the continued study of human placentation. Some researchers are even developing placenta-on-a-chip models using human iPSC-derived trophoblasts to study placental perfusion dynamics.25 “There’s a renewed interest in the field, an energy in the field, that will allow us in the next 10-20 years to make these breakthroughs and bring our understanding of the placenta up to speed with a lot of the other organs that we know so much about,” said Fogarty. “Just over half of all pregnancies are uncomplicated, normal pregnancies,” said Fogarty. The other half are affected by miscar-

1. Brosens I, et al. The “great obstetrical syndromes” are associated with disorders of deep placentation. Am J Obstet Gynecol. 2011;204(3):193-201. 2. James JL, et al. Modelling human placental villous development: Designing cultures that reflect anatomy. Cell Mol Life Sci. 2022;79(7):384. 3. Roberts RM, et al. The evolution of the placenta. Reproduction. 2016;152(5):R179-189. 4. Turco MY, Moffett A. Development of the human placenta. Development. 2019;146(22):dev163428. 5. Niakan KK, Eggan K. Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse. Dev Biol. 375(1):54-64. 6. Fogarty NME, et al. Genome editing reveals a role for OCT4 in human embryogenesis. Nature. 2017;550:67-73. 7. Horii M, et al. Modeling human trophoblast, the placental epithelium at the maternal fetal interface. Reproduction. 2020;160(1):R1-R11. 8. Okae H, et al. Derivation of human trophoblast stem cells. Cell Stem Cell. 2018;22(2):50-63.e6. 9. Jauniaux E, et al. Onset of maternal arterial blood flow and placental oxidative stress. Am J Pathol. 2000;157(6):2111-2122. 10. Moffett A, Colucci F. Uterine NK cells: Active regulators at the maternal-fetal interface. J Clin Invest. 2014;124(5):1872-1879. 11. Vento-Tormo R, et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature. 2018;563:347-353. 12. Turco MY, et al. Trophoblast organoids as a model for maternal-fetal interactions during human placentation. Nature. 2018;564:263-267. 13. Haider S, et al. Self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta. Stem Cell Reports. 2018;11(2):537-551. 14. Arutyunyan A, et al. Spatial multiomics map of trophoblast development in early pregnancy. Nature. 2023;616:143-151. 15. Thomson JA, et al. Embryonic stem cell lines derived from human blastocysts. Science .1998;282(5391):1145-1147. 16. Xu R-H, et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol. 2002;20:1261-1264. 17. Takahashi K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872. 18. Roberts RM, et al. Specification of trophoblast from embryonic stem cells exposed to BMP4. Biol Reprod. 2018;99(1):212-224. 19. Amita M, et al. Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4. Proc Natl Acad Sci USA. 2013;110(13):E1212-1221. 20. Horii M, et al. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease. Proc Natl Acad Sci USA. 2016;113(27):E3882-E3891. 21. Horii M, et al. An improved two-step protocol for trophoblast differentiation of human pluripotent stem cells. Curr Protoc Stem Cell Biol. 2019;50(1):e96. 22. Soncin F, et al. Derivation of functional trophoblast stem cells from primed human pluripotent stem cells. Stem Cell Reports. 2022;17(6):P1303-1317. 23. Horii M, et al. Modeling preeclampsia using human induced pluripotent stem cells. Sci Rep. 2021;11(1):5877. 24. Thornburg KL, Marshall N. The placenta is the center of the chronic disease universe. Am J Obstet Gynecol. 2015;213(4):S14-S20. 25. Lermant A, et al. Development of human iPSC-derived placental barrier-onchip model. iScience. 2023;26(7):107240.

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Unraveling the Mystery of Zombie Genes Digging into how and why some genes are resurrected after death sounds morbid, but it has practical applications. BY IRIS KULBATSKI, PhD

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

MODIFIED FROM © ISTOCK.COM, ILYA LUKICHEV

as stem cells, survive. 9 During this period of cellular afterlife, these cells release molecular distress calls in an ongoing display of death resistance. “Cells within tissues struggle to survive by changing their transcriptional programs to cause upregulation of developmental pathways,” Javan said.

Unwinding the clock

Cells don’t want to die. —Gulnaz Javan, Alabama State University

The will to live That’s life. But what about death? Less than a decade ago, researchers debunked the long-held assumption that gene expression—a hallmark of life—ceases at the time of death. While most gene activity is extinguished after an organism dies, certain zombie genes are reawakened, sometimes days later. Some of these are the very same genes that are active during development, then repressed throughout an organism’s lifetime. Death also activates other genes involved in mechanisms such as cell stress responses,, inflammation, immunity, and cancer.5,6 Why and how their resurrection occurs remains a mystery. Cell death is a natural and essential part of the biological life cycle. During the dance of development, cell death choreographs tissue maturation and corrects developmental errors.7 Cell death also plays an important role in the body’s response to cancer by mitigating genetic mutations and uncontrolled cell proliferation.8 Despite this, when an organism dies, cells rage against the process. “Cells don’t want to die,” said Gulnaz Javan, a forensic scientist at Alabama State University. Survival is programmed into their molecular makeup. While the moment of clinical death is absolute, some cells defy this moment. As postmortem time marches on, cells that remain stable in low-nutrient and oxygen conditions, such 34 T H E SC I EN T I ST | the-scientist.com

Noble and his team categorized these zombie genes into functional categories, including those that play a role in development, cancer, stress responses, inflammation, immunity, cell death, nutrient transport, and epigenetic processes. One of the key developmental genes activated was hypoxia inducible factor (HIF), which is part of a group of transcription factors that respond to low oxygen levels by regulating other oxygen sensitive genes that are active in early embryonic development and certain physiological and disease states.17 HIF transcription factors regulate the expression of hundreds of genes through various molecular signaling pathways that have far reaching roles in cell proliferation, growth, metabolism, and survival. Noble describes the zombie gene phenomenon as a genetic unraveling of the developmental clock. “After death, all hell breaks loose and just starts unwinding,” he said. The usual genetic and epigenetic brakes that silence developmental genes throughout an organism’s lifetime are released.

Beyond the veil As researchers unravel the secrets encoded in zombie genes, they also discover their far-reaching scientific relevance. “When we

MODIFIED FROM © ISTOCK.COM, ILYA LUKICHEV, LUCKYSTEP48; DESIGNED BY ERIN LEMIEUX

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ife begins and ends in low oxygen. Mammalian embryos are submerged in a hypoxic environment before the cardiovascular system and placenta develop. In this low oxygen state, embryonic stem cells hum with activity. They proliferate, activate developmental genes, and transcribe DNA in an intricate dance that choreographs the first buds of existence.1,2 When this early mass of pluripotent stem cells, the blastocyst, burrows into the lining of the uterus, access to higher oxygen levels through the maternal blood supply triggers stem cells to differentiate into cells that form the various tissues and organs.3,4 The genes that initiate and wind the clock of life eventually go dormant as key developmental milestones are reached.

Javan coined the term thanatotranscriptome, which derives from the Greek word for death, thanatos, to describe postmortem gene expression. Javan and her colleagues examined gene expression in postmortem human liver tissue and found a substantial increase in expression of a gene that promotes cell survival known as X-linked inhibitor of apoptosis protein (XIAP).10 They also found increased expression of XIAP and other prosurvival genes such as BAG1 and BCL2 in human prostate autopsy tissue.11 Peter Noble, adjunct professor of microbiology at the University of Alabama Birmingham, and his team examined gene transcription in zebrafish and mice in the days after death and made an unexpected discovery.5 “There was about one to two percent of the total transcriptome that was active,” Noble said. This included one thousand and sixty-three genes to be exact, some of which became active up to two days after death. Other researchers also discovered increased gene transcription after death in human tissues, including brain, blood, and skin samples.12-16

GENE ACTIVITY IN THE CELLULAR AFTERLIFE After an organism dies, most of its cells begin to extinguish activity and die shortly afterward. However, other cells exhibit a curious behavior. Instead of winding down their operations, certain gene activities resurrect.

LOW OXYGEN BEGINNINGS During the earliest stages of embryonic development, stem cells proliferate in a low oxygen environment. The genes that drive this stage are active for a short period of time. After the blastocyst implants into the uterus, eventually, oxygen levels increase and gene activity that was previously maintained by low oxygen levels silences.

SURVIVAL INSTINCT Cellular life after death may seem paradoxical, but sudden changes in oxygen levels trigger protective responses in cells. Genes that are transcribed during the low oxygen phase of embryonic development are reactivated when oxygen levels plumet after organismal death. Many emergency-mode genes also activate to support cell survival, including those involved in inflammation, immunity, stress responses, and cancer. Scientists studied this in zebrafish and mice, as well as human blood, prostate, liver, and brain tissue samples.

REAL-WORLD ZOMBIE GENES There are practical applications for understanding why and how genes are activated after death. For example, forensic scientists apply insights from postmortem gene transcription to estimate the time of death in criminal cases. Scientists also use information about cancer gene reactivation to improve organ transplant outcomes. Performing transplant surgery before these genes become active may help reduce the high incidence of cancer in organ transplant recipients.

Gulnaz Javan is a forensic scientist at Alabama State University who studies the practical applications of zombie genes.

continue to unravel the truth one gene at a time in their quest to uncover what lies beyond the veil. J

References

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1. Fathollahipour S, et al. Oxygen regulation in development: lessons from embryogenesis towards tissue engineering. Cells Tissues Organs. 2018;205(56):350-371. 2. Michiels C. Physiological and pathological responses to hypoxia. Am J Pathol. 2004;164(6):1875-1882. 3. Larsen’s Human Embryology—5th Edition. Available online: https://www.elsevier.com/books/larsens-human-embryology/ schoenwolf/978-1-4557-0684-6 (accessed on 6 October 2023). 4. Podkalicka P, et al. Hypoxia as a driving force of pluripotent stem cell reprogramming and differentiation to endothelial cells. Biomolecules. 2020;10(12):1614. 5. Pozhitkov AE, et al. Tracing the dynamics of gene transcripts after organismal death. Open Biol. 2017;7(1):160267. 6. Scott L, et al. Life and death: A systematic comparison of antemortem and postmortem gene expression. Gene. 2020;731:144349. 7. Arya R, White K. Cell death in development: Signaling pathways and core mechanisms. Semin Cell Dev Biol. 2015;39:12-19. 8. Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature. 2001;411(6835):342-348. 9. Cieŋla J, Tomsia M. Cadaveric stem cells: their research potential and limitations. Front Genet. 2021;12:798161. 10. Javan GT, et al. The apoptotic thanatotranscriptome associated with the liver of cadavers. Forensic Sci Med Pathol. 2015;11(4):509-516. 11. Tolbert M, et al. The thanatotranscriptome: Gene expression of male reproductive organs after death. Gene. 2018;675:191-196. 12. Dachet F, et al. Selective time-dependent changes in activity and cell-specific gene expression in human postmortem brain. Sci Rep. 2021;11(1):6078. 13. Antiga LG, et al. Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death. Sci Rep. 2021;11(1):16585. 14. Ferreira PG, et al. The effects of death and post-mortem cold ischemia on human tissue transcriptomes. Nat Commun. 2018;9(1):490. 15. Zhu Y, Wang L, Yin Y, Yang E. Systematic analysis of gene expression patterns associated with postmortem interval in human tissues. Sci Rep. 2017;7(1):5435. 16. Abouhashem AS, et al. The prolonged terminal phase of human life induces survival response in the skin transcriptome. Preprint. bioRxiv. 2023;2023.05.15.540715. 17. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012;148(3):399-408. 18. Hunter MC, et al. Accurate predictions of postmortem interval using linear regression analyses of gene meter expression data. Forensic Sci Int. 2017;275:90-101.

GULNAZ JAVAN

started this work, people thought we were nuts. They thought ‘who wants to study death?’” Noble said. “But it turns out that there are many practical reasons for doing so.” For example, zombie gene expression is being explored as a forensic tool to predict the postmortem interval—the time between death and the start of a criminal investigation—based on their precise and time-sensitive expression.18 Thanatotranscriptome research also informs cancer and organ transplant science. “When you transplant an organ, you're taking it from a dead donor. In some cases, there's an increase in cancer gene expression,” Noble said. This is important, given that the incidence of cancer in organ transplant recipients is significantly higher than the general population. “The common theme is that there is an immunological problem, but when you transfer a kidney or liver to a donor, the cancer genes have already been turned on in the dead person, and it's being transferred to the recipient,” Noble explained. Javan intends to further study postmortem decomposition of the prostate and liver, which are among the organs that remain intact the longest after death, to inform organ transplant research. “My team is assessing mRNA transcript abundance in postmortem prostate and liver tissues to obtain the list of candidate genes that can be used in the development of test kits to be used by organ transplantologists,” Javan said. Such biomarkers can improve the match between organ donors and recipients and reduce the rate of transplant rejection. Despite advances in understanding the thanatotranscriptome, the cellular afterlife remains shrouded in mystery. “When I was in college, I wondered what happens after we die. Does every cell in our body die at the same time or is there life that goes on?” Javan said. As scientists continue to unearth the answer to this question, zombie genes may hold the molecular keys to understanding far-reaching processes in the human body. The conserved molecular pathways that shape early embryonic development and resurrect postmortem gene activity suggest a continuum along the thin strand that binds cellular life from the cradle to the grave. “We really don't know what happens when an organism dies,” Noble said. How long certain genes remain active and whether they might stay dormant for protracted periods in cells that survive below the threshold of oxygen and nutrient availability that currently defines the needs of a living cell remains to be seen. In the meantime, scientists

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A Story of FIRE and Mice Studying how microglia control myelin growth and prevent its degeneration helps scientists better understand and address neurodegenerative diseases. BY NIAMH McNAMARA, PhD AND VERONIQUE MIRON, PhD

© ISTOCK.COM, BAWANCH AND KUDRYAVTSEV PAVEL; CARSTEN DITTMAYER; NIAMH McNAMARA

D

espite decades of research, scientists still grapple with the question of why neuronal health is compromised in neurodegenerative diseases. For a long time, scientists believed that Alzheimer’s disease resulted from toxic build-up of the proteins amyloid and tau, which normally aid neural growth, repair, and stability in the brain. Based on this idea, they developed therapies to break down these proteins, but those treatments did not completely improve patients’ cognitive abilities in clinical trials. This pushed scientists to explore other factors beyond protein toxicity that damage neuronal health. In 2004, George Bartzokis, a neuroscientist at the University of California, Los Angeles, first hypothesized that myelin, the protective membrane that wraps around axons, might be the key to understanding and treating Alzheimer’s disease.1 Myelin is essential for neuronal function. It provides axons with both the insulation and the nutrients they need to survive and thrive. Myelin is also central to efficient learning and memory encoding.2 In the early 2000s, researchers studied the brains of rhesus monkeys that were older than 30 years of age (their average lifespan is ~35 years) to better understand aging. They found that myelin in the brains of aged nonhuman primates showed structural changes and breakdown. Strikingly, these changes correlated with the degree of cognitive decline in the monkeys.3,4,5

Dysregulated myelin in FIRE mice indicates the essential role that microglia play in myelin stability.

When scientists conducted MRI studies in humans, they found that myelin shows damage up to 20 years prior to Alzheimer’s disease onset.6 Together, these studies hinted that myelin damage is intrinsically linked with cognitive decline and is an early event in Alzheimer’s disease progression. Despite this evidence, there were no follow up studies to validate this hypothesis for almost two decades, partially because researchers assumed that the myelin changes resulted from, rather than caused, neuronal loss. But recently, researchers at the Third Military Medical University and the University of California, San Francisco observed myelin degeneration coupled with a high rate of myelin repair in a mouse model of Alzheimer’s disease. Although the high repair rate could not compensate for the ongoing damage, they found that enhancing myelination improved cognition and neuronal function, irrespective of the amount of amyloid buildup.6,7 In fact, scientists at the Max Planck Institute for Multidisciplinary Sciences recently discovered that myelin damage itself can drive amyloid accumulation in mouse models of Alzheimer’s disease.8 These results finally provided evidence supporting Bartzokis’ hypothesis. 9,10

The microglia and myelination connection

Electron microscopy shows myelin overgrowth around axons in the white matter of a patient with ALSP. Myelin destabilization and eventual degradation are the hallmarks of neurogenerative disorders.

While these studies answered some questions, they raised several more. For example, if researchers could determine why myelin degenerates, they might be able to prevent dementia onset. The first step towards studying myelin degradation in diseases was to understand how myelin maintains its structure and function in a healthy brain. For this investigation, researchers turned to an internal partner: microglia. Microglia are a subset of glial cells that support neurons and keep the microenvironment in the central nervous system healthy and intact. Acting as soldiers on the brain frontlines, microglia function as immune cells, protecting the brain from pathogens, WINTER 2023 | T H E S C IE N T IST 3 9

MICROGLIA INFLUENCE MYELIN HEALTH In FIRE mice, the lack of microglia causes myelin overgrowth and eventual degeneration, indicating that microglia may contribute to age-related neurodegenerative diseases.

FIRE mice

Microglia absence

injury, or any harmful triggers. In the last decade, researchers have learned that microglia also contribute to healthy brain function. In fact, microglia consistently contribute to myelin health throughout the lifespan. Researchers recently found that young mice lacking microglia had less myelin in their developing brains than mice with intact microglia.11,12 This suggested that microglia mediated myelination in the developing brain by driving the production of oligodendrocytes, which produce myelin. However, there was one key challenge with these studies. The tools available for eliminating microglia at the time also targeted another small population of central nervous system immune cells known as border-associated macrophages, which are found in the meninges, surrounding blood vessels, and in areas that contact cerebrospinal fluid. Scientists did not know the potential contributions of these cells to myelination. In 2019, researchers at the University of Edinburgh created a game-changing new mouse model. By using CRISPR-Cas9, they deleted the FIRE sequence, which is a super enhancer in the gene encoding CSF1R, a receptor required for microglia development and survival.13 FIRE has redundant function in borderassociated macrophages, so FIRE knockout mice (or FIRE mice) lack microglia but retain border-associated macrophages. We used this new model to unpack the specific roles of microglia in myelination for the first time.

Microglia control myelin growth In our lab at the University of Edinburgh, we eagerly investigated myelin in these FIRE mice. We hypothesized that the lack of microglia in the FIRE mice would cause deficient myelination during early development, an idea in line with results from pre4 0 T H E SC I EN T I ST | the-scientist.com

Myelin overgrowth

Demyelination

vious studies that used global macrophage depletion models. But we were in for a surprise. In our analysis, we found no deficit in myelin formation in these mice. Perplexed, we spent months analyzing various myelin-associated proteins and characterizing oligodendrocyte numbers, but they all appeared unaffected. Finally, when we took a closer look at the ultrastructural level using electron microscopy, we had a breakthrough. We observed that myelin was present in FIRE mice, but in unexpected abundance. In the absence of microglia, young FIRE mouse brains formed excess myelin during development that remained in adult FIRE mice. These findings suggested that microglia actively engage in regulating myelin growth. To confirm our theory using another approach, we depleted microglia and macrophages in adult mice using the previous gold standard, a pharmacological drug that inhibits CSF1R. We saw the same result: FIRE mouse brains showed excess myelin formation after just one month of treatment. Next, we explored whether this phenomenon also occurred in humans. We obtained precious tissue from a rare human neurodegenerative disease known as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) from Werner Stenzel, a neuropathologist at Charité-Universitätsmedizin Berlin. In patients suffering from ALSP, heterozygous mutations in the CSF1R gene result in approximately half the normal amount of microglia, specifically in myelin-enriched areas in the white matter. Patients with ALSP typically present with early-onset dementia and usually pass away in their 40s or 50s. In brain samples from patients with ALSP, we observed myelin overgrowth similar to what we saw in FIRE mice.

This revelation meant that microglia not only control myelin formation during development, but they also regulate myelin growth into adulthood. Moreover, myelin overgrowth when microglia were absent or reduced in number looked remarkably similar to the disrupted myelin observed in the aged nonhuman primates with cognitive decline. This suggested that in the absence of healthy microglia, myelin may age prematurely and affect cognition. We next assessed cognitive function in the FIRE mice by running the Barnes Maze test. In this test, mice learn to locate an escape hole in a maze over several trials. After a few days, we relocate the escape chamber; how the mice adapt to the new situation reflects their cognitive flexibility. The FIRE mice showed deficits in cognitive flexibility, which is one of the first cognitive functions that declines with age.14,15 Previous studies have also suggested that the extent to which this ability is lost with age could predict development of Alzheimer’s disease.16

ILLUSTRATION BY © ASHLEIGH CAMPSALL, ADAPTED FROM A GRAPHIC BY NIAMH MCNAMARA; © ISTOCK.COM, NAEBLYS

Microglia prevent myelin degeneration Since aging perturbs myelin structure, and myelin degenerates in Alzheimer’s disease, we next wondered whether the myelin structural changes seen in the absence of microglia rendered myelin vulnerable to degeneration. In samples from patients with ALSP, we noticed some age-related differences in the myelin. A younger patient who died from an unrelated cause had abundant and extremely overgrown myelin. But in an older patient, much of the myelin had degenerated; any remaining myelin was overgrown. This led us to investigate the role of microglia in myelin maintenance. By six months of age in FIRE mice, we saw clear evidence of myelin degeneration. In the absence of microglia, myelin quickly went from overgrown to broken down. This was a strange phenomenon, and mechanistically, it didn’t make much sense. To find out whether myelin broke down due to the prolonged absence of microglia in FIRE mice, we pharmacologically depleted microglia and macrophages for one month in five-month-old wild type mice. We observed the same degenerated myelin in these mice, indicating that the function of microglia in maintaining myelin health is increasingly important as the brain ages.

Microglia burnout Burnout is characterized by utter exhaustion due to excessive workload and stress over a prolonged period of time. Microglia may also suffer from burnout, and understanding how this happens could be central to our fight against dementia. Numerous sophisticated single-cell transcriptomic sequencing studies have documented the appearance of a disease-associated microglia population in aging and in several disease contexts, including Alzheimer’s disease. This population may appear as a response to the increasing demands of the aging brain. With too many tasks to juggle, microglia may lose their ability to maintain myelin as well as they did when the brain was younger.

If we could rejuvenate microglia to their younger selves, perhaps we could prevent the degeneration of myelin and potentially the development of neurodegenerative disease in the aging brain. And if we can thwart this process, we might just be able to help our loved ones preserve their memories until well into their autumn years. J

Conflict of interest statement Veronique E Miron has received consultancy or research funds from Novartis, Biogen, GSK, Astex Pharmaceuticals, Clene Nanomedicine, ReWind Therapeutics. Niamh McNamara recently completed her PhD on microglial regulation of myelin integrity at the University of Edinburgh. She is now pursuing postdoctoral research at the Netherlands Institute for Neuroscience in Amsterdam. Veronique Miron is a neuroimmunology professor leading research laboratories at the University of Toronto and the University of Edinburgh that investigate what regulates myelin health and pathology across the lifespan.

References 1. Bartzokis G. Age-related myelin breakdown: A developmental model of cognitive decline and alzheimer’s disease. Neurobiol. Aging. 2004;25(1):5-18. 2. Xin W and Chan JR. Myelin plasticity: Sculpting circuits in learning and memory. Nat. Rev. Neurosci. 2020;21(12):682-694. 3. Peters A. The effects of normal aging on myelin and nerve fibers: a review. J. Neurocytol. 2002;31(8/9):581-593. 4. Peters A. The effects of normal aging on myelinated nerve fibers in monkey central nervous system. Front. Neuroanat. 2009;3. 5. Peters A and Sethares C. Aging and the myelinated fibers in prefrontal cortex and corpus callosum of the monkey. J. Comp. Neurol. 2001;442(3):277-291. 6. d’Arbeloff T, et al. White matter hyperintensities are common in midlife and already associated with cognitive decline. Brain commun. 2019;1(1). 7. Chen J-F, et al. Enhancing myelin renewal reverses cognitive dysfunction in a murine model of alzheimer’s disease. Neuron. 2021;109(14). 8. Depp C, Sun T, et al. Myelin dysfunction drives amyloid-` deposition in models of Alzheimer’s disease. Nature. 2023;618(7964):349-357. 9. Bartzokis G, et al. White Matter Structural Integrity in healthy aging adults and patients with alzheimer disease. Arch. Neurol. 2003;60(3):393. 10. Graff-Radford J, et al. White matter hyperintensities: Relationship to amyloid and tau burden. Brain. 2019;142(8):2483-2491. 11. Erblich B, et al. Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits. PLoS ONE. 2011;6(10). 12. Hagemeyer N, et al. Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood. Acta Neuropathol. 2017;134(3):441-458. 13. Rojo R, et al. Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations. Nat. Commun. 2019;10(1). 14. Boone KB, et al. Wisconsin card sorting test performance in healthy, older adults: Relationship to age, sex, education, and IQ. J. Clin. Psychol. 1993;49(1):54-60. 15. Moore TL, et al. Executive system dysfunction occurs as early as middle-age in the rhesus monkey. Neurobiol. Aging. 2006;27(10):1484-1493. 16. Ballesteros S, et al. Cognitive function in normal aging and in older adults with mild cognitive impairment. Psicothema. 2013;25(1):18-24.

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The Literature Rebranding Mitochondria As scientists realize the multifaceted role of mitochondria, some feel that the “powerhouse of the cell” analogy is out of date. BY DANIELLE GERHARD, PhD

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n a popular Indian parable, a few blind men interact with an elephant for the first time and imagine what it looks like. The man touching the tusk may describe the elephant as a spear, while the person tugging the tail may think that it’s a rope. All of them miss the big picture. The moral of the story is that narrow experiences can advance inaccurate perspectives. Martin Picard, a mitochondrial biologist at Columbia University, likened mitochondria to the elephant in the fable. “Mitochondria are diverse,” said Picard. “To some people, that’s a gentle reminder. For some people, that’s an eyeopening claim.” Mitochondria constantly churn out chemical energy to fuel the extensive network of biochemical reactions occurring throughout the cell, thus inspiring the universal aphorism of the “powerhouses of the cell.” However, the last few decades have ushered in a more nuanced understanding of the organelle with growing

understanding of the organelle, to mitochondrial biology. Considering this, Picard spearheaded two perspectives that he hopes will serve as an invaluable compendium on the organelle for experts and visitors to the

Mitochondria function like cellular processors, like little antennas that can receive information, integrate information, and then produce signals that influence the cell and the whole organism. —Martin Picard, Columbia University

field alike.1,2 In the first perspective, published in Cell Metabolism, Picard and Orian Shirihai, a mitochondrial biologist at the University of California, Los Angeles, made the case that the powerhouse analogy is dated; they instead focus on the organelle as the great communicator of the cell.1 “Mitochondria function like cellular processors, like little anten-

With a greater understanding of the many roles of mitochondria, the more precise you can be and the better and clearer the hypothesis you’ll come up with will be. —Mike Murphy, University of Cambridge

evidence that it contributes to a number of diseases, regulates several cellular processes, and plays multifaceted roles in cells. This attracted many scientists, even those who may have a narrow 4 2 T H E SC I ENTI ST | the-scientist.com

that direct output signals that orchestrate metabolic pathways, gene expression, and drive adaptive behaviors. Although Picard and his colleagues rebranded the organelle under the umbrella of communicators, they empha-

nas that can receive information, integrate information, and then produce signals that influence the cell and the whole organism,” said Picard. Inputs include hormones, metabolites, and nutrients

sized that context matters. Early in development, mitochondria diversify and specialize as different cell types and tissues emerge. Just as scientists continue to discover and define functionally and molecularly distinct cell types, Picard noted rising evidence of mitochondrial phenotypes, or mitotypes, that likely influence signal processing and mitochondrial communication.3 For example, Picard’s team and others showed that brain cells in mice exhibit regional and cell-specific functional differences, while human immune cells vary in ATP production and mitochondrial DNA copy number.3-5 “Now that we know this, we need a decent nomenclature system that will allow us to teach the next generation to formulate specific hypotheses and then design research to test that with a certain level of specificity,” said Picard. In their Nature Metabolism perspective, Picard and his colleagues proposed a terminol-

Mitochondria vary in their cell-dependent characteristics, such as topology and DNA copy number. Mitochondria are diverse and multifaceted, and they exhibit a high degree of heterogeneity across tissues.

Mitochondrial morphology and molecular composition, including their lipid, protein, and RNA profiles can differ even within the same cell.

Mitochondria undergo a range of cellular activities, including protein synthesis, metabolite and ion transport, and DNA repair.

ATP synthase

ADP

ATP

Mitochondria use a variety of functions, such as lipid synthesis, steroidogenesis, and energy production.

Endoplasmic reticulum

DESIGNED BY ASHLEIGH CAMPSALL

Mitochondria engage in different behaviors, like motility, fission and fusion, and communication with other mitochondria and organelles.

ogy system to increase specificity in the language of mitochondrial science.2 Their system distinguishes between the multitude of cell-dependent properties, molecular features, activities, functions, and behaviors employed by mitochondria. Mike Murphy, a mitochondrial biologist at the University of Cambridge who was not involved with writing the perspectives, agreed with Picard’s call for more precise language. “We’re using vague terms like mitochondrial dysfunction, and it’s not clear what that means,” said Murphy. Instead, descriptions should focus on the specific process that has gone awry, such as calcium homeostasis, oxidative phosphorylation producing ATP, or contributions to immune

signaling. “With a greater understanding of the many roles of mitochondria, the more precise you can be and the better and clearer the hypothesis you’ll come up with will be,” said Murphy. “I’m supportive of the goal, [but] I’m reluctant to go along with a rigid nomenclature,” said Murphy. Mitochondria are dynamic and constantly adapting in response to a changing environment, which could make it difficult to pigeonhole these shapeshifting organelles into one classification over another. Whether scientists adopt the proposed terminology system remains to be seen, but appreciation of the organelles’ incredible diversity is only growing. “In the world of mitochondrial biology, we’re in the same place as probably 200 years

Dynamic interplay across these domains allows these shape-shifting organelles to respond to their environments. In this evolving framework, mitochondria are viewed as cellular processors.

ago, when people realized ‘ooh, we’re made of cells,’” said Picard. J

References 1. Picard M, Shirihai OS. Mitochondrial signal transduction. Cell Metab. 2022;34(11):1620-1653. 2. Monzel AS, et al. Multifaceted mitochondria: Moving mitochondrial science beyond function and dysfunction. Nat Metab. 2023;5(4):546-562. 3. Rausser S, et al. Mitochondrial phenotypes in purified human immune cell subtypes and cell mixtures. eLife. 2021;10:e70899. 4. Rosenberg A, et al. Brain mitochondrial diversity and network organization predict anxietylike behavior in male mice. Nat Commun. 2023;14:4726. 5. Fecher C, et al. Cell-type-specific profiling of brain mitochondria reveals functional and molecular diversity. Nat Neurosci. 2019;22:1731-1742.

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THE LITERATURE

Biosensors for Colorectal Cancer Engineered bacteria sound the alarm on a common oncogenic mutation. BY HANNAH THOMASY, PhD

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

and Daniel Worthley at the Colonoscopy Clinic. The study authors hope that this technology will one day aid in the early diagnosis of colorectal cancer, one of the most common causes of cancer-related death globally. While scientists have previously engineered bacteria to detect inflammation or bleeding in the gut, this is the first bacterial biosensor that detects a specific DNA sequence from host tissues. To accomplish this feat, scientists leveraged Acinetobacter baylyi’s ability to take up extracellular DNA and integrate these sequences into its own genome. “Using living bacteria to sense things in the gut and detect disease is something that I find very exciting,” said David Riglar, a microbiome researcher at Impe-

Globally, colorectal cancer is the third most common cancer; bacterial biosensors may one day help with early detection of this disease.

rial College London who was not involved in the study. “Taking these naturally competent bacteria, detecting DNA changes, and then using that as a biosensor is a really cool advance.” In this study, researchers wanted to engineer A. baylyi to detect a common colorectal cancer marker: a mutation in codon 12 of the KRAS gene. “At the time, it seemed like a fairly far-fetched idea,” recalled Worthley. By bringing together an interdisciplinary team with expertise in synthetic biology and animal models of colorectal cancer, the researchers achieved this lofty goal.

© ISTOCK.COM, LUISMMOLINA

T

he human gut is awash in a sea of microbes that quietly ferment fibers, produce vitamins, and exchange information with the immune system.1 Now, scientists are tasking bacteria with yet another job as they spelunk their way through the digestive system: cancer detection. An international team of researchers engineered a bacterial biosensor capable of identifying a cancer-associated DNA mutation, which they published in the journal Science.2 The research team included molecular biologists Robert Cooper and Jeff Hasty of the University of California, San Diego and bowel cancer researchers Josephine Wright and Susan Woods at the South Australian Health and Medical Research Institute,

In their early proof-of-concept experiments, the researchers genetically tinkered with both A. baylyi and the tumor organoids that they wanted the bacteria to detect. They engineered tumor cells with a functional copy of the antibiotic

Taking these naturally competent bacteria, detecting DNA changes, and then using that as a biosensor is a really cool advance. —David Riglar, Imperial College London

resistance gene, kanR, flanked by KRAS homology arms. The bacteria had matching KRAS homology arms, plus two stop codons that prevented the expression of kanR. When the bacteria gobbled up the donor tumor DNA, the homology arms aligned the DNA sequences and the bacteria integrated the functional kanR into their own genomes, enabling them to grow on antibiotic-laced plates. To create bacteria that specifically detected mutant KRAS, the researchers harnessed the bacteria’s own CRISPR-Cas machinery, directing these molecular scissors to chop up wild-type, but not mutant KRAS. This would kill any bacteria that acquired the wild-type KRAS. The researchers then tested these bacteria against colorectal cancer organoids with and without the engineered donor DNA. Only the bacteria cocultured with the engineered tumors acquired antibiotic resistance, showing that the sensor bacteria could discriminate between normal and donor tumors. Next, the researchers tested the biosensors in vivo by administering the bacteria via enema to three groups: mice without tumors, mice with normal colorectal tumors, and mice with the engineered colorectal tumors. Again, only the biosensors administered to the mice with the engineered tumors grew in the presence of the antibiotic, confirm-

ing that the bacteria could be used to signal the presence of engineered colorectal cancer in mice. While these data were promising, human colorectal tumors don’t come engineered with a perfectly placed antibiotic resistance gene for bacteria to acquire. So, the researchers adjusted their strategy to detect natural tumor DNA with the KRAS mutation. This time, they placed a repressor gene inside the KRAS homology arms. This gene prevented the expression of a downstream kanR gene. When the bacteria swapped their KRAS DNA for the tumor’s KRAS, the repressor was lost, allowing the antibiotic resistance gene to be expressed. As before, wild type KRAS was targeted for destruction by the CRISPR-Cas system. In vitro, these new biosensors discriminated between mutant and normal KRAS by surviving and becoming antibiotic resistant only in the presence of the cancer-associated mutation. The team named this technique CATCH for cellular assay for targeted, CRISPR-discriminated horizontal gene transfer.

bacteria must be delivered orally, meaning that they will need to survive their journeys through the digestive system and be able to report their findings on the other side. Eventually, however, Worthley hopes that these biosensor bacteria will one day be used as point-of-care diagnostics in remote or low-resource areas such as the Australian outback. “Since we’ve engineered all the sophistication within the cell, we don’t need such a sophisticated laboratory outside the cell,” he said. Researchers hope for broader applications as well. Instead of turning on an antibiotic resistance gene when they sense tumor DNA, for example, the bacteria could be engineered to turn on production of a genotype-specific small-molecule therapeutic, delivering treatment precisely where it’s needed. Bacteria could be engineered to detect and respond appropriately to various oncogenic mutations, or even difficult-to-treat infections like Clostridium difficile. Worthley sees this potential to marry diagnosis and

Since we’ve engineered all the sophistication within the cell, we don’t need such a sophisticated laboratory outside the cell. —Daniel Worthley, Colonoscopy Clinic

Despite these preliminary successes, Riglar urged caution. “It’s important not to run too far ahead in terms of thinking that these systems are ready to go into the clinic,” he said. “This is absolutely not the endpoint,” Worthley agreed. The researchers are currently working on strategies to improve the biosensor’s sensitivity to natural tumor DNA in the complex environment of the colon. Due to concerns about administering antibioticresistant bacteria to humans, they are also developing other ways for the biosensors to signal the presence of mutant KRAS. To be commercially viable, the biosensor

therapy as the major advantage of these engineered bacteria. J

Conflict of interest statement: J.H., D.W., and S.W. have equity in GenCirq Inc., which focuses on cancer therapeutics. D.W., J.H., R.C., S.W., and J.W. have a provisional patent application on this technology.

References 1. Bull MJ, Plummer NT. Part 1: The Human Gut Microbiome in Health and Disease. Integr Med (Encinitas). 2014;13(6):17-22. 2. Cooper RM et al. Engineered bacteria detect tumor DNA. Science. 2023;381(6658):682-686.

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THE LITERATURE

Tracking Down Innate Immune Cells in Multiple Sclerosis A novel PET tracer targeting a receptor in myeloid cells can help monitor disease progression in a mouse model of multiple sclerosis. BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

of imaging agents for visualizing neuroimmune interactions. “We have never been able to do that before with such specificity.” James did not initially focus on TREM1. The membrane receptor caught her attention when she was looking at a transcriptomic data set from her colleague Katrin Andreasson, a neurologist at Stanford University and coauthor of the study. The pair noticed that the expression of TREM1 was upregulated only when there was a more harmful immune response. “We thought, ‘wow, this could be a really great biomarker to tell us when there is something really bad occurring with the innate immune system in the context of diseases,’” James recalled. To test this idea, the researchers focused on MS and used the experimental autoimmune encephalomyelitis (EAE) mouse model, which recapitulates important aspects of the disease such as muscle weakness and changes in the innate immune response. They developed a PET tracer by radiolabeling an anti-TREM1 antibody to specifically track down TREM1+ cells and used a PET imaging scanner to monitor the movement of myeloid cells through the animal’s body as disease progressed. TREM1 was selectively expressed on peripheral myeloid cells in the EAE mouse model, and the researchers observed central nervous system infiltration of TREM1+ cells even in early stages

We literally did very excited happy dances in the preclinical imaging facility because the clarity of the images was just outstanding. For PET imaging, it is kind of rare to be able to see such clear-cut images where you do not even need quantification to see what is going on. —Aisling Chaney, Washington University. 4 6 T H E SC I EN T I ST | the-scientist.com

PET imaging of TREM1 enables detection of infiltrating proinflammatory myeloid cells in early stage disease in a mouse model of multiple sclerosis.

of the disease, when mice showed few signs of loss of muscle function. “We literally did very excited happy dances in the preclinical imaging facility because the clarity of the images was just outstanding. For PET imaging, it is kind of rare to be able to see such clear-cut images where you do not even need quantification to see what is going on,” said Aisling Chaney, a neuroimaging biologist at Washington University and coauthor of the study. The team also found that TREM1 PET signal showed higher sensitivity in detecting myeloid cell infiltration in the central nervous system of EAE mice than the current gold standard PET tracer,

JAMES LAB, STANFORD UNIVERSITY

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ultiple sclerosis (MS) is a debilitating disease in which a patient’s immune system attacks the myelin protective coating around nerve cells, causing inflammation and disrupting communication to and from the brain. Evidence indicates that myeloid cells, components of the innate immune system, aid the initiation, progression, and remission of MS.1 However, scientists lacked methods to specifically track down myeloid cells as they turn from friend to foe and mount a more damaging immune response. To fill this gap, researchers led by Michelle James, a radiochemist and neuropharmacologist at Stanford University, developed a novel PET tracer that targets the triggering receptor expressed on myeloid cells 1 (TREM1). They showed that in vivo TREM1 PET imaging allowed early disease detection and treatment monitoring in a mouse model of MS.2 Their findings, published in Science Translational Medicine, posit that TREM1 is an early marker of maladaptive innate immune responses, and highlight the biomarker’s potential use for monitoring disease progression and response to treatment in patients with the disease. “We have a way of lighting up where inflammation is in the whole body and brain in the context of MS,” said James, whose work focuses on the development

which is widely used to detect neuroinflammation in vivo. Given the specificity of TREM1 to track harmful innate immune responses, the team next investigated if it could be used to indicate therapeutic response. They treated EAE-induced mice with the drug Siponimod and found a reduction in TREM1 PET signal in drug-treated mice. According to Chaney, these findings reveal that TREM1 could be used as a tool for screening different types of therapies, even those that do not directly target myeloid cells. Using TREM1 knockout animals as controls in the imaging experiments also revealed a biological role for TREM1, Chaney explained. “In the TREM1 knockout animals that we induced EAE in, 50 percent of them just did not get sick or they just did not get sick at the same time point that we were looking at in the wild type animals,” she said.

The team confirmed these observations by pharmacologically blocking TREM1 with LP17, a peptide decoy receptor known to attenuate TREM1 signaling, and observed reduced disease severity in EAE mice, suggesting therapeutic potential for targeting TREM1. To establish TREM1’s clinical relevance, the researchers looked at brain biopsy samples from two patients with MS and examined the presence of TREM1+ cells. Since patients would benefit the most by detecting and diagnosing MS at early stages, the team looked for treatment-naïve, early-stage samples, which are not easy to obtain, James explained. The team found a high number of TREM1+ immune cells in MS brain lesions compared to non-MS samples, indicating that TREM1 could be used to monitor disease progression in humans. The combination of multiple techniques strengthened the researchers’

findings, pointed out Daniele de Paula Faria, a molecular imaging researcher at the University of Sao Paulo, who was not involved in the research. “This is a complete study with promising results,” she added. James and Chaney plan to continue exploring TREM1’s role in neurological disorders. “TREM1 had actually not been looked at in a lot of neurological disorders before,” Chaney said. “It is opening up a whole area of peripheral immune cells and their implications in neurodegenerative disorders.” J

References 1. Mishra MK, Yong VW. Myeloid cells - targets of medication in multiple sclerosis. Nat Rev Neurol. 2016;12(9):539-551. 2. Chaney AM, et al. PET imaging of TREM1 identifies CNS-infiltrating myeloid cells in a mouse model of multiple sclerosis. Sci Transl Med. 2023;15(702):eabm6267.

SMART GATEWAYS INTO THE LAB OF THE FUTURE In this episode, Deanna MacNeil from The Scientist’s Creative Services Team spoke with Sofie Salama and David Haussler, professors at the University of California, Santa Cruz, to learn more about the smart technology behind growing brain organoids.

SOFIE SALAMA, PhD University of California, Santa Cruz

DAVID HAUSSLER, PhD University of California, Santa Cruz

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PROFILE

A Microbial Link to Parkinson’s Disease Haydeh Payami helped uncover the genetic basis of Parkinson’s disease. Now, she hopes to find new ways to treat the disease by studying the gut microbiome. BY MARIELLA BODEMEIER LOAYZA CAREAGA, PhD

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t the age of 19, Haydeh Payami, now a geneticist at the University of Alabama, Birmingham, left Iran and came to the United States with two suitcases and a fascination with genetics. In the following years, she pursued a doctoral degree in genetics and trailblazed her path as a researcher. Today, Payami leads a team of motivated researchers and combines her love of genetics with the microbial world to find ways to prevent and treat Parkinson’s disease. “Haydeh has certainly been a pioneer,” said Sarkis Mazmanian, a gut microbiome researcher at the California Institute of Technology, who believes that Payami’s work contributed greatly to instilling confidence in the idea that changes in the gut microbiome are not a secondary consequence of Parkinson’s disease, but may actually contribute to it. Payami’s interest in Parkinson’s disease did not begin with the microscopic inhabitants of the human gut. It started with a genetic investigation.

A genetic component of Parkinson’s disease?

4 8 T H E SC I EN T I ST | the-scientist.com

Haydeh Payami studies the interaction of the human genome, gut microbiome, and environmental factors in causation, progression, and treatment of Parkinson’s disease.

The reactions were mixed, she recalled. While some scientists resisted the idea that genes play a role in cases of Parkinson’s disease with unknown causes, others were more open and embraced the new discovery. Since the mid-1990s, Payami and others have linked 90 risk loci to Parkinson’s disease,3 but these genes still did not explain the whole story. So, Payami turned her attention to investigating how environmental factors and their interactions with genes influence the disease. In one study, for instance, her team conducted a genome wide association study (GWAS) to investigate how genes influence the protective effects of coffee in Parkinson’s disease. They found that heavy coffee drinkers who carried a variant of the gene GRIN2A, which encodes a glutamate receptor involved in excitatory transmission in the brain, had a lower risk for developing Parkinson’s disease.4 Although her findings added to other evidence that the environment influences this neurodegenerative disorder, a gene-environment interaction still did not provide all of the pieces of the puzzle. “What else is there if it’s not genes and environment?” Payami puzzled. Then, the boom in microbiome research in the 2010s gave her a new clue.

UNIVERSITY OF ALABAMA, BIRMINGHAM

In the early 1990s, Payami led a group at the Oregon Health and Sciences University that studied the genetic basis of Alzheimer’s disease. One day, an undergraduate student approached her with the idea of investigating the genetics of Parkinson’s disease. “I said, ‘Well, that’s fine. But choose either Parkinson’s or genetics because Parkinson’s doesn’t have a genetic component,’” Payami recalled. “This was 1992. Everybody was convinced that Parkinson’s was purely environmental.” At that time, the only well accepted case of a genetic link to Parkinson’s disease was an Italian family where individuals from different generations suffered from the disease.1 Motivated by the student’s determination to pursue the idea and the lack of knowledge about the role of genetics in Parkinson’s disease beyond that case, Payami and her colleagues conducted a family study to detect the presence of familial aggregation for the disease. The team looked at the family histories, including individual records of illnesses and health conditions along with records of parents and siblings of patients with Parkinson’s disease. Spouses or friends served as controls to help identify characteristic symptoms, including tremors, rigidity, and disease diagnoses. First-degree relatives of patients showed an increased risk for developing Parkinson’s disease, as indicated by a higher number of patients with a parent or sibling with symptoms or a diagnosis of the brain disorder.2 “We published that paper in 1994 and braced for the world to come down on us,” Payami said.

Haydeh has certainly been a pioneer. —Sarkis Mazmanian, California Institute of Technology.

Gut microbes in action The study of the microbiota traces back to the 17th century, when Antonie van Leeuwenhoek used his handmade microscopes to describe the animalcules he found in people’s mouths and stools. However, in the last 20 years, the human-associated microbiota research area has experienced a surge, as scientists have developed novel -omics approaches and bioinformatics tools to understand the impact and influence of these microscopic beings on the human body, including on the brain. In the mid-2010s, Mazmanian’s team uncovered a connection between the gut microbiome and hallmarks of Parkinson’s disease in a mouse model of the disease.5 In human studies, however, the association was less clear, and inconsistencies between studies made the link difficult to establish. “It was kind of a Wild West of a research area,” said Zachary Wallen, a medical bioinformatician at Labcorp Oncology and a former student of Payami’s. “There was no standardization of anything; there were no guidelines to go by. So, we just had to pave our own way to figure out how to tag the microbiome to Parkinson’s disease in the most robust way possible.” Payami’s team set out to determine if an imbalance, or dysbiosis, of the gut microbiome was present in patients with Parkinson’s disease. They collected stool samples from patients and neurologically healthy individuals, extracted DNA, and sequenced the 16S rRNA to determine the microbial composition. The team’s initial results provided evidence of an imbalance in the gut microbes of patients, uncovering an abundance of some bacterial taxa and a reduction in others.6 Next, the team characterized gut dysbiosis in Parkinson’s disease in more detail by using more advanced bioinformatics tools in their microbiome-wide association studies, which they modeled after the rigor and standards of GWAS. They identified an overabundance of opportunistic pathogens, bacteria that are normally harmless but can cause infections in immunocompromised individuals, in the microbiomes of patients with Parkinson’s disease.7 Although this was the first time that a group of potentially pathogenic microbes was found in samples from patients with this neurodegenerative disorder, the idea that a pathogen could trigger the disease was not entirely new. In 2003, the anatomist Heiko Braak and his colleagues proposed that nonfamilial forms of the disease could be initiated in the gut by an unknown pathogen.8 After entering the body through the nasal cavity and reaching the gut, this pathogen triggered the formation of abnormal aggregations of the alpha-synuclein protein, a hallmark of Parkinson’s disease. These aggregates then spread from the periphery to the brain via the vagal nerve and olfactory tract, causing progressive brain cell dysfunction and loss. The identification of these opportunistic pathogens is one of Payami’s major contributions to the field according to Wallen. It is still uncertain whether any of them is the disease trigger-

ing pathogen that Braak referred to, but revealing their identities will allow researchers to test their specific involvement in Parkinson’s disease. More recently, Payami’s group used a metagenomic approach instead of 16S rRNA sequencing to take a deeper look at the Parkinson’s disease gut microbiome. In this large-scale study, which included 490 patients with Parkinson’s disease and 234 neurologically healthy controls, they confirmed previous findings of an overabundance of opportunistic pathogens and uncovered microbial genes and pathways that may contribute to Parkinson’s disease pathology, such as dysregulation of the production and metabolism of neuroactive molecules and lower levels of anti-inflammatory molecules.9 Wallen, who spent his graduate and postdoctoral years under Payami’s supervision, remembers Payami’s determination to produce the best science and her collaborative nature the most. “She had an office; she hardly ever used it. We would just sit in the middle of our lab and talk because she just wanted to constantly talk and communicate,” he recalled. According to Mazmanian, human studies like those conducted by Payami’s team could provide clues to potential interventions. For example, the opportunistic pathogens identified by her team could be targeted and potentially removed from an individual suffering from Parkinson’s disease. Additionally, supplementing patients with the beneficial anti-inflammatory bacteria they lack might also prove to be a viable strategy, he said. Even though Payami’s main research goal is still to find a cure for Parkinson’s disease, she hopes that by looking at the microbiome, scientists can develop strategies to treat it. “There are so many people who are suffering with this disease. They’re not at an early stage, and they’re being ignored as far as clinical trials,” said Payami. “Their microbiomes are sick. We may not change their Parkinson’s, but we might be able to give them some relief or maybe even slow disease progression.” J

References 1. Golbe LI, et al. A large kindred with autosomal dominant Parkinson’s disease. Ann Neurol. 1990;27(3):276-282. 2. Payami H, et al. Increased risk of Parkinson’s disease in parents and siblings of patients. Ann Neurol. 1994;36(4):659-661. 3. Bandres-Ciga S, et al. Genetics of Parkinson’s disease: An introspection of its journey towards precision medicine. Neurobiol Dis. 2020;137:104782. 4. Hamza TH, et al. Genome-wide gene-environment study identifies glutamate receptor gene GRIN2A as a Parkinson’s disease modifier gene via interaction with coffee. PLoS Genet. 2011;7(8):e1002237. 5. Sampson TR, et al. Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson’s Disease. Cell. 2016;167(6):1469-1480.e12. 6. Hill-Burns EM, et al. Parkinson’s disease and Parkinson’s disease medications have distinct signatures of the gut microbiome. Mov Disord. 2017;32(5):739-749. 7. Wallen ZD, et al. Characterizing dysbiosis of gut microbiome in PD: evidence for overabundance of opportunistic pathogens. NPJ Parkinsons Dis. 2020;6:11. 8. Braak H, et al. Idiopathic Parkinson’s disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. J Neural Transm (Vienna). 2003;110(5):517-536. 9. Wallen ZD, et al. Metagenomics of Parkinson’s disease implicates the gut microbiome in multiple disease mechanisms. Nat Commun. 2022;13(1):6958.

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PROFILE

A Journey With Metabolism, Parasites, and Cancer Piet Borst led stellar work on cell organelles, trypanosomes, and cancer drug resistance during the golden age of biology. BY LAURA TRAN, PhD

Mitochondria and trypanosomes From an early age, Borst’s home was filled with discussions of scientific results and collaborators. His father was a professor of internal medicine and a clinical researcher. Following in his father’s footsteps, Borst studied medicine at the University of Amsterdam with the intent to pursue clinical research. 5 0 T H E SC I EN T I ST | the-scientist.com

For more than five decades, Piet Borst profoundly influenced discoveries in numerous fields and championed funding for science.

While waiting for his clinical internship, he stumbled upon an opportunity to conduct research in the biochemistry department with Bill Slater, an enterprising biochemist at the University of Amsterdam. “The Slater lab was fantastic,” Borst recalled. In fact, he was so impressed with Slater as a supervisor and researcher that when Slater later requested Borst’s support on a project, Borst happily accepted, despite being six months into his medical internship. Pausing his internship, Borst spent the next three years pursuing a PhD degree in Slater’s lab. At that time, he worked on mitochondrial properties in cancer cells and went on to uncover a metabolic

NETHERLANDS CANCER INSTITUTE

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or Piet Borst, a physician-biochemist and molecular biologist at the Netherlands Cancer Institute, success in science is a matter of luck. He described his career using the words “luck” and “accident,” but his research was linked by a common thread: scientific interest. Over 50 years, he passionately pursued opportunities that led him to make seminal discoveries in mitochondria, parasitology, and oncology.

cycle involving the mitochondria: the malate-aspartate shuttle, which helps cells extract energy from sugar.1 After he completed his internships, Borst wanted to become an endocrinologist. Because there was limited space at the endocrinology clinic, he had to wait two years before he could continue his training. So, Borst went to New York University School of Medicine and joined the Nobel laureate Severo Ochoa’s group as a postdoctoral researcher to study RNA phage replication in Escherichia coli.2 Slater offered Borst a chair position back at the University of Amsterdam. There, he combined mitochondria and nucleic acids and discovered circular mitochondrial DNA in vertebrates and in yeast.3 Borst noted that this circular DNA contained very little genetic information, and that all of the mitochondrial proteins must be encoded in nuclear genes and made in the cytosol before being imported into the mitochondria.4

If you want to discover new biology, it means that you cannot only do the obvious experiments. You must sometimes be a little more ingenious than the next person. So that means that you sometimes tackle projects that nobody has much faith in, and those projects often lead to real discoveries. —Piet Borst, Netherlands Cancer Institute

While working on mitochondrial DNA, he became interested in the unusual mitochondrial DNA of African trypanosomes, which cause sleeping sickness. Some of his most notable work was the discovery of an organelle called the glycosome, which contains glycolytic enzymes and his collaboration with parasitologist George Cross, now at the Rockefeller University, to elucidate the mechanism for antigenic variation in trypanosomes.5,6 Antigenic variation is a survival mechanism for trypanosomes that periodically alters the variant surface glycoproteins to evade the host immune response. Borst’s findings also elucidated a DNA transposition mechanism for antigenic variation and demonstrated trans-splicing as an essential step in synthesis of trypanosome mRNA.7,8 While working on antigenic variation, Borst investigated telomeres on chromosome ends of trypanosomes and found a repeated sequence that was later also identified in human telomeres.9 He also discovered an unusual base in trypanosome DNA called base J (named after his graduate student, Janet Gommers-Ampt, at the Netherlands Cancer Institute) and described its biosynthesis and function.10 Base J is a modi-

fied version of thymine and plays a role in terminating growing RNA chains in trypanosomes and similar parasites.11

Molecular pumps In 1983, Borst moved to the Netherlands Cancer Institute. As a physician, Borst was always interested in medical applications, so he started investigating genes involved with multidrug resistance (MDR) in cancer. He helped decipher the physiologic functions of several ABC transporters, which sit in cell membranes and transport compounds out of the cell to contribute to MDR. One of his notable discoveries was ABCB4, a phosphatidylcholine transferase that is essential for making bile.12 This was an unexpected discovery because at the time, scientists thought that bile salts passively extracted phosphatidylcholine into bile. Borst found that ABCB4 transports phospholipids actively into bile, and that the dysfunction of this transporter gene led to severe liver disease.13 Further studies demonstrated ABCB4’s ability to also transport some drugs.14 Borst discovered that one member of the P-glycoprotein family prevents entry of amphipathic toxins in the gut and the brain, which prevented certain oral medications from being absorbed.15 This insight helped guide strategies for more efficient drug delivery by inhibiting the molecular pump. Borst next turned to pseudoxanthoma elasticum, an inborn error of calcification due to the absence of ABCC6 in the liver.16 This genetic defect caused calcium to accumulate in the eyes, skin, and blood vessels. Borst found that without functional ABCC6, there is a lack of plasma pyrophosphate, which binds to calcium and prevents calcification. This work guided development of new strategies for overcoming drug resistance and improving therapeutic outcomes. “If you want to discover new biology, it means that you cannot only do the obvious experiments. You must sometimes be a little more ingenious than the next person. So that means that you sometimes tackle projects that nobody has much faith in, and those projects often lead to real discoveries,” Borst said.

Mentoring and advocating Borst was deeply invested in training and mentoring numerous students, many of whom have gone onto illustrious scientific careers. “That’s the nice aspect of working within science. It’s a combination of new biology and teaching biology. And I did a lot of teaching in my life,” said Borst. “It’s a joint effort to, on one hand, discover new things, and on the other hand, educate people to have useful careers in later life.” One of his previous graduate students, Titia de Lange, who is now a renowned cell biologist at the Rockefeller University, studied the genetic underpinnings of variant surface glycoproteins in trypanosomes under Borst’s guidance. During her time in Borst’s lab, she noted that he was exceptionally good at dissecting experiments and didn’t hold back from voicing WINTER 2 02 3 | T H E S C IE N T IST 51

PROFILE

his concerns. His deep commitment to science shone through his actions. “He is supremely adept at helping people achieve their best. He had endless patience to sit with people and go through their notebooks and look at the details of their experiments so he could help figure out what was going wrong. He never gave up on his trainees even though he was running the Dutch Cancer Center—a huge job,” said de Lange.

He is supremely adept at helping people achieve their best. He had endless patience to sit with people and go through their notebooks and look at the details of their experiments so he could help figure out what was going wrong. He never gave up on his trainees even though he was running the Dutch Cancer Center—a huge job.” —Titia de Lange, The Rockefeller University

Aside from teaching, Borst served as the director of research at the Netherlands Cancer Institute. At the time, the institution suffered from grant cuts, low productivity, and even lower morale. He stepped in to address the problems with some suggested changes. His vision to improve the institute faced stiff opposition. For instance, Borst wanted to eliminate tenure for graduate students because he thought that it locked people into positions that were unsustainable. It was an unpopular stance, but Borst insisted. This conflict took Borst to court where he appealed to the judges and convinced them that the institute could not function if graduate students worked in permanent jobs. Taking advantage of this momentum, Borst also began to implement other policies. To protect students, he established thesis committees to ensure the timely progression of students’ projects. He also implemented more stringent internal reviews of grant applications before they were submitted to dramatically improve funding success. Borst played a prominent role in engaging with the government on scientific matters. Funding for basic research paled in comparison to translational research. Borst appealed for more funding for fundamental basic research as a necessity to further exploit new knowledge that can be turned into practical applications. In addition, Borst firmly supported the need for science communication. He frequently appeared on TV and radio and wrote thought provoking columns in the Nieuwe Rotterdamsche Courant. “Effective communication is an important function of scientists,” Borst said. 52 T H E SC I EN T I ST | the-scientist.com

“It is important to stress the power of science to solve problems,” Borst said. “Our society is so completely dependent on science and technology that springs from that. If we don’t convince people that the scientific method is the way to get at the truth, then we won’t survive as humanity.” In his five decades of scientific work, Borst contributed to seminal discoveries in diverse topics, including mitochondria, trypanosomes, and molecular pumps involved in multidrug resistance. Borst’s deep passion for science extended beyond his scientific research; he mentored numerous notable scientists and played an active role in advocating for basic research and scientific integrity. For his exceptional career of scientific discovery, Borst received the 2023 Lasker~Koshland Award for Special Achievement in Medical Science earlier this year. J

References 1. Borst P. The malate-aspartate shuttle (Borst cycle): how it started and developed into a major metabolic pathway. IUBMB Life. 2020;72: 2241-2590 2. Borst P, Weissmann C. Replication of viral RNA, 8. Studies on the enzymatic mechanism of replication of MS2 RNA. Proc Natl Acad Sci. 1965;54:982-987. 3. Van Bruggen EF, et al. Circular mitochondrial DNA. Biochim Biophys Acta. 1966;119(2):437-439. 4. Borst P, Ruttenberg GJ. Renaturation of mitochondrial DNA. Biochim Biophys Acta. 1966;114:645-647. 5. Opperdoes FR, Borst P. Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett. 1977;80:360-364. 6. Hoeijmakers JH, et al. Novel expression-linked copies of the genes for variant surface antigens in trypanosomes. Nature. 1980;284:78-80. 7. Bernards A, et al. Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3’ end. Cell. 1981;27:497-505. 8. Van der Ploeg LH, et al. RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. Nucleic Acids Research. 1982;10:3591-3604. 9. Van der Ploeg LH, et al. Structure of the growing telomeres of Trypanosomes. Cell. 1984;36:459-468. 10. Gommers-Ampt JH, et al. beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei. Cell. 1993;75:1129-1136. 11. van Luenen H, et al. Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania. Cell. 2012;150(5):909-921. 12. Smit JJ, et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell. 1993;75(3):451-462. 13. Deleuze JF, et al. Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology. 1996;(4):904-908. 14. Smith AJ, et al. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping. J Biol Chem. 2000;275(31):23530-23539. 15. Schinkel AH, et al. Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc Natl Acad Sci. 1997;94(8):4028-4033. 16. Jansen RS, et al. ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report. Arterioscler Thromb Vasc Biol. 2014;34(9):1985-1989.

PROFILE

Making Standards Exceptional Samantha Maragh has taken on the difficult challenge of standardizing assays, data norms, and terminology in the ever evolving genome editing field. BY MEENAKSHI PRABHUNE, PhD

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hen Samantha Maragh, who now leads the Genome Editing Program at the National Institute for Standards and Technology (NIST), was in elementary school, she loved watching the Forensic Files documentary series on television with her father. Watching scientists, especially women, perform remarkable DNA analysis experiments to solve criminal cases was her first exposure to the profession. Maragh was inspired; the show sparked an early aspiration to become a scientist as an adult. “It’s a TV show, but it was really impactful to me,” Maragh recalled. Maragh grew up in Baltimore, Maryland, as a first-generation American to immigrant parents. Her parents, having moved from Jamaica to ensure opportunities for their children, prioritized her education throughout her school years. Maragh maintained a healthy interest in biology all those years, but when she took her first genetics course in her undergraduate studies at Loyola University, she knew instantly that she had found her calling. “As, Gs, Cs, and Ts and combining things together doesn’t make any logical sense, but it completely resonated with me, like I had found my science home,” she reminisced. Despite her desire to keep learning, Maragh did not pursue a master’s degree right away following graduation. Instead, she planned to first secure a job and then ask her employers to fund her continued education. Even though her friends were skeptical, Maragh remained hopeful. As luck would have it, Maragh secured a position as a technician at the NIST in May 2006. This job shaped the course of her career path.

SAMANTHA MARAGH

A tryst with NIST NIST is a nonregulatory federal agency under the US Department of Commerce. Set up in 1901, the organization sets standards for measurements for just about everything from atomic clocks to electrical outlets. While the NIST organization integrated physics, chemistry, and engineering into its working groups early on, the institution formulated biology specific divisions relatively recently in the 1990s. Maragh snagged a role at the Biochemical Sciences Division but knew nothing about NIST when she applied. However, she soon realized the value of the work done at the institute. One of NIST’s projects involved standardizing the short tandem repeat (STR) DNA analysis method used in forensics for reliably identifying an individual.1 Maragh recalled the surreal feeling when she realized that her workplace was integrally connected to the forensic science that inspired her years ago—a rare full circle moment.

Samantha Maragh leads the Genome Editing Program at NIST.

Maragh first worked on a collaborative project between NCI and NIST to study cancer biomarkers for early detection. Researchers reported new cancer biomarkers frequently, and yet the subsequent experiments with these markers weren’t panning out for some reason. Maragh took up the task of developing positive controls to ensure consistency and efficiency of assays, specifically sequencing-based ones for identifying mitochondrial DNA mutations, used across different research groups.2 “She was just learning and growing at such a rapid pace and had become a real thought leader,” recalled Laurie Locascio, who led Maragh’s division and oversaw the NCI collaboration projects at the time. Locascio is currently the director of NIST and the undersecretary of Commerce for Standards and Technology. “It was clear that she would be tremendously successful,” she added. Impressed by Maragh’s work excellence, her bosses at NIST supported her in taking night courses for continued education, WINTER 2 02 3 | T H E S C IE N T IST 53

PROFILE

Crispier than CRISPR During her graduate program, Maragh studied gene function in early cardiac development in zebrafish. She routinely knocked down protein expression using morpholinos, which are oligonucleotides that bind to mRNA and obstruct translation. Since knockdowns don’t guarantee complete protein inhibition, Maragh wanted to validate her results by suppressing protein expression at a genetic level. She first attempted to use the zinc finger nuclease (ZFN) system for genome editing but found it challenging. She then tried to knock out her desired gene using transcription activator-like effector nucleases (TALEN), a new genome editing tool at the time with growing popularity.3 Using TALEN, Maragh replicated the phenotype she had observed with RNA knockdown. Around that time, she heard about a new study where researchers had leveraged clustered regularly interspaced palindromic repeats (CRISPR) and an associated nuclease, which function as a natural immunity mechanism in bacteria, to create a programmable machinery to edit cells.4 Although Maragh did not get the chance to test the CRISPR system in the lab, she realized the power the flexible technology offered right away, especially having worked with the relatively more complicated ZFN and TALEN tools. As the list of CRISPR applications grew with researchers globally adopting the technique, she also saw the risk for inconsistencies and raised questions that not many were thinking about at the time. “What in the world are we doing to the genome? And how do we know what we are doing?” she recalled wondering. “My NIST brain went ‘controls, variability.’” When Maragh returned to NIST, she had the opportunity to propose a promising topic area that wasn’t on the organization’s radar at the time: CRISPR. “This is a totally new world and there’s a place for NIST here,” she thought. When she pitched her idea at her division’s proposal meeting to solicit peer feedback, a colleague asked, “Is there going to be something crispier than CRISPR one day?” That got her thinking. CRISPR was a promising tool, but ZFN and TALEN remained important players in the field. And given the rapid scientific advances in CRISPR-related technologies, it seemed likely that newer tools were on the horizon. 5 4 T H E SC I EN T I ST | the-scientist.com

When I heard about NIST and their mission and Samantha's interest in having NIST take a role in doing this, I thought, yes, that would be fantastic. —Keith Joung, Massachusetts General Hospital

Maragh broadened her scope to genome editing, successfully convinced her colleagues about the promising outlook of this area, and eventually started the Genome Editing Program at NIST in 2016. The program aims to apply the NIST lens of standards to genome editing research to ensure that scientists use consistent methodology, controls, data formats, and terminology to minimize variability in experiments. The first task for Maragh’s program team was to engage with the genome engineering community and demonstrate that NIST’s goals aligned with their needs. Around this time, Maragh serendipitously came across a short commentary written by an academic about the need for standards in CRISPR research.5 She knew right away that she had found a potential ally: Keith Joung, a genome editing pioneer and group leader at Massachusetts General Hospital.

A consortium is born Joung has been strategizing genome and epigenome editing technologies that could improve human health for almost 20 years. With the rapid growth in the sector at the time, Joung worried about experimental technique variability between groups. He saw a need for standardizing definitions and practices to establish a baseline for the researchers working in this emerging area. “When I heard about NIST and their mission and Samantha’s interest in having NIST take a role in doing this, I thought, yes, that would be fantastic,” he said. With Joung’s support, Maragh conducted her first workshop at the American Society of Gene and Cell Therapy Conference in 2016. Representatives from across the biotechnology sector, academia, pharmaceutical companies, and nonprofit organizations attended. Maragh sourced feedback regarding their challenges and interrogated where they needed data validation tools. “I heard a lot of positivity there. One organization was like, ‘You know, I can see their building, but I can’t go talk to them because of noncompete sort of things, and I need a mechanism that would allow us to be under the same umbrella,’” Maragh recalled. Maragh thought that a Maragh set up the genome editing consortium would be the program at NIST after completing perfect solution. However, on her graduate studies. soliciting further feedback,

NIST

and Maragh obtained her master’s degree in biotechnology from John Hopkins University in May 2008. However, Locascio and a few other NIST leaders had even bigger plans for her. Locascio felt that an advanced degree and exposure to life outside of NIST would enhance Maragh’s career options. Locascio’s boss back then, Willie May, agreed. May tapped her on the shoulder one day and said that she should pursue a PhD degree. Maragh could choose any competency for her graduate studies as long as she could apply that knowledge at NIST once she graduated. Three months after completing her master’s degree, Maragh enrolled in the human genetics PhD program at John Hopkins University, manifesting the master plan that she had conjured up as a student.

she realized that the genome editing community needed more than just a forum that convened to exchange ideas. They sought NIST’s support in making samples and executing experiments. When Maragh realized that this meant that NIST would almost function as their research arm, she worried about how she would find resources for these unconventional requirements. Determined to find a creative solution, Maragh conceptualized a new cost-sharing consortium model where every member contributed in some way. “If you are a sequencing company, maybe you can sequence. If you are a reagent maker, maybe you can provide reagents. If you are an engineering company, maybe you can support with some cell engineering, and if you are big pharma, maybe you just give money,” she explained. “It was very different for NIST. There was no such model; it took me a while. I had to create the model.” After months of grueling paperwork, Maragh officially launched the Genome Editing Consortium in October 2018 with 20 members from diverse institutions. The consortium comprised three working groups. The “specificity measurements” group worked on the reproducibility of on-target and off-target assays and related controls. The “data and metadata” group covered bioinformatics related topics such as evaluating different tools or generating high quality data sets that researchers could use as positive controls. Lastly, the “lexicon” team standardized the genome editing vocabulary. Members could join one or more groups based on their needs, and each group met monthly. Bringing together a disparate group of people who have different interests in working together is not an easy task, commended Joung. “I think she’s done a great job of that.” Locascio seconds that opinion. “I really give great credit to her for thinking about this and what was a very nascent field and building this consortium around what could be the difficulties in getting this implemented in the real world,” she said. The consortium has made big strides in several projects over the past few years. Maragh is particularly proud of completing the first version of a genome editing vocabulary standard that includes definitions of key terms used in the field. The team worked hard on curating the relevant terms, drafting the concise definitions, getting experts to review them, and finally seeking global feedback. After a regimented and rigorous review process, Maragh was thrilled when the document was finally approved by the International Standards Organization and published on their site. For her success in building the NIST Genome Editing Consortium as a public-private partnership, Maragh received the George A. Uriano Award in 2021.

Staying on target and under control Today, the genome editing consortium has grown to more than 40 members, and all three groups are still active. Joung enjoys watching Maragh in action bringing members with diverse interests to work together. “The advances come so quickly that to some degree, you have to be nimble and adjust or expand your expectations as far as the field’s,” he said. “I’m grateful that somebody like her is willing to undertake this type of effort.”

For instance, within just a few years after CRISPR being adopted in labs worldwide, the genome editing community welcomed new tools such as base editors and prime editors. So, the standardized lexicon document lacks the definitions for these technologies, which didn’t exist at the time of its creation, but are now crucial to the field. Maragh hopes that her teams will add suggested definitions for some of the missing terms to the glossary at some point. In fact, there is a long wishlist of goals she would like to accomplish in the near future. In one project, her team intends to generate some physical samples of engineered cells bearing arrays of edits for consortium members to use as positive controls for complex edits. Maragh spoke of another interesting consortium project that is currently underway. This Genome in a Bottle (GIAB) project is a blind inter-lab study to assess the accuracy of capabilities that consortium members are currently using for detecting DNA variants. The NIST team sent consortium researchers a mixture of cells or DNA with varying sizes and frequencies of mutations at different loci. The researchers will use the technology of their choice and expertise, such as sequencing, genome wide DNA imaging, and fragment analysis, and report back the variant size and frequency data. On comparing data from different research groups, Maragh hopes that the NIST team can zero in on the most accurate capabilities that will serve as the gold standard for detecting DNA variants moving forward. “The goal of setting standards for how things are measured in our field remains a very, very important one. And so, I would like to see them be a bit more vocal and proactive in trying to put these standards out there and trying to get people in the field to follow them,” Joung suggested. In addition to leading the Genome Editing Program, Maragh now comanages NIST’s Cancer Biomarker and Genomic Science Group, which includes programs on genome editing, cancer biomarkers, flow cytometry, and human genome sequencing. Although she does not have a master plan for the future anymore, she wants to continue working in the regenerative medicine and precision medicine application areas. “She is clearly a scientific leader, and now she leads an external facing consortium, but I definitely see her growing into larger roles and moving up the organization,” said Locascio. “She just has very unique characteristics that make her a natural leader.” J

References 1. Ruitberg CM, Reeder DJ, Butler JM. STRBase: a short tandem repeat DNA database for the human identity testing community. Nucleic Acids Res. 2001;29(1):320-322. 2. Jakupciak J, et al. Analysis of potential cancer biomarkers in mitochondrial DNA. Curr Opin Mol Ther. 2006;8(4):345-354. Accessed August 1, 2023. 3. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49-55. 4. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821. 5. Joung J. Standards needed for gene-editing errors. Nature. 2015;523:158

WINTER 2 02 3 | T H E S C IE N T IST 5 5

METHODS

Whenever, Wherever: Taking DNA Amplification Outside the Lab Recombinase polymerase amplification lets researchers rapidly replicate DNA in the clinic, in the field, or even in the International Space Station.

C

etus biochemist Kary Mullis’ invention of polymerase chain reaction (PCR) in 1985 revolutionized genetics and won him a share of the 1993 Nobel Prize in Chemistry.1 PCR was instrumental in the sequencing of the human genome, and today, it is used for everything from diagnosing genetic disorders to studying the evolutionary relationships between species.2,3 Despite its utility, PCR’s main limitation is that it requires precise cycles of heating and cooling to amplify DNA. The thermal cyclers that perform this operation are clunky, relatively expensive, and consume a lot of power, making them unsuitable for use in the field or in low-resource settings. In 2006, researchers Olaf Piepenburg, Colin Williams, and Niall Armes of ASM Scientific, which later became TwistDx, along with Derek Stemple, at the time a molecular biologist at the Wellcome Trust Sanger Institute and currently the chief science officer at Camena Bioscience, developed an alternative. They invented recombinase polymerase amplification (RPA), an isothermal amplification technique that functions at relatively low temperatures.4 “Just the warmth of your hand is enough to drive the reaction,” said Stemple. Since its invention, researchers have demonstrated a myriad of applications for this technology, including rapid identification of plant, animal, and human pathogens, including SARSCoV-2 , detecting antimicrobial resistance genes, and water quality monitoring.5–10

The beginnings of RPA The researchers didn’t originally set out to develop a new isothermal amplification method, said Piepenburg. Instead, they were trying to develop a new technique for single molecule sequencing. During this project, they became interested in ways to sequence specific sites within the genome, rather than random pieces. “Niall’s idea was to use recombinase proteins like RecA to target DNA primers to specific sites,” said Piepenburg. “This idea evolved into the notion that if you have two primers that are interacting with recombinase proteins and they move in opposite directions along a double-stranded piece of DNA, wouldn’t you start to get an amplification reaction?” RecA is a bacterial protein that is crucial for repairing double-stranded breaks in DNA via the process of homologous recombination. RecA binds to single-stranded DNA, searches along a separate piece of double stranded DNA for the cognate 5 6 T H E SC I ENTI ST | the-scientist.com

sequence, and then promotes DNA strand invasion.11 After testing various proteins in the RecA family, the researchers ultimately settled on the phage protein uvsX recombinase, which performs a similar function. In RPA, this recombinase protein binds to primers designed by scientists, then scans the DNA, and inserts the primers into complementary sites in the DNA. Then single-stranded binding proteins called gp32 bind to the displaced DNA strand to stabilize it. The recombinase disassembles, allowing a DNA polymerase to bind to the 3’ end of the primer, and DNA synthesis begins. “We tested a lot of different polymerases,” said Stemple. “The polymerases that worked the best were the ones that were able to displace the second strand, that don’t need single-stranded DNA to work from, like Taq polymerase.” The researchers eventually found a polymerase from Bacillus subtilis that was especially effective. “It’s like a cow catcher at the front of the train, separating double stranded DNA at the front of the polymerase,” said Stemple. This technique enables rapid amplification of the target DNA: the process takes just 20 to 40 minutes.12 Once the target DNA has been amplified, researchers can use a variety of strategies to detect the DNA, including lateral flow and fluorescence-based tests. The researchers also demonstrated the real-world utility of this technique. “We really wanted to show that we could detect disease-causing bacteria in very small numbers,” said Stemple. Indeed, by combining RPA with a fluorescent probe detection system, they detected just ten copies of DNA from methicillinresistant Staphylococcus aureus (MRSA), a pathogen responsible for an estimated 100,000 deaths per year.13 “After we published the paper, we got an email from Kary Mullis congratulating us,” said Stemple. “That was pretty cool.”

Detecting DNA from the cervix to outer space “A question that we faced early on when presenting at scientific conferences or talking to potential collaborators was, ‘Why not just use PCR?’” said Piepenburg. “PCR is excellent at what it does, but it is limited by the fact that you need a heavy instrument that takes a lot of energy to run. So, it’s very hard to see PCR ever reaching a stage where you can make a completely disposable diagnostic. And especially for rapid diagnostics, that’s really the holy grail.” With RPA in their toolkit, researchers around the world can now develop rapid tests for various plant, animal, and human

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BY HANNAH THOMASY, PhD

RECOMBINASE POLYMERASE AMPLIFICATION IN ACTION A rapid isothermal amplification technique enables pathogen identification and antibiotic resistance detection in low-resource settings.

Recombinase polymerase amplification (RPA) is a technique for rapidly copying segments of DNA. Unlike polymerase chain reaction (PCR), it does not require thermal cycling, so less equipment is needed.

HOW RPA WORKS

RPA APPLICATIONS

Researchers require three defining elements for RPA: uvsX recombinase proteins, singlestranded binding proteins, and strand-displacing DNA polymerases. They also need primers and nucleotides to amplify the target DNA.

Because RPA is rapid, portable, and runs at low temperatures it has diverse applications.

Recombinase proteins

Recombinaseprimer complexes

Optimal temperature 37-42° C

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Point-of-care testing for genetic and infectious diseases

DNA Strand-displacing DNA polymerase

Q 1 First, the primers bind to the uvsX recombinase proteins to form recombinase-primer complexes.

Q 4 The recombinase disassembles and the strand-displacing DNA polymerase binds to the 3’ end of the primer.

Water quality testing

Q 2 Then the recombinase inserts the primers into complementary sites in the DNA.

Q 5 The polymerase elongates the primer. Antibiotic resistance detection

Single-stranded binding proteins

Q 3 Single-stranded binding proteins bind to the displaced DNA strand and stabilize it.

Q 6 The target DNA strands are duplicated. Agricultural pathogen monitoring

METHODS

pathogens that can be used in places far afield from traditional laboratory settings, with important implications for both agriculture and human health.7,14,15 One such application is human papillomavirus (HPV) testing, which has long been difficult in low-resource settings. “There is a huge need for point-of-care technologies that can be used anywhere in the world; remote locations and developing countries are really in need of tests that can accurately detect HPV in women,” said Sylvia Daunert, a biochemist and molecular biologist at the University of Miami.

Just the warmth of your hand is enough to drive the reaction. —Derek Stemple, Camena Bioscience

The test also needs to be fast. In these settings, said Daunert, “if women leave the community health clinic without being treated, the likelihood that they will come back is very low.” “What makes RPA really good for this application is that it works at just one temperature, which is relatively low compared to other isothermal methods, some of which need to be kept at 65 degrees Celsius or require initial heating,” said Daunert. Daunert and her team designed consensus primers that could amplify any of the 14 high-risk HPV genotypes. They lyophilized the reagents and combined them with primers into one tube, reducing the number of steps and eliminating the need for precise pipetting. “We wanted anyone to be able to operate this test,” said Daunert. Their test detected high-risk HPV with a sensitivity of 96 percent and a specificity of 83 percent.7 Daunert’s next step will be testing the diagnostic on the ground in low-resource settings in regions like central Africa. Some researchers deploy RPA even farther afield. Gur Pines, who studies molecular diagnostics and detection at the Agricultural Research Organization–Volcani Institute, was working on a rapid molecular detection strategy for the peach fruit fly, an important agricultural pest, when he got the opportunity to send an experiment to space as part of Israel’s Rakia Mission. Pines studied a detection strategy that combined RPA and CRISPR technology. RPA amplifies the DNA, and when CRISPRCas12a binds to the target DNA, the Cas12a begins to indiscriminately chop up single-stranded DNA. (This nonspecific cutting of single-stranded DNA is unique to Cas12a and is not shared by the more well-known Cas9 protein.) Researchers use probes with a fluorophore and a quencher attached by single-stranded DNA, so when the target DNA is detected, Cas12a cleaves the probe, freeing the fluorophore and producing a fluorescent signal. This technique, dubbed DETECTR, was originally developed by Jennifer Doudna, a biochemist at the University of California, Berkeley and the codeveloper of CRISPR-Cas9 genome editing, and her team in 2018, but it had not previously been tested in microgravity.16 “We wanted to test whether this technology worked in microgravity because it is such an awesome technology,” said Pines. 5 8 T H E SC I ENTI ST | the-scientist.com

“It has so many potential applications and it could be ideal for long space missions. They don’t need to use fancy machines or send the samples back to Earth. They can have results almost in real time.” However, said Pines, “We don’t fully understand the effects of microgravity on biological reactions.” So, researchers designed a proof-of-concept study to test whether this technique could identify synthetic genomic sequences from three different species: a bacterium, a fungus, and an insect. The researchers found that the test was still extremely sensitive in microgravity: it identified the target DNA in attomolar concentrations.17 In the future, researchers could use this technology to design kits to diagnose diseases in astronauts, study how the microbiome of the station and its inhabitants changes over time, or monitor the health of space farming operations on long missions. J

References 1. The Nobel Prize in Chemistry 1993. NobelPrize.org. 2. Polymerase Chain Reaction (PCR) Fact Sheet. Genome.gov. 3. Suyama Y et al. Complementary combination of multiplex high-throughput DNA sequencing for molecular phylogeny. Ecological Research. 2022;37(1):171-181. 4. Piepenburg O et al. DNA Detection Using Recombination Proteins. PLoS Biol. 2006;4(7):e204. 5. Silva G et al. Rapid and specific detection of Yam mosaic virus by reversetranscription recombinase polymerase amplification. J Virol Methods. 2015;222:138-144. 6. Zhao G et al. Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis. BMC Veterinary Research. 2018;14(1):412. 7. Seely S et al. Point-of-Care Molecular Test for the Detection of 14 HighRisk Genotypes of Human Papillomavirus in a Single Tube. Anal Chem. 2023;95(36):13488-13496. 8. Nelson MM et al. Rapid molecular detection of macrolide resistance. BMC Infectious Diseases. 2019;19(1):144. 9. Luo N et al. Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick. Frontiers in Marine Science. 2022;9. 10. Liang L et al. Development of a multi recombinase polymerase amplification assay for rapid identification of COVID 19, influenza A and B. J Med Virol. Published online September 20, 2022:10.1002/jmv.28139. 11. Cox MM. Motoring along with the bacterial RecA protein. Nat Rev Mol Cell Biol. 2007;8(2):127-138. 12. Lobato IM, O’Sullivan CK. Recombinase polymerase amplification: Basics, applications and recent advances. Trends Analyt Chem. 2018;98:19-35. 13. Murray CJL et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet. 2022;399(10325):629-655. 14. Strayer-Scherer A et al. Recombinase Polymerase Amplification Assay for Field Detection of Tomato Bacterial Spot Pathogens. Phytopathology. 2019;109(4):690-700. 15. Conrad CC et al. A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease. Frontiers in Veterinary Science. 2020;7. 16. Chen JS et al. CRISPR-Cas12a target binding unleashes indiscriminate singlestranded DNase activity. Science. 2018;360(6387):436-439. 17. Alon DM et al. CRISPR-based genetic diagnostics in microgravity. Biosens Bioelectron. 2023;237:115479.

METHODS

Finding the One in a Million Phage display revolutionized peptide screening methods and unlocked opportunities in protein discovery and development. BY SHELBY BRADFORD, PhD

W

hen recombinant protein expression was introduced in 1977, screening clones was a laborious and timeconsuming process.1-3 While on a sabbatical at Duke University, George Smith, a biochemist at University of Missouri, reasoned that he could improve this process by leveraging a coat protein, called pIII, from the filamentous bacteriophage that he studied. He tested his initial design with two Duke University biochemists, Robert Webster and Paul Modrich. Their collaboration birthed phage display, a combinatorial marvel that Smith referred to in his Nobel lecture as “simple evolution in a Petri dish.” It completely changed the field of recombinant protein biology.

Use what you have In the early and mid-1980s, scientists used phages to express recombinant proteins. The protein was retained inside the virions, so screening required growing viral plaques, creating a stamp of these on a membrane, treating the membrane with antibody, and then mapping antibody labels on the membrane back to the exact viral plaques on the Petri plate. Smith’s vision for improving recombinant protein analysis was to express a foreign protein at one end of pIII and then select for it using an antibody affixed to a surface. In this way, scientists could retain only positive clones and immediately use their selected phage to generate more phages. This made screening higher throughput, as researchers could screen thousands or even millions of clones simultaneously.

It completely opened the door to making fully human antibodies as therapeutics.

© ISTOCK.COM, MIRROR-IMAGES

—John McCafferty, Cambridge Institute of Therapeutic Immunology and Infectious Disease

Additionally, because the protein would be bound to the bacteriophage with the inserted DNA, the product and instructions for it were obtained at the same time as a genotype-phenotype linkage. “You can then sequence that particular phage clone, and it will tell you the nucleotide sequence and therefore, the amino acid sequence of the peptide displayed on its surface,” said Jamie Scott, who joined Smith’s lab as a postdoctoral fellow in the late 1980s and is now a professor emeritus at Simon Fraser University. When Smith returned to his lab, he explored this method further, and in 1985, published his work on what he called fila-

Scientists use phage display to generate monoclonal antibodies.

mentous phage display.4 In subsequent years, Smith and his team developed a peptide library in which five to six amino acid residues in a peptide sequence were randomized.5 In this work, they demonstrated the value of phage display in selecting for highbinding peptides through a series of affinity purification steps, where bound phages were eluted, amplified in a bacterial host, and then reintroduced to their target ligand to select for binders. These libraries allowed for screening potentially billions of random epitopes simultaneously. While Smith and his colleagues’ peptide libraries demonstrated the potential of phage display, the sequences were randomly generated. Scientists did not have control over the amino acid frequencies, and because of redundancies in amino acids, could have overrepresentation of some residues. To improve control over the amino acids used and circumvent the introduction of stop codons, subsequent groups developed methods to synthesize codons individually.6 These presynthesized codons can be mixed at specific percentages to yield a library with the characteristics researchers are most interested in, such as polarity and binding affinities. “It’s not random,” said Anthony Kossiakoff, a protein engineer at the University of Chicago who studies protein structurefunction relationships. “There’s a lot of planning and designing WINTER 2023 | T H E S C IE N T IST 59

PHAGE DISPLAY ALLOWS RAPID SCREENING OF MILLIONS OF PEPTIDES A viral protein expression method links proteins and their coding instructions, enabling easier target identification for downstream analysis.

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Q 1 Scientists insert variable DNA sequences coding for their proteins of interest into plasmids that carry a phage coat protein gene, an antibiotic resistance gene, and a packaging signal. Then they transform the plasmids into a bacterial vector and infect it with a helper phage that supplies the other necessary viral proteins. This generates the phage library, where each virion expresses versions of the protein of interest on its coat protein and carries its genetic sequence in its genome. Q 2 In the next step, researchers isolate the phages expressing surface proteins using a surface ligand binding assay. The final eluted phages may bear a mix of different surface protein epitopes. Q 3 In the amplification step, researchers propagate the eluted phages in a bacterial culture, and may run additional rounds of affinity purification. Q 4 Researchers infect bacteria with the selected phages and culture them on plates containing antibiotics. In the final step, they pick resistant colonies to isolate the plasmids, which are then sequenced and cloned into the desired protein production vectors for various applications.

for how you can best utilize what you got.” Proficient structural biologists consider more than the library. “The biggest thing about any of these selections is the quality of the antigen,” Kossiakoff said. He explained that for phage display to yield usable products, teams using the method need to determine the stability of the antigen and ensure that it is in the desired conformation. Lastly, to select for high-affinity proteins, researchers must develop adequate stringency conditions in their affinity purification steps. This may include decreasing the concentration of ligand, introducing competing enzymes, screening against ligands in other conformations, or blocking potential binding sites of the target ligand during the maturation step.7 At the end, because of the genotype-phenotype linkage, a researcher can extract the protein’s genetic sequence for a variety of downstream purposes.

INFOGRAPHIC: DESIGNED BY ERIN LEMIEUX; © ISTOCK.COM, LOVE EMPLOYEE

Opening the Door This linkage of the instructions with the binding product, coupled with the ability to evaluate huge volumes of candidates, became an attractive model for many research applications, but particularly for antibody research and production. While Smith’s method generated random peptide sequences and tested them against known antibodies, other groups considered alternative approaches. “Wouldn’t it be really cool if we could do that the other way around?” said John McCafferty, currently a biologist at the Cambridge Institute of Therapeutic Immunology and Infectious Disease and chief executive officer of Maxion Therapeutics. He and his postdoctoral mentor, Greg Winter, a molecular biologist currently at the Medical Research Council Laboratory of Molecular Biology, used phage display to produce antibodies against known targets. By cloning the variable regions of the heavy and light chains of antibodies from immunized blood donors together

on a single coding region, the two produced a fusion protein with all of the antigen binding properties of a full antibody, which they called single chain Fv (scFv). This scFv was expressed by the bacteriophage and could be affinity selected analogously to Smith’s peptides by using the immobilized peptide as a binding target for the antibodies. From the inception of this idea to the publication of the first successful antibody took one year8—“A feat that I’ve tried to repeat ever since without success,” McCafferty said. Producing monoclonal antibodies was previously possible using hybridoma methods.9 However, these methods could be laborious and take months to immunize animals, isolate and culture B cells that were fused to immortalized cells, and then screen them. With phage display, it was possible to isolate the genetic material from B cells from immunized animals or humans and immediately begin amplifying it in bacteriophages. Not only was this faster, it also simplified the process of studying the genetic sequence of these antibodies. Additionally, it alleviated the problems of hybridoma technology for producing animal antibodies that had to be humanized. The binding sequences identified through phage display could be isolated, reinserted into an existing human IgG backbone, and expressed in bacterial or mammalian cells. “It completely opened the door to making fully human antibodies as therapeutics,” McCafferty said. This method led to the development of the world’s first antibody produced by phage display, the anti-TNF antibody adalimumab, more commonly known as Humira, which was produced by McCafferty’s and Winter’s company Cambridge Antibody Technology with the help of Baden Aniline and Soda Factory (BASF). Coupled with next-generation sequencing, phage display allows for the rapid production and assessment of antibodies

Filamentous phages are used to express recombinant proteins in phage display.

METHODS

It’s an extremely powerful technique to do all kinds of things.

from multiple snake genera.24,25 “It’s an extremely powerful technique to do all kinds of things,” Kossiakoff said. J

—Anthony Kossiakoff, University of Chicago References

against an array of targets, including those that would otherwise be impossible to produce in a human system. “Because in a synthetic library, it has no basis for tolerance,” said Scott.

An extremely powerful technique Phage display, like most technologies, has evolved. In the world of antibody production, researchers explored display vehicles such as nontraditional viruses and bacteria and also used eukaryotic cells, such as yeast and mammalian cell lines.10,11 “In some ways, a phage display system is a bit of a black box,” McCafferty said. “You’ve got your library; you’ve got your antigen; you’ve been through a process; and out comes stuff at the other end.” While one can easily find a slew of binding candidates, it’s often more than can be easily or desirably assessed for detailed binding affinity or cross-recognition with other potential targets. While yeast and mammalian cell display methods do not offer the scale of library potential that phages do, these approaches are compatible with flow cytometry and cell-sorting applications, which allow researchers to explore far more of their protein-expressing clones and their biophysical properties. Researchers also use alternative backbones for their peptides beyond the traditional immunoglobulin scaffolds. These include small proteins or even domains of proteins, such as fibronectin binding domain, which can support the introduction of variable peptide regions.12 These new scaffolds can be smaller than immunoglobulin and overcome structural limitations, such as the immunoglobin’s dual-chain design, a double cysteine bond, and necessary post-translational modifications.13,14 Modified selection methods also expand the capacity to select displayed proteins with desired characteristics. This includes in vivo selection, where candidates are delivered to a model animal and their organ distribution is assessed, and whole cell culture, where the library can be screened against multiple cell types to determine receptor recognition.15-17 Beyond antibodies, researchers use phage display to explore and manipulate protein interactions, either to study protein functions or to apply toward novel drug discovery.18,19 “You can ask questions that are very outside the box about the energetics of interfaces and understand, as evolution has put these things together, what was really important, and why it’s important and conserved,” Kossiakoff said. Phage display was used to identify rare or even unnatural peptides for cancer therapeutics and B cell epitopes in disease and to screen against allergens to characterize them or chemical compounds to study their mechanism of action.20-23 It’s also being explored for identification and production of antivenom antibodies, some of which are cross protective against venom 6 2 T H E SC I ENTI ST | the-scientist.com

1. Itakura K, et al. Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science. 1977;198(4321):1056-1603 2. Mierendorf RC, et al. Gene isolation by screening gt11 libraries with antibodies. Methods Enzymol. 1987; 152: 458-469 3. Young RA & Davis RW. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci. 1983;80(5):1194-1198 4. Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228(4705):1315-1317 5. Scott JK & Smith GP. Searching for peptide ligands with an epitope library. Science. 1990;249(4967):386-390 6. Virnekas B, et al. Trinucleotide phosphoramitides: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 1994;22(25):5600-5607 7. Pande J, et al. Phage display: concept, innovations, applications, and future. Biotechnol Adv. 2010;28(6):849-858 8. McCafferty J, et al. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990;348:552-554 9. Mitra S & Tomar PC. Hybridoma technology; advancements, clinical significance, and future aspects. J Genet Eng Biotechnol. 2021;19(159) 10. McMahon C, et al. Yeast surface display platform for rapid discovery of conformationally selective nanbodies. Nat Struct Mol Biol. 2018;25:289-296 11. Robertson N, et al. Development of a novel mammalian display system for selection of antibodies against membrane proteins. J Biol Chem. 2020;295(52):18436-18448 12. Gilbreth RN & Koide S. Structural insights for engineering binding proteins based on non-antibody scaffolds. Curr Opin Struct Biol. 2012;22(4):413-420 13. Binz HK & Plückthun. Engineered proteins as specific binding reagents. Curr Opin Struct Biol. 2005;16(4):459-469 14. Skerra A. Imitating the humoral immune response. Curr Opin Chem Biol. 2003;7(6):683-693 15. Kolonin MG, et al. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004;10:625-632 16. Pasqualini R & Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature. 1996;380:364-366 17. Barry MA, et al. Toward cell-targeting gen therapy vectors:selection of cellbinding peptides from random peptide-presenting phage libraries. Nat Med. 1996;2:299-305 18. Sidhu SS, et al. Exploring protein-protein interactions with phage display. Chembiochem. 2003;4(1):14-25 19. Krumpe LRH & Mori T. Potential of phage-display peptide library technology to identify functional targeting proteins. Expert Opin Drug Discov. 2007;2(4):525-537 20. Brown KC. Peptidic Tumor Targeting Agents: The Road from Phage Display Peptide Selections to Clinical Applications. Curr Pharm Des. 2010;16(9):1040-1054 21. Dybwad A, et al. Identification of new B cell epitopes in the sera of rheumatoid arthritis patients using a random nanopeptide phage library. Eur J Immunol. 1993;23(12)3189-3193 22. Rhyner C, et al. Cloning allergens via phage display. Methods. 2004;32(3)212-218 23. Van Dorst B, et al. cDNA phage display as a novel tool to screen for cellular targets of chemical compounds. Toxicol in Vitro. 2010;24(5):1435-1440 24. Ahmadi S, et al. An in vitro methodology for discovering broadly-neutralizing monoclonal antibodies. Sci Rep. 2020;10:10765 25. Ledsgaard L, et al. Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain-_-neurotoxins from snakes. Nat Commun. 2023;14:682

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