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OUR INNER
NEANDERTHAL
ANCIENT SECRETS IN THE
HUMAN GENOME
DRUG TRIALS
FOR FRAGILE X
DOPING ATHLETES
VS ANTI-DOPING
OFFICIALS
RETHINKING
ANIMAL RESEARCH
PLUS
NEW MODEL
ORGANISMS ON
THE BLOCK
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SEPTEMBER 2019
Contents
THE SCIENTIST THE-SCIENTIST.COM VOLUME 33 NUMBER 09
© ISTOCK.COM, SIMONKR; © SCIENCESOURCE, S. ENTRESSANGLE AND E. DAYNES
Features ON THE COVER: © SCIENCESOURCE, S. ENTRESSANGLE AND E. DAYNES
30
The Doping Arms Race
36
Our Inner Neanderthal
44
A Broken X
Athletes have devised ways to outsmart From skin color to immunity, human Despite a solid understanding of the
tests for performance-boosting drugs, but biology appears to be linked to our biological basis of fragile X syndrome,
scientists are working hard to get ahead. archaic ancestry. researchers have struggled to develop
BY ANNA AZVOLINSKY BY JEF AKST effective treatments. But recent trials
offer hope.
BY RANDI HAGERMAN
09. 201 9 | T H E S C IE N T IST 3

SEPTEMBER 2019
Department Contents
13 59
16 FROM THE EDITOR
We’re Different, We’re the Same
LAB TOOLS
Genetics’ Next Top Models
Diversity is a strength. And so is a You’re well-acquainted with
full appreciation of our similarities. Drosophila, C. elegans, and the
BY BOB GRANT humble lab mouse, but lesser-known
model systems are poised to make
16 NOTEBOOK their mark.
Impervious; Stellar Influence?; BY AMBER DANCE
Help for Hellbenders; Complex
Inheritance 63 CAREERS
Fixing Animal Research
28 CRITIC AT LARGE Many in vivo preclinical studies are
Diversify Our Human Genomic Data poorly designed and generate
To better understand the biology irreproducible data, but efforts to
of our species and to make address the problem are on the rise.
individualized medicine accessible BY DIANA KWON
to all, we need to consider the
52 comprehensive genetic makeup of a 67
DEWALD KLEYNHANS, UNIVERSITY OF PRETORIA; © ISTOCKPHOTO.COM, RALWEL; © KEN RICHARDSON PHOTOGRAPHY
READING FRAMES
wider swath of humanity. Meeting Our Maker
BY CHARLES LEE Humans are the first species to
decipher the genetic code that
28 MODUS OPERANDI underlies our biology, but does it
Optogenetics Sans Genetics hold all the answers?
DNA-coated gold nanorods enable BY BILL SULLIVAN
cells to be activated by light without
genetic manipulation. 72 FOUNDATIONS
BY RUTH WILLIAMS Modern Synthesis, 1937
BY CHIA-YI HOU
52 THE LITERATURE
A synthetic ion channel makes plants IN EVERY ISSUE
grow faster; a new family of viruses; 10 CONTRIBUTORS
potential biomarkers of suicidal 15 SPEAKING OF SCIENCE
thoughts in PTSD patients 71 THE GUIDE
57
ANSWER
54 PROFILE PUZZLE ON PAGE 15
Defining Rare Disorders
Judith Hall was instrumental in L UN AM O T H R A S H
incorporating genetics into medical I I I O N O
MA N I C UR E R A I N
practice, describing previously
E E R R J T E
unknown disorders.
T O X I CO L O GY
BY ANNA AZVOLINSKY
C W F D S M B
A N I MA L S H R I K E
57 SCIENTIST TO WATCH T N U S U C E
Cigall Kadoch: Unraveling Cancer A L GON QU I A N
BY CHIA-YI HOU R S A N T C T
A L P S S T A R GA Z E
C A A E V N
T UN A I N H E R E N T
09. 201 9 | T H E S C IE N T IST 5

SEPTEMBER 2019
Online Contents
THIS MONTH AT THE-SCIENTIST.COM:
VIDEO VIDEO VIDEO

Fragile Xpert Genomic Medicine’s Future Neanderthals in Our Family Tree
The MIND Institute’s Randi Hagerman Charles Lee, scientific director of Vanderbilt University’s Tony Capra, who
discusses symptoms and treatment in the Jackson Laboratory for Genomic is quoted in this month’s feature article on
children with fragile X. Medicine, talks about his vision for Neanderthal DNA in modern genomes,
how genomic discovery will fuel discusses the effects of interbreeding
individualized medicine. between ancient hominin groups.
AS ALWAYS, FIND BREAKING NEWS EVERY DAY ON OUR WEBSITE.
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SEPTEMBER 2019
Contributors
After finishing undergraduate studies at the University of California, Davis, in 1971, Randi Hagerman received
her medical doctorate from Stanford Medical School and went on to specialize in pediatrics and neuroscience.
She now does clinical work as a developmental behavioral pediatrician and is a leading researcher of fragile X
syndrome, a condition where genetic mutations on the X chromosome result in varying levels of intellectual dis-
ability. Hagerman is a distinguished professor of pediatrics and the medical director of the MIND Institute at
the University of California, Davis. She is currently running screening studies for fragile X syndrome in Mexico,
the Philippines, and Colombia. Hagerman and her colleagues have also been involved in training programs for
doctors and health-care professionals on neurodevelopmental disorders. In this issue of The Scientist (page 44),
Hagerman writes about our current understanding of fragile X syndrome and the experimental treatments going
through clinical trials.
Scientist and science communicator Bill Sullivan has been transfixed by the parasite Toxoplasma gondii since pursu-
ing his PhD with David Roos at the University of Pennsylvania in 1994. After completing postdocs at the pharmaceuti-
cal company Elanco and at the Indiana University School of Medicine (IUSM) in Indianapolis, Sullivan established his
own lab at IUSM in 2003 and continues to study T. gondii. The organism, which lives out its reproductive stage in cats,
causes infected rodents to lose their fear of their natural feline predators. The striking survival tactic piqued
Sullivan’s interest in potential effects of T. gondii on the behavior of humans, about a third of whom are infected with
the protozoan. “This parasite has figured out how to tweak the brain of a rodent to get it into a cat’s belly, where it wants
to go,” he says. “It begs the question, if it can change the behavior of rodents, can it change the behavior of people?”
So began Sullivan’s deep dive into the forces that shape human behaviors and personalities, namely genetics,
epigenetics, the environment, and microbiota. His new book, Pleased to Meet Me: Genes, Germs, and the Curious
Forces That Make Us Who We Are, untangles the biological underpinnings of both our “goofy quirks and dark sides”
and poses the question of whether we are really in total control of our own behaviors, he says.
Sullivan has a long-held passion for communicating science in accessible, engaging language, kick-started by his
blog THE 'SCOPE. His byline can be found in publications such as Scientific American, COSMOS magazine, and the
PLOS SciComm blog, for which he is a writer and editor. He also promotes public engagement in science through the
science/art organization The da Vinci Pursuit, and in his spare time he loves rocking out with his band Open Cage.
COURTESY OF UC DAVIS HEALTH; ERIC SCHOCH; THE JACKSON LABORATORY

During a research elective in the last year of his undergraduate degree at the University of Alberta in 1990,
Charles Lee coauthored a scientific publication and became enamored with the excitement of research. As a
result, he chose to forego medical school and instead commit to graduate school.
During his master’s research, also at the University of Alberta, he studied the genome of the Indian muntjac
deer, which has the smallest chromosome number among mammalian species, to understand the genesis of their
six giant chromosomes. Fortuitously, in the course of this work, Lee accidentally forgot to bake the chromosomes
at 60 °C prior to performing fluorescent in situ hybridization, an error that became the basis of a new discovery:
skipping the baking increased the binding efficiency of the molecular probes to the chromosomes. The improved
probe binding clearly revealed many DNA sequences within the giant chromosomes of the Indian muntjac deer
that were remnants of chromosome fusion events in ancestral deer species. After completing his Masters degree in
1993, Lee stayed on at the University of Alberta for his PhD, studying repetitive DNAs in the centromeres of human
chromosomes and completing his doctorate in 1996.
Lee has since directed research units at Brigham and Women’s Hospital, Harvard Medical School, and
the Harvard Cancer Center. He is now the scientific director of the Jackson Laboratory for Genomic Medicine
and president of the Human Genome Organization. He is also a distinguished professor at Ewha Womans
University in South Korea as well as at the First Affiliated Hospital of the Xi’an Jiaotong University in China.
Throughout his career, he has actively worked towards identifying different types of genetic variation among
diverse human populations. On page 26, Lee writes about the need for including greater diversity in cataloged
human genomic data.
1 0 T H E SC I EN T I ST | the-scientist.com
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FROM THE EDITOR
We’re Different, We’re the Same

Diversity is a strength. And so is a full appreciation of our similarities.
BY BOB GRANT
O
ne of my favorite books to read to my children is DNA makes humans
titled “We’re Different, We’re the Same: And We’re different from one
Wonderful.” It features a cast of cartoon humans another. In study
and Sesame Street characters, and on each pair of pages a after study, research-
different aspect of physical appearance is highlighted. For ers have found that
example: “We’re different. Our noses are different.” The the genetic ambigu-
accompanying illustrations show people and Muppets with ity between “races”
proboscises of varying sizes, shapes, and colors. Turn the far outweighs any
page, and the text reveals the characteristics that bind all clustering of genes
of these diverse noses together: “We’re the same. Our noses capable of defining a
are the same. They breathe and sniff and sneeze and whiff.” particular race. But
Big Bird and Elmo are delivering a direct, timeless there are discernible
message: even though individuals look different, we are genetic differences
all, at a basic level, similar. The denizens of Sesame Street that track with ances-
are of course trafficking in phenotypes, but the book’s try. For example, peo-
moral assumes fresh meaning when viewed in the con- ple whose ancestors
text of our expanding knowledge of human genetics. come from sub-Saharan Africa or Northern Europe have
This simple message, cast in a new light, came to mind a statistically higher risk of sickle-cell disease or of cystic
as I edited this month’s Critic at Large essay (see page 26), a fibrosis, respectively, than people with other geographic
piece from Jackson Laboratory Scientific Director Charles origins. We really are different.
Lee on the need to inject large-scale genomic databases with The genetic differences that researchers are burrow-
sequences from a wider swath of humanity. Diversifying ing into with each new study can help inform our concept
our cataloging and curation of human genome sequences, of humanness and heal our ills. But we are the same. The
Lee argues, will help us better understand the genomic tap- fact of our predominant genetic similarities, in addition
estry that makes up our species. For example, researchers to drawing us together and driving home the realization
now know that human genomes differ from one another by that we are all truly related, can inform the development
about 0.6 percent—as much as six times greater than esti- of a new era in medicine. Our sameness can aid us in mak-
mates broadly agreed upon during the early 2000s, as the ing therapies that are specific to genomic profiles shared
Human Genome Project was wrapping up—and that there by groups of people.
is a lot of nuance tucked into these bits of variable DNA. To ensure that they do not repeat the shameful mis-
“As we obtained more whole-genome sequence data,” Lee use of science that plagued the study of human genetics in
writes, “we realized that individual genomes differed much decades past, researchers must communicate the unfurl-
more than we initially appreciated.” ing genomic landscape in a way that is clear and accurate.
So, modern genomic science is increasing our appre- Doing so will help science and medicine incorporate this
ciation of the uniqueness of the genetic blueprints that complexity and use it to everyone’s advantage. And paint-
build wonderfully individual human beings. We’re differ- ing a factual picture of our genetic commonalities and
ent. At the same time, cutting-edge research is reinforcing differences can ultimately assist humanity in celebrating
the fact that we humans share a remarkable similarity in both the ties that bind us and the uniqueness that makes
our DNA. We’re the same. each of us (at least potentially) wonderful. g
What we do with this knowledge is extremely impor-
tant. History is littered with examples of people and gov-
ANDRZEJ KRAUZE
ernments trying to tip the perceived balance between

humans’ biological sameness versus differentness in
their favor. Even today, after decades of widespread
misuses of genetic science, such as eugenics, there are Editor-in-Chief
voices that seek to misconstrue the degree to which eic@the-scientist.com
09. 201 9 | T H E S C IE N T IST 1 3

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QUOTES
Speaking of Science
1 2 3 4 5 6 7 These observations represent yet
another warning to translational
medical researchers that lab mice
8 9 are very imperfect predictors of
10
results’ translation to humans. But
there is a silver lining that, now, a
11 better mouse model is available to
12 13 confirm or refute promising results
Note: The answer grid will include every letter of the alphabet.
in traditional lab mice.

14 15 —Franck Carbonero, Washington State University
microbiome researcher, speaking to The Scientist about a
16 new study that used rodents with microbiomes similar to
those of wild mice to accurately predict the failure of two
17 candidate drug treatments in humans (August 1)
18 19 It makes me wonder if they’re

20 21 trying to make some sort of
intellectual claim rather than
move the science along, or
22 23 they’re trying to be the first,
generate hype, or somehow
BY EMILY COX AND HENRY RATHVON attract more money.
—New York University bioethicist Arthur Caplan talking
to Gizmodo about the recent announcement from an
international team of researchers who claim to have created
human-monkey chimeric embryos in China (August 2)
ACROSS DOWN
1. Saturniid with 1-Down wings (2 1. Shade of green
wds.) 2. Band sum for Texas armadillos
5. Hives, for example 3. Tiny 14-Across forms like nematodes
8. Job for one acquainted with 4. Zone straddling the equator
alpha-keratin 6. Relating to an organism’s bodily
9. Atacama rarity structure
11. Study of the safety or danger of 7. Apis mellifera member
doses 10 Yucca plant iconic in the Southwest
14. See 3-Down (2 wds.)
15. Hook-billed bird with a shrill cry 12 Opacity in the lens of the eye
17. Big family of Native American 13 Unbeatable feature of a wandering
languages albatross
20. Mountains where edelweiss is wild 16 Result of exposure to ultraviolet
21. Try to eye Rigel, say radiation
JONNY HAWKINS
22. Bluefin or skipjack 18 Typical spot for a vesper bat

23. Existing by nature rather than 19 Caterpillar’s cocoonlike home
nurture
Answer key on page 5
09.2019 | T H E S C IE N T IST 1 5
NEWS AND ANALYSIS
Notebook SEPTEMBER 2019
Impervious pepper. The mole rats turned out to be

similarly nonchalant when exposed to
BITE ME: Highveld mole rats are indifferent
to several chemicals that cause pain in other
DEWALD KLEYNHANS, UNIVERSITY OF PRETORIA
P
animals—an advantage when rooming with an
ain, unpleasant though it may be, dilute hydrochloric acid. “We wondered,
aggressive ant species.
is essential to most mammals’ sur- first of all, how they became insensitive
vival, a warning to back off before to these things,” says Lewin, who heads
we lose a limb or worsen a wound. So up a lab at Berlin’s Max Delbrück Center species, plus the more distantly related
it was curious when, in a 2008 study, for Molecular Medicine. East African root rat. They found that,
molecular physiologist Gary Lewin and The team took an evolutionary in addition to the naked mole rat, the
his colleagues found that, unlike most approach to finding the answer. Several Natal mole rat was insensitive to cap-
mammals, naked mole rats (Hetero- group members traveled to the naked saicin, while the Cape mole rat and
cephalus glaber) didn’t lick or flick a mole rat’s native territory of East Africa the root rat didn’t seem to feel a burn
limb that had been injected with a small to try out three common pain-causing from the hydrochloric acid. Most star-
amount of capsaicin—the hot in hot chili substances on seven other mole rat tlingly, one species, the highveld mole
rat (Cryptomys hottentotus pretoriae), Lewin and colleagues were left with these biting ants, I guess their best
didn’t flinch when injected with a few the question of why highveld mole rats option is to eventually become insensi-
milliliters of a highly diluted solution of had evolved not to mind such a nox- tive to their stings,” she says. Were they
an irritant present in mustard and was- ious substance. Daniel Hart, a post- to live in different environments with
abi known as AITC—an agent that even doc at the University of Pretoria who higher risk of predation or other harm,
the naked mole rat reacted to. trapped some of the animals used in “pain would actually be more of a bene-
When team member Karlien Debus the study, suggested an answer: high- fit to them and they might not have that
donned a gas mask to inject a similar veld mole rats frequently live in bur- advantage anymore.”
amount of 100 percent AITC under rows alongside Natal droptail ants The study’s combination of field-
the skin of a highveld mole rat, there (Myrmicaria natalensis), an aggres- work, molecular biology, and physiol-
was still no response. “Probably the sive species that chomps animals with ogy makes for “a fascinating story,” says
AITC was the most interesting because its mandibles and then injects them Paul Heppenstall, a neuroscientist at
AITC is a substance that actually every with a venom made in its abdomen. the European Molecular Biology Labo-
[other] animal in the entire animal Could the highveld mole rats’ surfeit ratory in Rome who did a postdoc with
kingdom avoids,” Lewin says. An elec- of NALCN ion channels in its dorsal Lewin but was not involved in the new
trophilic compound, AITC can cross- root ganglia be a way of enabling them study. The identification of the NALCN
link an animal’s proteins and damage to live comfortably alongside these channel as being involved in pain sens-
its cells. unpleasant roommates? ing, in particular, is novel, he says,
Intrigued, the researchers took a look at To find out, the researchers ground and blocking the channel with a drug
the highveld mole rats’ TRPA1 gene, which up the abdomens of Natal droptail ants to determine its involvement “was just
codes for the receptor that detects AITC, to make what Lewin terms “ant juice” such a clear, beautiful experiment that
and found some differences in sequence for use in a new pain test, and injected it showed that this was the main hinge
compared with the same gene in other into highveld mole rats. They also tried which these mole rats were using to reg-
rodent species. But they suspected these formic acid, which works similarly to ulate the pain sensitivity.”
differences couldn’t completely explain AITC and which many ant species use Indeed, the study’s authors suggest
the level of insensitivity they saw. So the for defense. Sure enough, the rodents that the channel, whose sequence is
researchers compared the sequences and remained stoic, while other mole rat highly conserved among mammals, could
expression levels of more than 6,000 of species reacted in the same way they be a target for the development of new
the highveld mole rat’s genes with those had to AITC. pain therapies. “It’s now actually quite
of the other, AITC-sensitive species the conceivable that you could design a drug
team had studied. One hit was a gene that that would bring NALCN channels more
codes for a sodium channel protein called We wondered, first of all, to the membrane in a mouse or human”
NALCN: while the sequence was nearly how they became insensitive to shut down pain responses, Lewin says.
identical among the species, the highveld to these things. Heppenstall agrees this is an exciting
mole rat was churning out far more RNA angle to explore, though he concedes it’s
—Gary Lewin, Max Delbrück Center for
transcripts from the gene in a pain-sensing Molecular Medicine not yet clear whether NALCN mediates
region of the spine known as the dor- types of pain beyond those caused by the
sal root ganglia. Unlike some ion chan- compounds tested in the study. “A big
nels, NALCN channels are always open, The researchers suspect that insen- question is: What else is [overproduction
Lewin says, allowing sodium ions into sitivity to the ants’ sting “would prob- of this channel] doing? . . . I don’t think
the cell. This means that in neurons that ably give this particular species an that they really answered that.”
overproduce the channel, “the membrane advantage because it was able, then, Jianguo Cheng, an anesthesiolo-
potential is slightly depolarized, and it’s to populate areas of Africa which are gist and pain researcher at the Cleve-
also harder for electrical stimuli to excite infested with the Natal droptail ant, land Clinic who was not involved in the
action potentials.” which other mole rat species would study, also sees potential in the NALCN
To test whether NALCN was really basically avoid,” Lewin says. finding for new, safer avenues toward
key to the species’ insensitivity to AITC, Lindsey Macpherson, a taste treating pain. “It’s exciting because it
the researchers injected the agent into researcher at the University of Texas demonstrates [that] evolution has fig-
highveld mole rats that had been given at San Antonio who has studied the ured out a way to render [a] certain
a drug to block the channel. These ani- TRPA1 protein but was not involved pain insensitivity that still enables the
mals displayed normal pain behaviors, in the study, agrees with that interpre- animals to survive and have a normal
confirming the channel’s role (Science, tation. “If they have to live there and function.”
364:852–59, 2019). they have no choice but to interact with —Shawna Williams
09. 201 9 | T H E S C IE N T IST 1 7

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the_scientist_magazine
Stellar ism,” he says, “because we had to get from

one tree to another.”
Johannesburg, writes in an email to The
Scientist. But gathering convincing evi-
Influence? In May, Melott and Brian Thomas of

Washburn University in Topeka, Kan-
dence to support any bipedalism hypoth-
esis is extremely difficult, he notes.
Millions of years ago, hundreds of light- sas, published a paper detailing the pro- Owen Lovejoy, an anthropologist at
years from Earth, massive stars started to posed connections between these stellar Kent State University in Ohio, is more
explode. As they died, the stars spewed explosions, which started roughly 7 mil- blunt, calling the hypothesis both “bizarre”
energetic particles into space, many of lion years ago in a nearby part of the Milky and “weird.” It doesn’t match the fossil
which rocketed toward Earth, tearing Way, and the bipedalism transition (J Geol,
through the atmosphere and causing a 127:475–81, 2019). The authors’ argument
surge in the electric charge of the layers of ties in with an existing idea, known as the
Bipedalism is a horrible
air closest to the planet’s surface. Research- savannah theory, that a change from forest
ers studying the Earthly effects of these to grassland could have spurred our hom- form of locomotion. We are
stellar explosions hypothesize that this inin ancestors in Africa to stand up to see miserably adapted to it,
electric boost triggered lightning storms what tree to walk to next and to check for even after 6 million years of
and sparked wildfires that burned the for- predators that might be lurking among the doing it.
ests of Africa, turning them to grasslands. grasses. By providing data on what caused —Owen Lovejoy, Kent State University
One of these researchers, University our ancestors’ landscape to change, the
of Kansas physicist Adrian Melott, says team’s study helps flesh out this particular
he thinks that these lightning storms and hypothesis, Melott says. record and can’t account for just how signif-
wildfires also could have played a role in The study is just the latest of many to icant an adaptation bipedalism was at the
hominins’ transition to walking on two feet. tackle the origins of bipedalism—but it time it arose, he says. According to the fos-
According to Melott, supernovae-induced hasn’t particularly convinced anthropolo- sil record, hominins were definitely walk-
fires would have left just a few trees scat- gists. “It is an interesting hypothesis,” Ber- ing upright by the time of Australopithecus
tered across the landscape. For our tree-liv- nhard Zipfel, an expert on human evolu- roughly 4 million years ago, and possibly
ing ancestors, “[this] encouraged bipedal- tion at the University of the Witwatersrand, as early as the time of Sahelanthropus, 7
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09. 201 9 | T H E S C IE N T IST 1 9

NOTEBOOK
million years ago. And many of the fossils dence that early bipedal hominins may not covery when she went to check on the ani-
showing evidence of bipedalism have been have been monogamous, and there may mals. Although all of the nearly 100 boxes
found alongside those of woodland animals, be more than one reason for hominins to she’d installed several months earlier in
suggesting that the behavior evolved while have stood up and started walking. In his the Little Tennessee River watershed were
our hominin ancestors still lived in forests. mind, still, “the drivers of human bipedal- still in place, not one housed a hellbender.
This evidence would rule out the savan- ism remain largely elusive.” Closer investigation revealed the popula-
nah theory and, in turn, the supernova- —Ashley Yeager tion was gone.
lightning hypothesis, he says. Hellbenders inhabited these streams
But if bipedalism evolved before our as recently as 2015. Nobody knows exactly
ancestors left the forests, what might have
driven its spread? Part of the reason anthro- Help for when they left or why, but Diaz fears they
died out. Because hellbenders only live in
pologists and evolutionary biologists have
trouble coming up with answers is that, Hellbenders fast-moving, clean mountain streams, she
speculates that changes in land use around
from many perspectives, bipedalism “is a When Clemson University master’s stu- the Little Tennessee River might be affect-
horrible form of locomotion,” Lovejoy says. dent Lauren Diaz set out to study the ing the water quality, although she hasn’t
“We are miserably adapted to it, even after ecology of hellbender salamanders (Cryp- yet tested that hypothesis.
6 million years of doing it.” Walking upright tobranchus alleganiensis) in the streams This population’s disappearance is just
can be slow and unsteady, and it leaves us of western North Carolina, she anticipated one of the latest blows for the salamanders,
prone to falls. For bipedalism to take hold, some challenges. Although they can grow whose numbers are believed by research-
he says, it had to have huge advantages. to two feet long, hellbenders—also known ers to be in decline across their range from
Researchers have proposed several as snot otters, devil dogs, and Allegh- New York to Alabama and Mississippi and
ideas, ranging from better presentation of eny alligators—are difficult to find in the as far west as Missouri. To the surprise
sexual display signals to the ability to wade wild due to effective camouflage and their of many biologists and conservationists,
in water to improved vigilance. Bipedal- habit of hiding under rocks. And it’s not however, the US Fish and Wildlife Service
ism could also have helped ancient homi- unusual for the nest boxes that research- in April denied the eastern hellbender (C.
nins find and transport food, an idea sup- ers install as habitat for the amphibians to alleganiensis alleganiensis) federal pro-
ported by the observation that chimpanzees get washed away or blocked by sediment. tection under the Endangered Species Act.
stand up and walk on two legs when they But last spring, Diaz made a startling dis- The agency cited a paucity of data: there
are carrying rare and coveted food, freeing
their hands to hold more of it (Curr Biol,
22:PR180–R181, 2012).
Lovejoy supports this hypothesis, but
says he thinks the story might be more
complex. He and his colleagues propose
hominin bipedalism arose as part of a
reproductive adaptation, perhaps as our
ancestors evolved from polygamous soci-
eties toward more monogamous ones
(PNAS, 115:E1108–16, 2018). The fossil
record shows that, around the time that
researchers think the transition to biped-
alism was happening, apes seemed to be
evolving traits associated with less aggres-
sion, such as smaller canine teeth. Lovejoy
says he thinks that this was partly due to
sexual selection by females for males that
were better providers instead of better
aggressors. Because walking upright might
have allowed males to carry more food, this
sexual selection therefore could also have
LAUREN DIAZ
I AM A ROCK: Hellbenders are difficult enough to study

favored bipedalism. when they’re adults (above). Hardly anything is known
It’s an interesting hypothesis and has about their lives as larvae (inset).
merit, Zipfel says. However, there’s evi-
wasn’t enough information available about hood, but we don’t understand the mecha- the North Carolina Wildlife Resources
the past and current status of hellbenders nisms of why these occur,” says Jachowski. Commission, is pursuing research into the
to make an informed assessment. artificial shelters Diaz used in the Little
Diaz, along with her advisor, Clem- Tennessee River, both as a monitoring tool
son University ecologist Cathy Bodi- When a stream is full of and as habitat enhancement. Essentially
nof Jachowski, is working to address shoebox-sized concrete containers with
sediment, and water quality
this data deficiency while investigating a tunnel, these shelters mimic the natu-
what’s causing hellbender populations to
declines, it’s not just hell- ral crevices that hellbenders use as homes
suffer. For example, Jachowski recently benders that suffer. and nests while also giving researchers
completed a study examining how land —Cathy Bodinof Jachowski easy access via a removable lid on top.
Clemson University
use upstream from hellbender habitat “Lifting rocks is an effective way to
influences abundance and survival of find hellbenders, but we’re increasingly
the animals (Biol Conserv, 220:215–27, aware that it can actually damage the
2018). She found that forest cover in the Herpetologist Brian Miller of Mid- microhabitat beneath,” says Diaz. “With
upstream watershed was associated with dle Tennessee State University has also the shelters, we can just lift the lid off to
more hellbenders downstream. observed declines in hellbender popula- find them instead of altering the habi-
What’s more, in heavily forested water- tions. He agrees with Diaz that a decrease in tat they’re using.” Diaz is now comparing
sheds, hellbenders were not only more water quality may be a contributing factor, different designs to identify which offer
abundant, but their populations showed and also proposes a potential role for dis- the best resilience to disturbances such
higher rates of reproduction and greater eases such as the fungal disease chytridio- as flooding, are least likely to be buried
numbers of young animals reaching adult- mycosis. But he emphasizes that “the cause by sediment, and are most attractive to
hood. “It seems that animals in degraded or causes of the declines are unknown.” home-seeking hellbenders.
watersheds have trouble reproducing, or To better investigate what’s going on, The team is also ramping up research
young animals are not surviving to adult- Jachowski’s laboratory, with funding from on larval hellbenders, which are only two
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NOTEBOOK
inches long when they leave their natal In 2011, Srivastava and collaborators The researchers decided to experi-
nest. “Larval ecology of hellbenders is like used whole-exome sequencing to search mentally investigate whether the com-
a black box of research,” says Kirsten Hecht, for genetic variants in Tatiana, by then bination of the parents’ mutated genes
a doctoral student at the University of Flor- three years old, and her parents. It turned might be responsible for the children’s
ida and one of the few scientists study- out that Tatiana’s father had mutations in symptoms. They used CRISPR technol-
ing larval hellbenders, although she isn’t MYH7 and MKL2 (also known as MRTFB ogy to recreate the same genetic muta-
involved in the Clemson University group’s or myocardin related transcription factor tions in mice and found that, although
work. “We’re trying to conserve this animal, B), genes important for heart and muscle animals with just one of the mutations
but there are five to seven years of its life development. Her mother, meanwhile, had normal phenotypes, mice with
that we know very little about.” had a variant of the NKX2-5 gene, which all three mutations had heart pathol-
Diaz says she and Jachowski hope to encodes a cardiac-specific protein involved ogy similar to the children’s (Science,
identify the resources young hellbend- in regulating embryo development. Each 364:865-870, 2019). The team also
ers need, information vital to help guide mutation led to a single amino acid differ- created induced pluripotent stem cells
monitoring efforts, stream restoration ence at the protein level. from the parents and Tatiana, and dif-
attempts, and potential reintroductions. The researchers examined the parents’ ferentiated them into beating heart
In the meantime, conservation efforts hearts and found that the father showed cells in vitro. When compared with cells
should focus on the animals’ habitat, says subtle signs of lowered heart function, from the father or mother, cells derived
Bill Hopkins, an ecologist at Virginia Tech while the mother was apparently unaf- from Tatiana’s tissue showed reduced
who supervised Jachowski’s PhD. “I think fected by her mutation. The research- adhesion to the cell-culture dish, along
one of the most important things that ers also used whole-exome sequencing to with lowered expression of adhesion-
can be done is addressing landscape-level scan the genomes of Tatiana’s older sis- related genes and higher expression of
changes that affect stream quality, sedi- ter, Anna, and of tissue from the failed genes associated with immature heart-
mentation in streams, and the physical pregnancy that was sampled during an cell stages.
microhabitat of streams,” he says. autopsy. They found that all three chil- It seems that the genetic muta-
Jachowski says that even if people dren had inherited all three genetic mutation the children inherited from their
don’t view hellbenders as beautiful crea- tions—and, on further examination, that mother acts as a modifier for the father’s
tures the way she does, the amphibians are they all had similar heart problems. Each mutations, says Srivastava. The NKX2-5
still valuable as indicators of stream health child’s left ventricle had failed to mature variant appears to have exacerbated the
and water quality. “When a stream is full properly, and their hearts were thus abnormal development caused by the
of sediment, and water quality declines, unable to pump blood effectively. father’s mutations, leading to a pheno-
it’s not just hellbenders that suffer,” she
says. “In that sense, hellbenders are a sen-
tinel of environmental quality. If they are
declining, that should send off warning
bells to all of us.” —Mary Bates
Complex
Inheritance
In 2008, pediatric cardiologist Deepak
Srivastava treated a newborn baby who
suffered from acute heart failure and
had to be put on life support. At the time,
the Gladstone Institutes physician-
researcher didn’t know what had caused
© ISTOCK.COM, SUDOK1
the emergency. But when he found out

that the baby’s parents had previously
lost a child at 24 weeks into the preg-
nancy, Srivastava became suspicious
that there was a genetic component to
the disease.
type that is more severe in the children ease mechanism, which I think will be Each child’s left ventricle had
than in either of the parents. very important,” as it could guide the failed to mature properly,
The phenomenon of a handful of development of therapeutics. and their hearts were thus
genes together determining a phe- With advances in techniques such as unable to pump blood
notype is known as oligogenic inher- gene editing, researchers will increas-
effectively.
itance, and it’s not a new theory for ingly be able to focus on the functional
disease mechanisms. Traditionally, effects of mutations in multiple genes,
“we’ve be able to understand human says Nicholas Katsanis, a geneticist at ics approaches are able to identify genes
disease through [single gene] disor- Lurie Children’s Hospital and North- that have strong effects on phenotype,
ders that are relatively rare but eas- western University who was also not says Kim, but struggle to detect those
ier to detect,” says Srivastava. Yet the involved in the work. “As more papers with subtle effects that are common in
fact that many genetic variants are not like this one come out, we’ll all come to oligogenic disorders.
deterministic of disease “would sug- accept the idea that you cannot explain To test more combinations of gene
gest most disease is a combination of a disease by a single allele of a gene.” variants, Srivastava and collaborators
genes,” he tells The Scientist. Picking apart multiple genes’ effects are now developing CRISPR-based
“What is important here is that is challenging, however. With some dis- single-cell technologies to help them
they used different techniques to show eases, the genes involved could num- assay hundreds of gene variants in
that this is really oligogenic inheri- ber in the dozens or more. “We are not thousands of cells. Ultimately, says
tance,” says muscle researcher Nyam- able to experimentally validate all pos- Srivastava, identifying additional
khishig Sambuughin of the Uniformed sible candidates for such oligogenic genetic modifiers like the mother’s
Services University of the Health Sci- inheritance patterns,” says Artem Kim, variant in this study may be key to
ences who was not involved in the a geneticist and bioinformatician at understanding and developing thera-
study. “It not only explains genetic Université de Rennes who did not take pies for oligogenic diseases.
mechanism, but it also explains dis- part in the work. Current bioinformat- —Chia-Yi Hou
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CRITIC AT LARGE
Diversify Our Human Genomic Data

To better understand the biology of our species and to make individualized medicine accessible
to all, we need to consider the comprehensive genetic makeup of a wider swath of humanity.
BY CHARLES LEE
S
ince the Human Genome Project was completed, scien-
tists around the world have worked tirelessly to populate the
sequence and variant databases that have become the crown
jewels of genomics research. These databases are now brimming
with genomic information, but unfortunately, they are greatly biased
towards individuals of European descent. For example, 70 percent
of the data stored in the Genome-wide Association Study (GWAS)
Catalog, a publicly available resource that contains manually curated
array-based data from more than 2,800 published studies, is from
individuals of European descent. The other 30 percent comes from
individuals with Asian ancestry. Similarly, the database of Geno-
types and Phenotypes (dbGaP) and the Genome Aggregation Data-
base (gnomAD) are lacking data from individuals hailing from the
Middle East, Central Asia, Oceania, and Africa.
European countries such as Iceland, Estonia, and the UK are are accompanied by demographic and/or phenotypic data, this ano-
among the first to launch countrywide whole genome sequenc- nymization is not 100 percent effective. Second, there are inconsis-
ing efforts. Hence, it’s no surprise that these databases are skewed tent policies surrounding genomic data sharing. The Fort Lauderdale
accordingly. What matters now is that we recognize this as a prob- Agreement supports free and unrestricted use of genomic sequencing
lem that needs to be addressed. data prior to scientific publication. On the other hand, some fund-
This is not just an academic issue. People whose lineages are well ing entities restrict genomic data sharing in the interests of national
represented in genomic databases will benefit the most from preci- security.
sion medicine approaches that rely on genetic markers to diagnose,
understand, and treat a patient’s disease. Individuals from underrep- New references
resented ethnicities are more likely to get inaccurate diagnoses from The human reference genome represented a major achievement,
genetic tests, which may have negative consequences. For instance, the and thanks to ongoing improvements, continues to be the founda-
identification rate of genetic pathogenic variants for nonsyndromic tion for much of biomedical research. Scientists built this reference
hearing loss (NSHL) has consistently been lower in people of African genome with about two-thirds of the data coming from one individ-
ancestry than in European individuals because the lack of data limits ual’s genome—ironically an African American—during a time when
clinical geneticists’ ability to diagnose this condition in patients of Afri- it was thought that human genetic diversity was much more limited.
can ancestry. Similarly, researchers recently showed that a genetic test We learned a tremendous amount from sequencing and assem-
for cardiomyopathy yielded six false positive results among approxi- bling that first-ever human reference genome, and technical improve-
mately 230 patients of African ancestry because the variants in ques- ments in sequencing technology subsequently made it easier to
tion were more common among healthy control individuals of African determine the DNA sequences of many additional individuals. As we
descent than among healthy control individuals of European descent. obtained more whole-genome sequence data, we realized that indi-
Of course, the availability of sequencing data from diverse human vidual genomes differed much more than we initially appreciated.
populations depends on the regular deposition of DNA sequences Indeed, the sequencing and de novo assembly of a Korean individual
from individuals of different populations in shared and open data- was used to fill more than 100 gaps in the human reference genome
bases. However, there can be several impediments to open sharing and identify many variants specific to people of Asian descent.
of such data. First, while most DNA sequencing data obtained these As more genomes from different populations are sequenced,
© ISTOCK.COM, GRIVINA
days are from research programs enlisting properly consented indi- population-specific reference genomes are subsequently being devel-
viduals, it is estimated that by 2022, more than 80 percent of human oped. For example, scientists in Japan have integrated three newly
genome sequence data will ultimately come from health-care sys- assembled genomes into a population reference with detailed analy-
tems. This will bring technical challenges in accessing data from dis- ses of genetic variants. These, and other new genome assemblies, will
parate sources and respecting regulatory barriers: patient protection serve as excellent foundations on which to establish individualized
is facilitated by anonymizing sequencing data, but when these data medical treatments for those populations.
Diversifying types of genetic variants
Do
Just as we must look at genomes broadly across populations, so
too must we look broadly at variant types within and between
world populations. Structural variants (SVs)—which include dele-
tions, duplications, insertions, inversions, and translocations—are
what
important for precision medicine, but our ability to identify classes
of SVs has consistently lagged behind our ability to identify the eas-
ier-to-find single nucleotide polymorphisms (SNPs).
Fortunately, that’s starting to change. For example, my team
matters
previously worked with scientists on the 1000 Genomes Project. Its
goal was to catalog 99 percent of the SNPs seen in at least 1 per-
cent of 26 world populations (6 populations from the Americas—
including 2 African-descendant populations—5 populations from
for
Africa, 5 European populations, 5 South Asian populations, and 5
East Asian populations). In total, the project sequenced and analyzed
the genomes of 2,504 individuals (about 100 genomes from each of
the chosen populations) and identified more than 88 million vari-
your
ants, including 60,000 SVs.
As we further understand the extent of human genetic diver-
sity, it will be critical that more researchers consider this variation
when designing studies to test and model human diseases. Experi-
matter
menting on cells from an individual of one ethnic background lim-
its the conclusions to individuals of that population. Establishing a
standardized and well-characterized panel of induced pluripotent
stem cell lines, made from individuals representing myriad popu-
lations, could be an excellent resource for recapitulating human
genetic diversity in future experimental work. The use of differ-
entiated cells from genetically diverse individuals could help us
Neurological research relies heavily
determine which populations a particular conclusion applies to.
Similarly, when conducting in vivo experiments, researchers
on quality antibodies for isolation,
should use animals with different genetic backgrounds to more characterization, and localization of
fully represent genetically diverse humans, their complex traits, target proteins. Choose from our
and their susceptibility to diseases. In mice, the “collaborative extensive collection of multi-assay
cross” and the “diversity outbred” panels (developed in part by, and validated neuroscience antibodies to
available from, the Jackson Laboratory) are being used to show a
propel your research further than
wide variety of phenotypic outcomes for a given mutation depend-
ing on genetic background, and can be a powerful platform for ever before.
identifying mutation modifiers.
The lack of diversity in our databases and genomic studies Learn more at: rockland-inc.com/neuro
should be considered a problem for the entire life-science commu-
nity, and together we must strive to address it—not just by sequenc-
ing more people of different backgrounds, but by taking such vari-
ation into account in all our future research. I urge scientists to
face the challenge and consider how they can contribute to a more
comprehensive solution that will prevent persistent inequality in
the availability of individualized medicine. g
Charles Lee is director of the Jackson Laboratory for Genomic

Medicine in Farmington, Connecticut. He is also a distin-
guished professor at Ewha Womans University in South Korea
as well as at the First Affiliated Hospital of the Xi’an Jiaotong
University in China. Lee is currently president of the Human
Genome Organization.
MODUS OPERANDI
Optogenetics Sans Genetics

DNA-coated gold nanorods enable cells to be activated by light without genetic manipulation.
BY RUTH WILLIAMS
Nanorods
O
Injured muscle
ptogenetics involves engineering
cells to make them light-responsive
and then using a laser to control their
activity—whether in a dish or a live animal. As
a research tool, optogenetics is unquestionably Differentiating
muscle cell MET dimer
powerful. But the technique requires genetic complex
manipulation, which makes it less favorable for MET protein
certain clinical uses.
“[Optogenetics] is super cool for animal
work,” says Marta Cerruti, who studies
biosynthetic interfaces at McGill University
in Montreal, “but I think it would be more
easily ethically approved” to control cells if SHINE A LIGHT ON ME: Aiming an infrared laser beam at the site of a muscle injury on the leg of a
it didn’t include genetic tinkering. In devising mouse activates DNA-loaded gold nanorods that have been injected into the tissue, spurring growth
and differentiation of muscle cells. The laser beam heats the nanorods, thereby unzipping the double-
a new technique involving gold nanorods,
stranded DNA helices bound to them and releasing a single-stranded DNA targeted to the receptor
biochemical engineer Zhou Nie of Hunan protein MET. When this DNA binds to MET receptors on the muscle cells, the complex triggers
University in China and colleagues were able downstream signaling, which in turn activates the growth of new muscle tissue.
to control cells with light but without genetic
manipulation. “That’s what I thought was from its gold-bound sister, is designed to bind the rods induced MET signaling as well as
really interesting,” says Cerruti. and activate a particular cell-surface receptor. migration and proliferation in cultured cells,
Nie knew gold nanorods had potential. After delivering the rods to cultured cells or the researchers injected the nanorods into
These nanometer-sized particles heat up injecting them into animals, the particles the leg muscles of live mice that had been
when irradiated with near-infrared light, and are hit with infrared light, which heats and injured with liquid nitrogen. Because infra-
this photothermal response is already being denatures the double helix, releasing the red light has a slightly deeper tissue penetra-
explored for, among other things, light- unfused strand to do its work. tion than the blue light commonly used for
controlled drug release for cancer therapy. In proof-of-principle experiments, optogenetics, the researchers were able to
Nie and colleagues decided to use the same Nie’s team loaded nanorods with a DNA use external lasers to activate the nanorods,
approach, but instead of loading their sequence designed to activate the MET pro- which were approximately 5 mm below the
nanorods with drugs, they packed them with tein, a receptor present on certain stem and skin. Simply focusing a laser beam at the
double-stranded DNA. progenitor cells that drives cell migration animals’ damaged limbs stimulated the pro-
The tail of one strand is fused to the gold and proliferation, among other processes. duction of new muscle cells. (Nano Lett, 19:
particle. The other strand, when released After showing that infrared irradiation of 2603−13, 2019) g
AT A GLANCE
CELL MANIPULATION HOW IT WORKS IN VITRO AND GENETIC MANIPULATION CELL TYPES
TECHNIQUE IN VIVO USE? NEEDED?
Optogenetics A light-sensitive ion channel from green algae, archaea, or bacteria Yes Yes Excitable cells
is expressed on the surface of an animal cell. Exposure to light allows
© GEORGE RETSECK
ions to flow into the cell, activating it.
DNA aptamer–coated Gold nanorods are coated with a receptor-specific DNA strand Yes No MET-expressing cells tested
nanorods kept inert via hybridization to a complementary sequence. Infra- so far, but in principle any cell
red light heats the nanorods, unzipping the DNA and releasing type can be targeted with a
the receptor-targeting strand to induce downstream signaling. custom DNA strand
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The Doping
Arms Race
Athletes have devised ways to outsmart tests for performance-boosting
drugs, but scientists are working hard to get ahead.
BY ANNA AZVOLINSKY
W
hen he worked for the Cali- that year’s Winter Olympic Games, a local Pearlmutter. “It’s the reason why the PCC
fornia-based biotechnology lab used the test to analyze athletes’ samples, exists. If doping were static then anti-doping
company Amgen in the 1990s, and late in the Games, three skiers—Johann could be static.”
Steve Elliott was instrumental in the devel- Mühlegg of Spain and Olga Danilova and
opment of darbepoetin alfa (Aranesp), a Larissa Lazutina of Russia—tested positive Establishing a baseline
drug to treat anemia in people with chronic for darbepoetin and were disqualified. For athletes who want to cheat, the most
renal failure. Approved by the US Food and But the number of athletes charged with attractive substances are those that are
Drug Administration in 2001, darbepoetin darbepoetin doping dropped off quickly already found in the body. Besides EPO,
is a second-generation version of recombi- after that, as some athletes switched to blood these include growth hormone, steroids
nant human erythropoietin (EPO) puri- transfusions or started repeatedly taking such as testosterone, and blood transfusions.
fied from Chinese hamster ovary cells that much smaller amounts of darbepoetin, called Because exogenous and endogenous biomol-
express the modified gene. microdoses, to keep levels below detectable ecules can be challenging to distinguish and
EPO is a cytokine produced by the kid- limits—a strategy they already employed for because there is a wide range of “normal” lev-
neys that sends a signal to the bone mar- other forms of recombinant EPO. Another els for these compounds and their associated
row to increase production of red blood cells approach to hide their drug use was to infuse metabolites, these agents present a particular
(RBCs). For anemic patients, the drug can recombinant EPOs directly into circulation, challenge to anti-doping watchdogs.
be lifesaving. But for people with normal red rather than injecting it below the skin, so
blood cell counts, it offers something else: a that the molecules are cleared more quickly
boost in physical performance by increasing from the body. Athletes also started supple- A common misconception is
the amount of oxygen the blood can carry menting their recombinant EPO molecule of that when an athlete’s sample
to muscles. As Elliott his colleagues devel- choice with iron, which is a key component is collected that it’s ana-
oped the drug, “it was apparent that Aranesp of hemoglobin. The additional iron increases
lyzed with a simple dipstick
would be used for doping,” he says. the effectiveness of the EPO, meaning that
Sure enough, after darbepoetin was an athlete can take lower doses. The iron also
and that it’s either positive or
approved by the US Food and Drug Admin- masks EPO-related biomarkers that are used negative.
istration (FDA), it became a tool for perfor- as indirect tests of EPO use. —Matt Fedoruk, US Anti-Doping Agency
mance enhancement among some athletes. Today, “we see that no one is taking just
Possibly assuming that a test to detect it erythropoietin,” says Michael Pearlmutter,
would not yet be available at the 2002 Win- executive director of the nonprofit research “For most of the tests, there is quite a
ter Olympics in Salt Lake City, Utah, some collaborative Partnership For Clean Compe- lot of variability in the biomarkers from one
athletes dosed themselves with darbepoe- tition (PCC), for which Elliott now consults. individual to another,” says endocrinologist
tin leading up to and during the Games. But “They are combining it with iron or other Richard Holt of the University of South-
there was a test. Elliott and his colleagues had substances, which makes the doping more ampton in the UK.
developed the detection method in parallel difficult to detect.” The problem for athletes— To make the testing results for steroids
with the drug’s coming to market, but they an opportunity for anti-doping labs—is that and other doping agents less ambiguous
did not publicly announce the test’s existence taking iron alters blood-borne biomarkers and to allow for more effective and sensitive
prior to the start of the Games. of EPO use that officials are now testing for monitoring, the World Anti-Doping Agency
Compared to recombinant human EPO alongside the various forms of the recombi- (WADA) introduced the Athlete Biological
already on the market for medical use, the nant hormone itself. Passport (ABP) to track biomarkers in indi-
protein that Elliott and his colleagues had And so continues the cat-and-mouse vidual athletes over the long term. In 2009,
engineered had a longer half-life, allowing game between athletes and anti-doping offi- WADA formally launched the ABP’s blood
for less-frequent dosing of anemia patients. cials. As athletes experiment with drugs that module, which tracks markers of blood dop-
This feature also gave anti-doping enforcers can help give them an edge over their coming in circulation. Levels of hemoglobin, red
a leg up, because the molecule lingered for petition, they tweak their strategies to try blood cells, and other blood-borne markers
longer in the body, giving them more time to to evade detection. Meanwhile, anti-dop- of EPO and whole-blood doping are moni-
detect it. Moreover, the unique combination ing researchers are continuously develop- tored. In urine samples, peptide hormones,
of sugar molecules on the surface of darbepo- ing assays and other methods that are more including growth hormone, are also tracked.
© ISTOCK.COM, SIMONKR
etin distinguished it from endogenous EPO sensitive to low levels of doping agents that Five years later, WADA added the
and from other recombinant EPOs. accumulate in urine or blood, or that extend steroid module to the ABP: urine tests
In 2002, an anti-doping lab at the Uni- the detection window to secure a positive test to specifically check for testosterone and
versity of California, Los Angeles, deployed from a cheating athlete. other steroid doping by measuring not
the urine-based test, which could detect dar- “Athletes are continuing to become more only the steroids themselves, but also
bepoetin’s distinct glycosylation profile. At sophisticated in their doping efforts,” says immediate precursors or derivatives, and
09. 201 9 | T H E S C IE N T IST 3 1

secondary molecules such as metabolites “A common misconception is that when tute in Sweden have demonstrated that,
that fluctuate with steroid levels. An ath- an athlete’s sample is collected that it’s ana- by establishing a baseline, the ABP could
lete’s passport is flagged for further eval- lyzed with a simple dipstick and that it’s either enable detection of a single 100-mil-
uation, and possibly more testing, when positive or negative,” says Matt Fedoruk, ligram dose of testosterone gel to the
levels of these biomarkers are outside of scientific director at the US Anti-Doping skin—what would be considered a micro-
the athlete’s 99.5th percentile reference Agency (USADA). “Rather, we are collecting dose—as long as seven days after appli-
range, calculated with a mathematical blood and urine, and there is a high level of cation in healthy male volunteers.1 In
model that is based on a population of sophistication that is applied to detect hun- another study, Mullen’s team found that
healthy individuals and an individual’s dreds of prohibited substances. The analyses injection of a 125-milligram microdose of
own results. As an athlete accumulates are quite challenging and complex.” testosterone enanthate, a drug intended
CREDIT LINE
more test results, her individual refer- Anti-doping researcher Jenny Mullen to boost levels of testosterone among
ence range narrows. and colleagues at the Karolinska Insti- males with low levels of the steroid, could
THE DOPING-
DETECTION
PASSPORT
The World Anti-Doping Agency recently
developed and instituted the Athlete
Biological Passport (ABP), a tools
that helps anti-doping organizations
track levels of various molecules in
competitive athletes. In 2009, WADA
published formal guidelines on how
to conduct standardized testing for
evidence of blood doping, including how
to track athletes’ longitudinal data as
part of the ABP’s blood module, which
includes 14 bloodborne biomarkers to
indicate misuse of erythropoietin (EPO),
blood transfusions, or other forms of
doping using blood samples.
In 2014, WADA added the steroidal
module to track the levels of testosterone
and other steroids. These sets of tests
can detect exogenously administered
steroids, their various metabolites,
precursors, and related molecules to
better nail down doping as well as other
anabolic agents. Tracking these markers
over time is also a way to identify
samples that may have been tampered
with or exchanged with the urine sample
of another individual, as happened with
the Russian Olympic team during the
2014 Winter Olympics in Sochi.
be detected using the same methods for Elusive testosterone exogenous testosterone. Precursor levels rel-
as long as eight weeks.2 In addition to tracking biomarker levels in ative to the levels of testosterone in the body
“WADA has rightly set the bar very athletes over time, anti-doping officials are over time can indicate if something unnatu-
high for [doping] detection, but that working to understand what the relative lev- ral is going on. In 2007, researchers devised
means we don’t always have the sensi- els of various testosterone precursors and a statistical technique to detect individuals’
tivity we would like for our tests,” says related markers can tell them about an ath- abnormal steroid values,3 and WADA added
Holt. “The ABP is a mechanism by which lete’s normal physiological levels and doping the steroid module to the ABP in 2014.
athletes who travel around the world to status. Such comparisons are critical to the But there can still be some ambigu-
compete can be tested by different labs, identification of exogenous testosterone use: ity as to what these test results mean. If the
and those results are comparable among numerous precursors of the steroid hormone, variability of steroid biomarkers or their
those labs and centrally stored as part of as well as epitestosterone, an isomer of tes- ratios are suspicious to the anti-doping sci-
the athlete’s history.” tosterone, are suppressed in the presence of entist doing the assessment at a WADA-
ERYTHROPOIETIN (EPO)
A circulating glycoprotein cytokine and essential hormone for red blood cell production in the bone
marrow. In response to cellular hypoxia, the kidneys and liver secrete EPO. Doping with recombinant EPO
or other agents that stimulate the production of red blood cells increases the availability of oxygen to
muscles, which boosts performance and enhances aerobic power for endurance sports.
Testing for EPO is part of the blood module: Detected in urine by distinguishing the glycosylation profiles
of naturally occurring EPO from the various versions of recombinant EPO using protein electrophoresis.
Also detected indirectly in the blood by measuring related markers including hemoglobin, hematocrit, and
reticulocytes.
BLOOD TRANSFUSIONS
Similar to EPO, blood transfusions increase the availability of oxygen to the muscles to boost performance and enhance aerobic power
for endurance sports by increasing the amount of oxygen-carrying hemoglobin in the body. Transfusions can be with an individual’s own
blood, known as an autologous transfusion or a homologous transfusion using donor blood. Monitoring for blood abnormalities due to
blood transfusions is another part of the blood module.
Testing for homologous blood transfusions: A blood test looks for multiple red blood cell populations by identifying different antigen molecules.
Testing for autologous transfusions: Available tests are imperfect and indirectly measure biomarkers such as the number of immature
red blood cells known as reticulocytes. Plasticizer tests can also be performed to look indirectly for the remnants of plastics used in blood
storage bags.
TESTOSTERONE
A steroid hormone taken by athletes to enhance protein synthesis and muscle growth. Testosterone is
naturally synthesized from cholesterol in men and is present in smaller amounts in women.
Testing for testosterone is part of the steroid module: Typically measured in the urine in relation
to related molecules such as the testosterone isomer epitestosterone. The naturally occurring ratio
of testosterone to epitestosterone is about (1:1). When athletes fall outside of their normal range,
labs conduct further analysis using isotope-ratio mass spectrometry to confirm exogenous steroid
administration. Four other testosterone-related markers are tested as part of the steroidal module.
THE SCIENTIST STAFF
GROWTH HORMONE (GH)
A naturally occurring peptide hormone made by the pituitary gland that boosts muscle strength, can
increase sprint capacity, and is thought to help muscle recovery.
Testing for GH is not currently part of the ABP, but an endocrine module is currently in development:
A blood-based isoform test detects the ratio of various GH isoforms. A second blood test measures
two GH-related biomarkers that increase following a GH dose. These two tests, along with measures
of additional biomarkers, will form the basis of the future ABP endocrine module.
certified lab, or if steroid biomarker values new detection strategies are of utmost impor- growth hormone positives globally, further
are too high based on set rules set by WADA, tance,” says Fedoruk. research into increasing the sensitivity of cur-
the testing authority can order a pricier but Adaptability is also the strategy for offi- rent growth hormone detection methods is
definitive test that combines mass spectrom- cials deciding which Olympics-bound ath- warranted,” says Fedoruk.
etry and gas chromatography to measure the letes to test for doping agents and how often. To overcome detection challenges, Holt
ratio of two carbon isotopes, carbon-12 and “All testing is intelligence-based and strategi- led the development of a second test to
carbon-13, in urine samples. These carbon cally-driven to maximize the deterrence and indicate the hormone’s misuse. Called the
isotopes in endogenous testosterone come detection of performance-enhancing drugs,” GH-2000 score, it relies on immunoassays
from the individual’s diet and differ from the says Fedoruk. The USADA relies on recent and mass spectrometry methods to measure
carbons found in lab-made hormone, which doping trends, the history of doping in a spe- levels of two proteins—insulin-like growth
has a lower carbon-13:carbon-12 ratio. If an cific sport, the ABP, and tips received on pos- factor-1 (IGF-1) and N-terminal propep-
athlete’s sample exceeds WADA-determined sible doping situations to determine the tests tide of type III collagen (P-III-NP)—that
thresholds, they are considered to have tested athletes undergo. increase following growth hormone dos-
positive for testosterone doping. ing. The test is approved by WADA and was
Growth hormone launched in the run-up to the 2012 London
Another naturally occurring molecule Olympic and Paralympic Games.
To have a conclusive test, abused by athletes is human growth hor- But this biomarker test hit a manufac-
we need to make sure the mone, which naturally increases in the turing setback when one of the makers of
body as a result of exercise, can make mus- one of the assays withdrew its kit from the
markers we are measuring
cles grow, and allows faster recovery after market. So Holt and others are now work-
are not affected by training exertion. If taken during training, recom- ing with alternative assays to measure both
and changes in diet, injury, or binant growth hormone can both decrease IGF-1 and P-III-NP. One of the newer assays
pathology. fat and increase lean body mass. By the end involves mass spectrometry to measure IGF-
—Richard Holt, University of Southampton of the 1980s, growth hormone was among 1. Approved by WADA in 2014,4 it is now
the substances prohibited by the Interna- routinely used in combination with P-III-
tional Olympic Committee, yet scientists NP immunoassays. But researchers are still
Anti-doping watchdogs measure other only launched the test to detect the com- working to develop a reliable P-III-NP mass
biomarkers that can influence an athlete’s pound in 2004. spectrometry method.
urine levels of steroid hormones to reach The majority of the endogenous protein And challenges face the existing tests.
stronger conclusions on the likelihood of dop- that circulates in the body is 22 kilodaltons in Holt and his colleagues have accumulated
ing. For example, an individual’s alcohol con- size, but an alternative splicing pattern of the evidence that “neither the isoform nor the
sumption or the presence of bacteria in the messenger RNA can result in a 20-kilodalton biomarker test is performing as well as we
bladder or in the stored sample can cause an form. The lab-made product consists of only had hoped,” he says. The biomarker test
increase in testosterone levels, so labs look for the 22-kilodalton molecule, and taking exog- has a longer window of detection than the
bacteria and alcohol-specific metabolites in enous growth hormone blocks the natural isoform test—about one to two weeks—
urine. And most recently, scientists involved production of growth hormone by the pitu- but levels of the biomarkers can differ
in a PCC working group rolled out a liquid itary gland, explains Holt. “[This] changes widely between individuals, making it dif-
chromatography– and mass spectrometry– the ratios of the various isoforms, which can ficult to draw concrete conclusions about
based test that achieves greater sensitiv- be picked up by the current assays.” an athlete’s potential doping behavior. “To
ity in detection of testosterone doping with But interpreting the ratio has its chal- have a conclusive test, we need to make
repeated testing of the steroid’s levels in blood lenges. Although each individual has a sta- sure the markers we are measuring are not
serum, rather than urine. This test, adminis- ble ratio of the isoforms, and deviations sig- affected by training and changes in diet,
tered by WADA-accredited laboratories just nal that doping is likely, those deviations injury, or pathology,” says Holt.
before the 2016 Olympic Games in Rio de are short-lived because endogenous growth Growth hormone detection is not cur-
Janeiro, caught two female Ukrainian track hormone production rapidly resumes in the rently part of the ABP, but WADA is con-
and field athletes who, the Court of Arbitra- pituitary gland after doping, providing only sidering adding either the isoform or bio-
tion for Sport confirmed in April, had illegally a short window of 12 to 24 hours for authori- marker test despite the flaws of both. One
used testosterone. ties to detect misuse. Moreover, athletes con- issue is that the ABP’s steroid module mea-
Still, researchers know they need to con- ceivably could continue to use growth hor- sures steroids in urine, but growth hor-
stantly improve tests and testing strategies. mone sourced from human cadavers, which mone requires a blood draw: the molecule
“We need to be aware that athletes will con- contains all of the endogenous isoforms and is larger, and therefore not readily passed
tinue to adapt their doping behavior; there- cannot be picked up using the isoform test. through the kidneys into the urine. Blood
fore, ongoing efforts to develop and refine “Judging by the relatively low number of tests are generally done less frequently, as
they are less athlete-friendly than easier-to-

administer urine tests.
“These tests are expensive, on the order
of $50 a test,” says Holt, “and so it’s not a triv-
ial decision to keep adding additional tests
without demonstrating their effectiveness in
detecting growth hormone misuse.”
Boosting with blood

Another performance-boosting tactic that
can be challenging to detect is a blood
transfusion. By increasing the volume of
RBCs, athletes can carry more hemoglo-
bin, allowing the blood to transport more
oxygen to the muscles, similar to the effect
of EPO. The transfusions can come from
either a donor or the athletes themselves,
with the former being easier to detect. In
2004, testing labs implemented a method
to detect blood transfused from a donor by that the lab is now working on a single-cell with interpretation of the findings so the
looking for changes in RBC populations. approach to identify RBC microdosing. results management process is fair and just
The test can detect antigen differences for the athlete.”
on the cells, and it caught many cheaters, Getting ahead of users Researchers continue to delve into the
including Tyler Hamilton, an elite cyclist As researchers continue to develop more- biology of how the body processes exog-
who coauthored a tell-all of his own dop- sensitive assays, they can better equip enous versions of naturally occurring hor-
ing experiences. anti-doping officials to keep athletes on mones and other molecules in order to
To indirectly detect autologous blood a level playing field. Yet results of highly develop more-sensitive and more-accu-
transfusions, WADA has developed a test sensitive tests can also underscore gaps rate tests, Pearlmutter, Elliott, and Holt all
for plasticizers, such as di-2-ethylhexyl in knowledge on how performance- acknowledge. And the PCC is specifically
phthalate (DEHP), that can leach from enhancing substances are metabolized working on dried plasma and saliva-based
storage bags into the blood, but plasticizer and cleared from the body. tests, which could circumvent costly trans-
detection is not a widely used method. A In December 2018, for example, port of temperature-controlled blood sam-
major issue is that plasticizer detection UFC fighter Jon Jones tested positive for ples and the need for a phlebotomist to be
is not reliable because these compounds trace amounts of a long-term metabo- present at testing sites. But details of this
are also found in other common products lite of a common doping steroid called development are closely guarded secrets.
including upholstery, food packaging, and oral turinabol (the “little blue pill” used “You obviously don’t want to tell athletes
children’s toys, and can leach into the envi- by the East German state-sponsored dop- exactly what testing is coming next,” says
ronment and ultimately into athletes’ bod- ing scheme in the 1970s and ’80s), raising Elliott. “Then you get behind.” g
ies through their food and water. the issue of whether a detected metabolite
As another option, Jen-Tsan Chi’s lab is a sign of recent doping or a remnant of References
at the Duke University School of Medicine, long past use. Jones had tested positive for 1. J. Mullen et al., “Sensitivity of doping biomarkers
with funding from WADA and the PCC, turinabol back in July 2017 and had been after administration of a single dose testosterone gel,”
looked for changes in RNA expression as suspended by the California State Ath- Drug Test Anal, 10:839–48, 2018.
2. J. Mullen, “Urinary steroid profiles in doping testing,”
RBCs aged for up to 42 days—the longest letic Commission for 15 months. After the
Thesis, Division of Clinical Pharmacology, Karolinska
time US blood banks allow blood to be December 2018 test result, the USADA
© ISTOCK.COM, SOUTH_AGENCY
Institutet, Stockholm, Sweden, 2018.

refrigerated for transfusions. The results, concluded that because only the long- 3. P.-E. Sottas et al., “Bayesian detection of abnormal
published last October, revealed a distinct term metabolite and not the steroid itself values in longitudinal biomarkers with an application
increase in two microRNAs and a drop in was detected, the most likely scenario is to T/E ratio,” Biostatistics, 8:285–96, 2006.
4. J.K. Powrie et al., “Detection of growth hormone
two others during these six weeks of stor- that Jones had not ingested oral turinabol
abuse in sport,” Growth Horm IGF Res, 17:220–26,
age.5 “The abundant and diverse species of recently. Ultimately, he was allowed to con- 2007.
RNA in the mature RBC offer a novel way tinue to fight. 5. W.H. Yang et al., “Angiogenin-mediated tRNA
to characterize the changes in RBC prop- The tests are getting better, says cleavage as a novel feature of stored red blood cells,”
erty during storage,” says Chi, who adds Fedoruk, but then “the challenge becomes Brit J Haematol, 185:760–64, 2019.
09. 201 9 | T H E S C IE N T IST 3 5

Our Inner
Neanderthal
From skin color to immunity, human biology appears to
be linked to our archaic ancestry.
BY JEF AKST
A
fter the 2010 publication of the Neanderthal draft als. The endeavor was further powered by the first high-quality
genome sequence, evolutionary biologist Joshua Akey, Neanderthal genome sequence, which gave the duo confidence
then at the University of Washington in Seattle, and his that the sequences they’d identified were indeed of archaic ori-
© SCIENCESOURCE, S. ENTRESSANGLE AND E. DAYNES

graduate student Benjamin Vernot began looking into its most gin. Still, in the back of Akey’s mind, he had doubts about the
provocative implication: that the ancient hominins had bred with research. “I remember telling Ben [when] we were working on
the ancestors of modern humans. Neanderthals had been living in this, ‘I wake up every day in a cold sweat that this is all just
Eurasia for more than 300 millennia when some human ances- incomplete lineage sorting’”—a methodological artifact that
tors left Africa some 60,000–70,000 years ago, and according to would undermine their conclusions about Neanderthal ancestry,
the 2010 publication, in which researchers compared the Nean- meaning the sequences were the result of the common ancestry
derthal draft genome with modern human sequences, about 2 the two groups shared.
percent of the DNA in the genomes of modern-day people with Then, as Vernot and Akey were getting ready to submit their
non-African ancestry is Neanderthal in origin.1 work for publication, their department got a visit from Svante
To investigate the archaic ancestry of the living human pop- Pääbo, a geneticist at the Max Planck Institute for Evolutionary
ulation, Akey and Vernot set to work searching for Neanderthal Anthropology who had pioneered techniques for extracting and
DNA in modern genomes. They developed a statistical approach analyzing DNA from ancient specimens and had led the early
to identify genetic signatures suggestive of Neanderthal ancestry Neanderthal genome efforts. They spoke with him about their
in the genomes of 379 European and 286 East Asian individu- ongoing project, and Pääbo noted that his collaborator, David
Reich at Harvard Medical School, was pursuing a very similar the genomes from living humans they studied. The regions that
line of research. So Akey gave Reich a call. contained high frequencies of Neanderthal sequences included
“The end result [of the conversation] was we agreed to coor- genes that could yield clues to their functional effect. Base-pair
dinate publication,” Akey recalls. “We also agreed not to even differences between Neanderthal and human variants rarely fall
look at each other’s papers because we didn’t want to influence in protein-coding sequences, but rather in regulatory ones, sug-
the results in any way.” (See “Simultaneous Release,” The Scien- gesting the archaic sequences affect gene expression. (See “Den-
tist, June 2014.) isovans in the Mix” on page 39.)
Vernot and Akey submitted to Science;2 Reich and his col- A number of segments harbor genes that relate to skin biol-
leagues submitted to Nature.3 The two journals synchronized pub- ogy, such as a transcription factor that regulates the develop-
lication of the papers at the end of January 2014. The day they went ment of epidermal cells called keratinocytes. These variants may
live, Akey anxiously began to read the paper from the Reich group. underlie traits that were adaptive in the different climatic con-
“I remember sitting in my office, reading it, and really sort of just ditions and lower levels of ultraviolet light exposure at more
going through the checklist” of the key results, he says. Quickly, the northern latitudes. Reich’s group similarly found genes involved
relief set in. “We essentially said the exact same thing,” Akey recalls. in skin biology enriched in Neanderthal ancestry—that is, more
“Usually when you publish something, it’s years before you see vali- than just a few percent of people carried Neanderthal DNA in
dation. . . . This was sort of instant gratification.” these parts of the genome.
The two groups had used different statistical approaches to It was unclear, however, what specific effect the Neanderthal
identify Neanderthal DNA in modern human genomes, putting variants had on phenotype. For that, researchers needed phenotypic
to bed any skepticism about the history of hominin group inter- data on many different kinds of traits, paired with genetic informa-
breeding. “[It was] the final nail on the coffin that it couldn’t tion, for thousands of people. Vanderbilt University evolutionary
be anything else,” says Janet Kelso, a computational biologist at geneticist Tony Capra has access to such a resource: the Electronic
the Max Planck Institute for Evolutionary Anthropology and a Medical Records and Genomics (eMERGE) Network. Right around
collaborator on Reich’s publication. the time the scientific community was beginning to map Neander-
With the issue of Neanderthal/modern human mating settled, thal DNA in the genomes of living people, eMERGE organizers were
scientists could focus on a new goal, says Akey, now at Princeton compiling electronic health records and associated genetic data for
University. Namely, what was the consequence of this interbreed- tens of thousands of patients from nine health-care centers across the
ing? “Was it just this curious feature of human history that didn’t US. “We felt like we had a chance to evaluate some of those hypoth-
have an impact, or did it alter the trajectory of human evolution?” eses [about functionality] on a larger scale in a real human popula-
In the past five years, a flurry of research has sought to tion where we had rich phenotype data,” says Capra.
answer that question. Genomic analyses have associated Nean-
derthal variants with differences in the expression levels of
diverse genes and of phenotypes ranging from skin and hair Was it just this curious feature of
color to immune function and neuropsychiatric disease. But human history that didn’t have an
researchers cannot yet say how these archaic sequences affect
impact, or did it alter the trajectory of
people today, much less the humans who acquired them some
50,000–55,000 years ago. human evolution?
“So far I have not seen any convincing functional studies —Joshua Akey, Princeton University
where you take the Neanderthal variant and the human vari-
ant and do controlled experiments” to identify the physiological In collaboration with Akey and Vernot, who helped iden-
consequence, says Grayson Camp, a genomicist at the Institute tify Neanderthal variants in the genetic data included in the
of Molecular and Clinical Ophthalmology Basel (IOB) in Swit- database, Capra’s group looked for links between the archaic
zerland. “No one has actually shown yet in culture that a human DNA and more than 1,000 phenotypes across some 28,000 peo-
and Neanderthal allele have a different physiological function. ple of European ancestry. They reported in 2016 that Neander-
That will be exciting when someone does.” thal DNA at various sites in the genome influences a range of
immune and autoimmune traits, and there was some association
Neanderthal in our skin with obesity and malnutrition, pointing to potential metabolic
Most Neanderthal variants exist in only around 2 percent of effects. The researchers also saw an association between Nean-
modern people of non-African descent. But some archaic DNA derthal ancestry and two types of noncancerous skin growths
is much more common, an indication that it was beneficial to associated with dysfunctional keratinocyte biology—supporting
ancient humans as they moved from Africa into Eurasia, which the idea that the Neanderthal DNA was at one point selected for
Neanderthals had called home for more than 300,000 years. its effects on skin.4
In their 2014 study, Vernot and Akey found several sequences “This was crazy to me,” says Capra. “What these other
of Neanderthal origin that were present in more than half of groups had predicted based on just the pattern of occurrence—
the presence and absence of Neanderthal ancestry around cer- DENISOVANS IN THE MIX
tain types of genes—we were actually seeing in a real human Neanderthals thrived in Eurasia as a dominant hominin group
population, that having Neanderthal ancestry influenced traits for hundreds of thousands of years and have long been a focus
related to those types of skin cells.” What remains unclear, of scientific inquiry. But less than a decade ago, researchers
however, is what the benefits of the Neanderthal sequences discovered that there was another group of archaic hominins
were for those early humans. that coexisted with Neanderthals and the ancestors of mod-
At the same time, Kelso and her postdoc Michael Dannemann ern humans. DNA collected from a single finger bone and two
were taking a similar approach with a relatively new database teeth appeared to be neither Neanderthal nor human, and sci-
called the UK Biobank (UKB), which includes data from around entists named a new group, the Denisovans, after the Siberian
half a million British volunteers who filled out questionnaires cave in which the remains were found in 2008.
about themselves, underwent medical exams, and gave blood Once researchers reconstructed the entire high-quality
samples for genotyping. Formally launched in 2006, the UKB Denisovan genome in 2012 (Science, 338:222–26, 2012),
published its 500,000-person-strong resource in 2015, and it became clear that, like Neanderthals, Denisovans had
Kelso and Dannemann decided to see what information they interbred with modern humans during the time that they
could extract. Conveniently, the genotyping data specifically coinhabited Eurasia, with analyses suggesting that the
includes SNPs that can identify variants of Neanderthal origin, introgressed DNA likely came from multiple Deniso-
thanks to Reich’s group, which provided UKB architects with a van populations within the last 50,000 years, sometime
list of 6,000 Neanderthal variants. after mixing occurred between Neanderthals and human
Among the many links Kelso and Dannemann identified ancestors (Cell, 173:P53–61.E9, 2018; Cell, 177:P1010–21.
as they dug into data from more than 112,000 individuals E32, 2019). Denisovan DNA makes up 4–6 percent of the
in the UKB was, once again, an association between certain genomes of people native to the islands of Melanesia, a
Neander thal variants and aspects of skin biology.5 Specifically, subregion of Oceania, and to a lesser extent they left their
the archaic sequences spanning the BNC2 gene—a stretch of genetic mark in other Pacific island populations and some
the genome that Vernot and Akey had identified as having modern East Asians, while it is largely absent from the
Neanderthal origin in some 70 percent of non-Africans—were genetic code of most other people. As with Neanderthal
very clearly associated with skin color. People who carried introgression, the question that remains to be answered is:
Neanderthal DNA there tended to have pale skin that burned What effect did these variants have on our own lineage—
instead of tanned, Kelso says. And the stretch that included and are we still experiencing Denisovans’ genetic influence?
BNC2 was just one of many, she adds: around 50 percent of As with Neanderthal DNA, experts have identified regions
Neanderthal variants linked with phenotype in her study have of modern human genomes that are significantly depleted of
something to do with skin or hair color. Denisovan DNA, and they saw that these “deserts” were the
The effect that Neanderthal DNA might have on skin appear- same ones that lacked Neanderthal sequences—indications
ance and function is “fascinating,” says Akey. “Something that we’re of selection against deleterious variants (Science, 352:235–39,
still really interested in and starting to do some experimental work 2016). “That’s as close as you can get to sort of a replication
on is: Can we understand what these genes do and then maybe what in this type of work,” says Princeton University evolutionary
the selective pressure was that favored the Neanderthal version?” biologist Joshua Akey. In terms of introgressed bits of Deniso-
van DNA that might have been beneficial to modern humans,
Neanderthal-derived immunity researchers have found links to toll-like receptors and other
Another area of human biology tightly linked to Neanderthal vari- contributors to immune function, similar to links found with
ants in the genome is the immune system. Given that human Neanderthal variants.
ancestors were exposed to a menagerie of different pathogens— Denisovan DNA may have also offered some unique ben-
some of which came directly from the Neanderthals—as they efits to ancient humans. One scientific team identified Deniso-
migrated through Eurasia, the Neanderthal sequences intro- van variants in the genomes of Greenland Inuits that include
gressed into the human genome may have helped defend against genes involved in the development and distribution of adipose
these threats, to which Neanderthals had long been exposed. tissue, perhaps pointing to advantages in cold tolerance and
“Viral challenges, bacterial challenges are among the stron- metabolism (Mol Biol Evol, 34:509–24, 2017). And maybe
gest selective forces out there,” says Kelso. Unlike changes in the strongest suggestion of beneficial Denisovan introgres-
other environmental conditions such as daylight patterns and sion comes from a 2014 study in which researchers linked the
UV exposure, “pathogens can kill you in one generation.” archaic sequences with high altitude adaptation among popu-
Hints of archaic DNA’s role in immune function surfaced as lations that live in the Tibetan highlands (Nature, 512:194–97,
early as 2011, as soon as the Neanderthal genome was available 2014). The particular variant they focused on was so highly
for cross-referencing with sequences from modern humans. A selected, notes Kelso, that “almost everyone living on the pla-
team led by researchers at Stanford University found that certain teau carries this piece of Denisovan DNA.”
A MIXED HISTORY
Some 350,000 or more years ago, the group of hominins that
would evolve to become Neanderthals and Denisovans left
Africa for Eurasia.
A few hundred millennia later, about 60,000 to 70,000
years ago, the ancestors of modern non-Africans followed a
similar path out of Africa and began interbreeding with these
other hominin groups. Researchers estimate that much of the
Neanderthal DNA in modern human genomes
came from interbreeding events that took
place around 50,000 to 55,000 years ago in
350,000+ YEARS AGO
the Middle East. Thousands of years later, Neanderthal and Denisovan
humans moving into East Asia interbred ancestors leave Africa and
with Denisovans. migrate throughout Eurasia
1 2
60,000 TO 70,000
YEARS AGO 50,000 TO 55,000
Some human ancestors YEARS AGO
leave Africa Neanderthals and human
ancestors interbred
3 4 5
NOTABLE HOMININ GENOMES
45,000–50,000 YEARS AGO 1 The 50,000- to 65,000-year-old remains of

Denisovans and human a Neanderthal in Vindija Cave, Croatia, gave
ancestors interbred researchers their first look at the Neanderthal
genetic code.
The genome of a 40,000-year-old human

2
whose remains were unearthed in Romania
harbored 6–9 percent Neanderthal DNA.
Teeth dating to some 80,000 years ago yield

3
DNA from a Denisovan individual in Denisova
Cave in Siberia confirmed the existence of a
third group of ancient hominins that coexisted
with Neanderthals and human ancestors.
The 120,000-year-ago remains of a

4 Neanderthal individual was also discovered in
Denisova Cave.
5 The 90,000-year-old remains of a Denisovan-

Neanderthal hybrid in Denisova Cave laid bare
the fact that the two groups interbred.
THE SCIENTIST STAFF
human leukocyte antigen (HLA) alleles, key players in pathogen David Enard and colleagues found that one-third of Nean-
recognition, held signs of archaic ancestry—from Neanderthals, derthal variants under positive selection were linked to genes
but also from another hominin cousin, the Denisovans.6 “It’s a encoding proteins that interact with viruses.9
cool paper and one that contributed to a lot of people thinking Researchers have also identified several less-easily explainable
about the effects of introgression,” says Capra. phenotypic associations with Neanderthal introgression. In their
Several other studies since then have strengthened the 2017 analysis, for example, Kelso and Dannemann found that
link between archaic DNA and immune function, branching Neanderthal variants were associated with chronotype—whether
out from the HLA system to include numerous other path- people identify as early birds or night owls—as well as links with
ways.7 For example, multiple labs have tied Neanderthal vari- susceptibility to feelings of loneliness or isolation and low enthu-
ants to altered expression levels of genes encoding toll-like siasm or interest. The associations with mood-related phenotypes
receptors (TLRs), key players in innate immune responses. In jibe with what Capra’s group had found the year before in its data-
2016, Kelso, Dannemann, and a colleague found that pathogen set of medical information, which linked Neanderthal variants to
response and susceptibility to develop allergies were associ- risks for depression and addiction. “These were associations that
ated with Neanderthal sequences that affect TLR production.8 were quite strong,” says Capra. “And when we looked at genes
Viruses, in particular, appear to be potent drivers of adap- where these regions of Neanderthal ancestry fell, in many cases
tation. Last year, University of Arizona population geneticist they made sense given what we already know about those genes.”
ALL ABOUT REGULATION

PERCENTAGE OF ALLELES OF
In their seminal 2014 studies, the groups of David Reich of Harvard Medical School and
NEANDERTHAL ORIGIN THAT ARE
Joshua Akey, then at the University of Washington, noted that the Neanderthal variants FOUND IN >10% OF MODERN EURASIANS
that correlated with human phenotypes did not appear in coding regions. Two years later, a
genome-wide analysis published by investigators in France found that Neanderthal ancestry
was enriched in areas tied to gene regulation (Cell, 167:643–56.e17, 2016). The implication Non-
functional
was that sequences that originated in Neanderthals tend to have “less impact through pro-
regions
tein and more impact through gene expression,” says coauthor Maxime Rotival, a geneticist
at the Pasteur Institute in Paris.
To ask this question more directly, Akey turned to the Genotype-Tissue Expression
(GTEx) Project, which has cataloged gene expression data from roughly 50 tissues for
Promoters
each of 10,000 individuals. “It’s this really fine-scale record of gene expression,” says Akey.
His then-postdoc Rajiv McCoy, now an assistant professor at Johns Hopkins University,
developed a method to assess messenger RNA levels based on which allele was being
expressed—the one from an individual’s father or mother—and the researchers applied this
AM J HUM GENET, DOI:10.1016/J.AJHG.2019.04.016, 2019; THE SCIENTIST STAFF

approach to people in the GTEx database who were heterozygous for a particular Neander- Enhancers
thal variant. Comparing expression levels based on which allele was being expressed, the
researchers found that a quarter of the stretches of Neanderthal DNA in human genomes
affect the regulation of the genes in or near those stretches (Cell, 168:P916–27.E12, 2017).
“We’ve known for a long time that gene expression variation is an important source of
miRNA
phenotypic variation within populations and phenotypic divergence between species,” says
binding sites
Akey. “We were interested in asking whether Neanderthal sequences make any contribu-
tion to gene expression variability.” The answer was a resounding yes.
Earlier this year, Rotival and two colleagues calculated ratios of Neanderthal to non-
Neanderthal variants across the genome and compared those ratios for protein-coding
Coding
regions and various regulatory sequences, specifically enhancers, promoters, and regions
microRNA-binding sites. Consistent with previous results, they found a strong depletion
of Neanderthal variants in coding portions of genes, and a slight enrichment of the archaic
sequences in regulatory regions (Am J Hum Genet, doi:10.1016/j.ajhg.2019.04.016, 2019).
“What we see is that in coding regions, the ratio of archaic to non-archaic variants is much 15% 25% 35%
smaller than the ratio outside of coding regions,” says Rotival.
ANCESTRAL ANALYSIS: Sequences of Neanderthal
“This is not at all a surprise,” says Vanderbilt University’s Tony Capra, whose lab has gen-
origin in people of Eurasian descent are more
erated similar findings in people of Eurasian descent, “but it’s really nice to see it quantified common in nonfunctional and regulatory regions of
very comprehensively.” the genome than in coding regions.
Why these associations exist is still a mystery. Kelso suspects really hard to estimate,” he says. “I think the field is kind of stuck
that light might be a unifying factor, with both changes in day- right now.” But he’s hopeful that as more genomes from various
length patterns and UV exposure reductions as they moved to archaic hominin groups and from modern humans come online,
more-northern latitudes. But that’s just a hunch, she emphasizes. researchers’ power to model how all of these groups interbred
“It’s fun speculating about how [Neanderthal introgression] will strengthen. A second high-quality Neanderthal genome was
could have been advantageous, or how variants that make us published in 2017 (Science, 358:655–58), and researchers now
depressed in the modern environment could have been bene- have the genome of a 40,000-year-old human who had a Nean-
ficial,” says Capra. “I don’t really even know what depression derthal ancestor just a few generations back. Last year, research-
meant 40,000 years ago. That’s both the challenge and the fun, ers published the sequence of a first-generation hybrid of Den-
provocative part about all this.” isovans and Neanderthals.
Those data will likely yield some surprises. Capra has found
evidence, for example, that some of the Neanderthal segments
What we think we were seeing is that correlated with modern phenotypes may not affect those
phenotypes directly. His work has uncovered cases in which the
strong circumstantial evidence that
correlation was driven by sequences close enough in the genome
there was natural selection to remove to Neanderthal variants that the two always appear together.
Neanderthal DNA. These sequences were carried by the common ancestor of Nean-
—David Reich, Harvard Medical School derthals and modern humans but were missing from the group
of humans who founded the modern Eurasian population. These
variants, which had been retained by Neanderthals, were then
A question of functionality reintroduced to the ancestors of modern non-Africans during
Even with more straightforward associations, such as with skin periods of interbreeding.10 “These genetic variants existed in
traits or immune responses, conclusions thus far are drawn modern [Eurasians only] in the Neanderthal context, but these
from correlations between genotypes and phenotypes. While were not of Neanderthal ancestry,” Capra says.
such genetic and statistical approaches can conceptually link Akey has come upon another interesting twist: Africans
Neanderthal introgression with phenotypes and hint at why such do have Neanderthal ancestry. Unpublished work from his
sequences may have been selected for in humans’ early history, group points to the possibility that some of the ancient modern
researchers have not yet published in vitro validation studies. humans that bred with Neanderthals migrated back to Africa,
“Studying Neanderthal DNA more closely on a molecular level where they mixed with the modern humans there, sharing bits
in the lab is pretty tricky,” says Dannemann. Neanderthal variants of Neanderthal DNA. If true, that would mean that Africa is not
tend to come in packages, and the linkage between the variants devoid of Neanderthals’ genetic influence, Akey notes. “There’s
makes it difficult to identify the function of each one, he explains. Neanderthal basically all over the world.” g
That challenge hasn’t stopped researchers from trying. As
a postdoc in Pääbo’s lab in Germany, Camp, along with Vernot,
References
Kelso, and Dannemann, established a handful of brain organ-
1. R.E. Green et al., “A draft sequence of the Neandertal genome,” Science,
oids from induced pluripotent stem cell lines of modern Europe- 328:710–22, 2010.
ans who vary in their Neanderthal-derived genetics, and tracked 2. B. Vernot, J. Akey, “Resurrecting surviving Neandertal lineages from modern
single-cell transcriptomes as the cultured cells matured. The human genomes,” Science, 343:1017–21, 2014.
early data suggest that the Neanderthal variants affect gene 3. S. Sankararaman et al., “The genomic landscape of Neanderthal ancestry in
present-day humans,” Nature, 507:354–57, 2014.
expression in the same way as documented by previous work,
4. C.N. Simonti et al., “The phenotypic legacy of admixture between modern
validating the model. humans and Neandertals,” Science, 351:737–41, 2016.
But such research is still in the proof-of-principle stage, says 5. M. Dannemann, J. Kelso, “The contribution of Neanderthals to phenotypic
Camp, who is continuing this work in his own lab in Switzerland. variation in modern humans,” Am J Hum Genet, 101:P578–89, 2017.
“Now you just need to increase throughput. You need to do this 6. L. Abi-Rached et al., “The shaping of modern human immune systems by
multiregional admixture with archaic humans,” Science, 334:89–94, 2011.
for 100 or 200 individuals.” Even then, he adds, the conclusions
7. H. Quach et al., “Genetic adaptation and Neandertal admixture shaped the
researchers will be able to draw will be limited. “I am a bit cautious immune system of human populations,” Cell, 167:643–56.e17, 2016.
and maybe pessimistic [about whether] you can really identify . . . 8. M. Dannemann et al., “Introgression of Neandertal- and Denisovan-like
impacts [of Neanderthal variants] on some physiological outcomes.” haplotypes contributes to adaptive variation in human toll-like receptors,” Am
There are other fundamental questions that are proving diffi- J Hum Genet, 98:P22–33, 2016.
9. D. Enard and D.A. Petrov, “Evidence that RNA viruses drove adaptive
cult to answer about Neanderthal introgression, says Akey, from
introgression between Neanderthals and modern humans,” Cell, 175:P360–
the number of hybridization events to the timescale over which 71.E13, 2018.
those events took place, and whether there was sex bias in pat- 10. D.C. Rinker et al., “Neanderthal introgression reintroduced functional alleles
terns of gene flow. “There are all these important things that are lost in the human out of Africa bottleneck,” bioRxiv, doi:10.1101/533257, 2019.
09. 201 9 | T H E S C IE N T IST 4 3

MODFIED FROM © ISTOCK.COM, DR_MICROBE; THE SCIENTIST STAFF

A Broken X
Despite a solid understanding of the biological basis of fragile X syndrome,
researchers have struggled to develop effective treatments. But recent
trials offer hope.
BY RANDI HAGERMAN
W
hen I visited Ricaurte, similar social deficits. I had never seen so accepted. Their behavior does not seem
Colombia, in 2016, I was many individuals with FXS all together. unusual to those living in the village.
surrounded by men with I thought to myself: This is ground zero Relatives who have moved away from
long faces and prominent ears. As we for FXS. Ricaurte and then subsequently have
spoke, they would ask repetitive ques- Likely because the founding fam- had a child with FXS will move back to
tions while mumbling and failing to ilies of this small village had one or this town for the acceptance and sup-
maintain eye contact, and when they more carriers of the causative mutation, port they find there. This pattern fur-
shook my hand, they turned their body Ricaurte has the highest known preva- ther enhances the genetic cluster of
away from me. They were interested in lence of FXS in the world. Last year, our FXS-causing mutations in this area.
me but were too shy to interact. This type team published the results of genetic Initial chromosome testing docu-
of anxiety-related approach-withdrawal testing of almost all of the inhabitants mented the fragile site on the X chro-
behavior is typical of those with fragile in this village. We found that nearly 5 mosome, and we saw the disease’s high
X syndrome (FXS), a well-characterized percent of male and more than 3 per- prevalence as an advantage in our quest
genetic disease that is the most common cent of female inhabitants of Ricaurte to study the hard-to-treat condition.
inherited form of intellectual disability have FXS, 1 compared to around 0.02 While research of the past three decades
and the most common single-gene cause percent of people living in the US and has detailed the underlying genetic and
of autism. Even many of the Ricaurte in Europe. In Ricaurte, the residents molecular causes of FXS, it has yet to
women, who usually have at least one are supportive of these individuals, who yield a treatment that can reverse the
good copy of the X chromosome, showed work in the community and are well associated neurobiological abnormalities
09. 201 9 | T H E S C IE N T IST 4 5

in the brain. By analyzing the genomes unknown reasons looked “fragile” when higher your IQ. Paradoxically, premuta-
of FXS patients in the village, we hope their cells were studied in culture media tions result in higher FMR1 mRNA lev-
to learn more about the founders and to deficient of folic acid. In 1991, research- els than are found in wildtype individu-
investigate the background allelic vari- ers identified an expansion of the DNA als, and these RNAs can cause problems
ations that facilitate the appearance of sequence CGG in the promoter region of even if FMRP protein levels are normal.
new FXS mutations. We are also investi- the FMR1 gene as the underlying cause (See “Sidebar” on page 51.)
gating individual variability and whether of the X chromosome’s broken appear- If women carrying the premutation
environmental toxicity can exacerbate ance. This gene codes for fragile X men- pass it on, it can expand into the full
FXS patients’ conditions. tal retardation protein (FMRP), which mutation during egg development.
At the same time, we are conduct- controls the translation of hundreds of Once the CGG repeats number more
ing several controlled trials of promis- messenger RNAs into various proteins than 100, every time that X chromo-
ing FXS drug candidates at the MIND that are important for neuronal function. some is passed from mother to child it
Institute at the University of California, FMRP is the main controller of synap- will carry the full mutation, as the CGG
Davis, Medical Center. The candidates tic plasticity in early brain development repeats apparently expand during either
include cannabidiol as well as metfor- and is needed throughout life to make oogenesis or embryonic development.
min, a type 2 diabetes medication that new neurons. Recent research has found The details of this expansion are still
can also help obesity. We are hopeful that the protein also controls a protein murky. Male carriers of the premutation
that our work in Ricaurte, along with the essential for epigenetic changes across will pass it on to all of their daugh-
work of other teams investigating popu- the genome, perhaps explaining FMRP’s ters. If a man carries a full mutation,
lations with high rates of FXS in Indo- wide-ranging effects. it reverts back to a premutation in his
nesia, Israel, and on the Spanish island The mutated FMR1 gene itself har- sperm, such that all of his daughters
of Mallorca, among other places, will bors epigenetic differences. While inherit the premutation. Again, the
inform the development of better treat- the wildtype gene lacks methylation, reason for and mechanisms of this back
ments for patients all over the world. sequences with more than 200 CGG mutation are unclear. (See illustration
repeats—considered the full muta- on page 49.)
What we know about fragile X tion—are heavily methylated, which Despite questions about how the
Fragile X syndrome was named for a deli- thereby blocks transcription of the FXS locus shuffles CGG repeats in a
cate site at the bottom of the X chromo- gene. In males, this leads to a complete somewhat predictable manner, the
some of patients with the disease that for absence of FMRP, while females typi- underlying molecular mechanisms of
cally have one X with the wildtype the disease are fairly well characterized,
gene that provides some thanks to copious research on FMRP’s
amount of the pro- role in the cell. This has led to human
tein. As a result, trials of several treatments that target
their symptoms the relevant molecular pathways, but
are typically less unfortunately, they’ve had little suc-
severe. cess. While there are therapies to man-
Some indi- age symptoms, there is currently no
viduals carr y cure for FXS.
what is called
the premutation, Our failure in treatment trials
characterized by There are many ways to cure a mouse
a CGG expansion of FXS. For example, inhibitors of the
MODFIED FROM © ISTOK.COM, DR_MICROBE
of 55 to 200 repeats. metabotropic glutamate receptor 5
Premutation genes (mGluR5) pathway, which is involved
with fewer than 120 CGG in neural signaling and is known to be
repeats typically result in nor- overactive in FXS, can relieve mouse
mal FMRP levels, while those with models of all symptoms of the disease.
more repeats have lowered FMRP But when such therapies moved into
production, leading to relatively human trials a few years ago, adoles-
minor learning problems or, rarely, cents and adults with FXS experienced
intellectual disabilities. Up to no benefit beyond what the placebo
a normal threshold, generally group enjoyed. Similarly, agonists that
the more FMRP produced, the target the neurosignaling GABA system,
which is underactive in FXS, succeeded als that used multiple behavioral ques- tute, for example, my colleagues and I
in alleviating symptoms in mice but in tionnaires, did in fact improve FXS tested long-term low dosing of a selec-
humans proved no better than placebo.2,3 patients’ ability to look into the eyes of tive serotonin reuptake inhibitor (SSRI)
Regardless of whether patients got the a person’s photograph as measured by called sertraline in two- to six-year-old
mGluR5 inhibitor, GABA agonist, or the an infrared gaze-tracker.8 children with FXS. Serotonin levels are
placebo, between 20 percent and 30 per- Armed with more-objective mea- low in the brains of young children with
cent of patients showed improved behav- sures, researchers are hoping to autism spectrum disorder, and an SSRI
ior and their families were feeling posi- improve the track record of FXS clinical functions to increase serotonin at the
tive about the outcome.4,5 trials. The US pharmaceutical company synapse. In 2016, we published results
Given the high expectations among
patients and their families for targeted
FXS treatments, such high placebo
responses are common. There is thus
For the foreseeable future, treating FXS will
a need for quantitative outcomes that be a matter of managing symptoms and
measure patient responses to treatment
through electrophysiological measures.
working through therapy to support healthy
In the last six years, such measures have development.
been developed. For example, event-
related potentials (ERPs), patterns
of activity visible on EEG readouts in Ovid, for example, is currently trialing demonstrating significant benefits in
response to sensory cues, can reveal a GABA agonist at multiple centers in fine motor, visual reception, and expres-
the failure of FXS patients to habitu- the US for FXS patients ages 13 to 22 sive language skills,9,10 leading doctors to
ate to repeated stimuli such as repeti- years. The outcome measures include a begin using the drug to treat FXS. Now,
tive sounds or visual stimuli, leading to specific quantitation of behaviors typi- the FX-LEARN study—currently enroll-
hyperarousal. Another option is to track cal for FXS, electronic measures such as ing patients at several US centers, includ-
patients’ eye movements to monitor eye tracking, and molecular biomarkers. ing the MIND Institute—will test the use
their ability to make eye contact. There Meanwhile, Pennsylvania-based of an mGluR5 antagonist in combina-
are also new molecular biomarkers that pharma company Zynerba has been test- tion with an educational intervention in
reflect a reversal of the neurobiological ing its transdermal ointment of pure can- three- to six-year-old children with FXS,
changes in FXS. For example, a pro- nabidiol (CBD), a nonpsychotropic com- using ERPs, eye tracking studies, and
tein called matrix metallopeptidase-9 ponent of marijuana, in FXS patients at other quantitative outcome measures.
(MMP-9) is elevated in the blood of multiple sites in the US, Australia, and Another treatment that we’re begin-
FXS patients, and a targeted treatment New Zealand, using eye tracking and ning to test in children with FXS is met-
such as metformin lowers the MMP-9 molecular biomarkers alongside question- formin. In FXS, metformin has been
level in mice and patients with FXS. naires measuring patients’ anxiety. Pre- shown to lower activity in the mTOR
These new outcome measures are liminary studies demonstrated improve- pathway, which is overactive in FXS
now being employed in targeted treatment in several behaviors on standardized patients. It is inexpensive and available
ment trials for FXS. In 2013, Andrea questionnaires in children with FXS when in its generic form. In 2017, after the
Schneider at the MIND Institute, in the ointment was administered topically drug treated the behavioral, cognitive,
collaboration with me and other col- on the shoulder over a 12-week period. and neuroanatomical features of FXS
leagues, used ERPs to determine that This positive open-label study where all in FMR1 knockout mice and in a Dro-
auditory habituation to repetitive patients received the medication, not pla- sophila model of FXS, 11,12 my colleagues
sounds improved in children with FXS cebo, is still ongoing in Australia and has at the MIND Institute and I tested met-
who received minocycline, which lowers led to a controlled trial of this CBD oint- formin in obese patients with FXS. We
MMP-9, compared with those receiv- ment, rubbed twice a day on the shoulder, found that after just a few months, not
ing a placebo.6 Patients on minocycline at multiple clinics in the US and Australia only did it reduce weight gain, families
also had lower levels of an MMP-9 pro- for children ages 3 to 18. said that their children could communi-
tein biomarker that is elevated in FXS.7 This follow-up study points to cate better and carry on a conversation
And earlier this year, the MIND’s David another trend in FXS research: the treat- when they previously could not.13 This
Hessl and colleagues demonstrated that ment of young FXS patients. As children, prompted us to launch a controlled trial
an mGluR5 antagonist that had shown patients’ brains have more time, and with patients 6 to 25 years old with mul-
promise in mouse models but failed may be better equipped, to build syn- tiple electrophysiological outcome mea-
to beat the placebo in controlled tri- aptic connections. At the MIND Insti- sures, including eye tracking and ERPs
09. 201 9 | T H E S C IE N T IST 47

THE FRAGILE X
MUTATION
Fragile X syndrome is caused by an expansion of CGG nucleotide repeats in
the FMR1 gene at the end of the long arms of the X chromosome. To identify
the mutation, researchers culture cells in media deficient in folic acid, which
causes the ends of the X chromosome to appear as though they are about to
break off. Before molecular testing, this was the only way to see the mutation.
The FMR1 gene encodes the fragile X mental retardation protein (FMRP),
which regulates gene expression and protein translation in the brain. FMRP is
important for maintaining synaptic plasticity and the ability to make new neu-
rons. Levels of FMRP associated with disease severity in patients with FXS.
Normal X chromosome Fragile X chromosome
NORMAL PREMUTATION FULL MUTATION

CGG repeats < 55 55 ≤ CGG repeats ≤ 200 CGG repeats > 200
FMR1 GENE
FMR1 mRNA
X
FMR PROTEIN
X
FXTAS, FXPOI,
TYPICAL FXS
NEUROPYSCHIATRIC PROBLEMS
Premutation: The FMR1 gene has 55 to 200 CGG repeats that are not methylated. Pre- Full mutation: The FMR1 gene has more
mutations with fewer than 120 CGG repeats typically lead to normal FMRP levels; more than 200 repeats of the nucleotides CGG
THE SCIENTIST STAFF
repeats lead to lowered FMRP production, though this doesn’t necessarily translate to that are heavily methylated. Males com-
more severe disease. Premutations of any number of repeats can result in higher FMR1 pletely lack FMRP, while females typi-
mRNA levels, which can cause problems for the individual even if FMRP protein levels cally have some protein produced from
are normal. Collectively these problems are referred to as fragile X–associated disor- the FMR1 gene on the healthy X chromo-
ders; they include fragile X–associated tremor/ataxia syndrome (FXTAS), primary ovar- some. Individuals carrying a copy of the
ian insufficiency (FXPOI), and neuropsychiatric problems. full mutation present with FXS.
INHERITANCE at the MIND and at two sites in Canada.

IN MALES: Full mutations revert back to premutations during development of X chromo- The trial is ongoing, but already some
some–carrying sperm. Thus, men with the full mutation or the premutation pass the premuta- doctors are beginning to prescribe met-
tion on to their daughters. Because it is X-linked, they cannot pass it on to their sons. formin for some FXS patients.
IN FEMALES: Premutations of 100 CGG repeats or more convert to the full mutation during
A new way ahead
egg development or in the embryo. Thus, women with the full mutation or a premutation with
For the foreseeable future, treating FXS
100 repeats or more on one of their X chromosomes pass on the full mutation to their children
will be a matter of managing symptoms
approximately 50 percent of the time. Women with a premutation of fewer than 100 repeats
and working through therapy to support
will pass on a full mutation less often.
healthy development. There are a few
Wildtype (WT) gene: < 55 CGG repeats Long premutation: > 100 CGG repeats early-stage trials for FXS treatments,
Short premutation: ≤ 100 CGG repeats Full mutation: > 200 CGG repeats and one, the FX-LEARN trial, involves
targeted therapies that could reverse
some of the pathways that are dys-
functional due to the absence or defi-
MAN WITH ANY ciency of FMRP. However, because so
WILDTYPE WOMAN
FORM OF MUTATION
many pathways are problematic in FXS,
it is likely that more than one targeted
treatment will need to be utilized in the
treatment of FXS. Thus, a cure for the
disease remains a long way off.
Continued clinical work has improved
the educational and behavioral interven-
tions that can help young children with
Premutation in daughters Y chromosome to sons FXS. One example developed by Len
Abbeduto and his team at the MIND
Institute, is teaching parents to work
WOMAN WITH FULL
directly with their children on language
WILDTYPE MAN MUTATION OR LONG development. We are currently using the
PREMUTATION so-called parent-implemented language
intervention (PILI) in our FX-LEARN
study, and we have demonstrated effi-
cacy with PILI alone to improve lan-
guage skills in kids with FXS.14
We are making progress, but we have
a long road to travel and will need to
involve multimodal interventions for the
Full mutation in 50% of children
best effect. As we improve our treatments
for FXS, we want to apply these treat-
ments to those in Ricaurte, Colombia, and
elsewhere. We have returned to Colombia
WOMAN WITH SHORT
WILDTYPE MAN and found other centers where fragile X
PREMUTATION
is common, so there is much work to be
done regarding further testing and treat-
ment endeavors. We must use the tools we
have today while pursuing the knowledge
that will yield the cure. g
Randi Hagerman is a Distinguished

Professor of Pediatrics at the MIND
Some form of premutation or full mutation in 50% of children
Institute and Department of Pediatrics,
University of California, Davis, Medi-
Occasionally, if both parents carry premutations or full mutations, their daughters can
cal Center, Sacramento.
inherit two fragile X chromosomes: one with a premutation from the father and the
other with either a premutation or a full mutation from the mother.
09. 201 9 | T H E S C IE N T IST 49
References CONSEQUENCES OF THE FRAGILE X PREMUTATION

1. W. Saldarriaga et al., “Genetic cluster of fragile X
Patients with the fragile X premutation have normal to low levels of the fragile X men-
syndrome in a Colombian district,” J Hum Genet,
63:509–16, 2018.
tal retardation protein (FMRP) and are usually unaffected intellectually. Counterintui-
2. E. Berry-Kravis, “Mechanism-based treatments tively, however, they actually exhibit increased FMR1 mRNA expression, and the excess
in neurodevelopmental disorders: Fragile X transcripts often lead to health problems, from neuropsychiatric disorders to repro-
syndrome,” Pediatr Neurol, 50:297–302, 2014. ductive issues.
3. E.M. Berry-Kravis et al., “Drug development for
The RNA itself can be toxic to neurons, leading to dysregulation of calcium levels
neurodevelopmental disorders: Lessons learned
from fragile X syndrome,” Nat Rev Drug Discov,
and mitochondrial dysfunction that cause the cells to die more easily in culture. More-
17:280–99, 2018. over, elevated levels of FMR1 mRNA can sequester proteins that are important for neu-
4. E. Berry-Kravis et al., “Mavoglurant in fragile X ronal function in the nuclei of neurons and astrocytes throughout the brain and periph-
syndrome: Results of two randomized, double- eral nervous system. This sequestration dysregulates the levels of certain miRNAs and
blind, placebo-controlled trials,” Sci Transl Med,
somehow leads to increases in calcium ions in neurons, upping the likelihood that the
8:321ra5, 2016.
5. E.A. Youssef et al., “Effect of the mGluR5-NAM
cells will fire an action potential. These changes somehow cause mitochondrial dys-
basimglurant on behavior in adolescents and function. Lastly, there is evidence that translation begins at an aberrant start site in the
adults with fragile X syndrome in a randomized, FMR1 mRNA, leading to the production of a protein called FMRPolyG that is toxic to
double-blind, placebo-controlled trial: FragXis the neuron.
phase 2 results,” Neuropsychopharmacology,
How these issues relate to one another and affect patient outcomes is unclear.
43:503–12, 2018.
6. A.Schneider et al., “Electrocortical changes
People with the premutation suffer from high rates of anxiety and depression, along
associated with minocycline treatment in fragile with other neuropsychiatric conditions such as attention deficit/hyperactivity disor-
X syndrome,” J Psychopharmacol, 27:956–63, der, chronic fatigue syndrome, and migraines. The label of fragile X–associated neu-
2013. ropsychiatric disorders (FXAND) has been given to these conditions.15 The fragile X
7. M. Dziembowska et al., “High MMP-9 activity
premutation is also the most common genetic cause of early menopause, before age
levels in fragile X syndrome are lowered by
minocycline,” Am J Med Genet A, 161:1897–903,
40, and about 20 percent of female carriers suffer from this condition, called fragile X–
2013. associated primary ovarian insufficiency (FXPOI).
8. D. Hessl et al., “Effects of mavoglurant on visual The most severe problem associated with the premutation is fragile X–associ-
attention and pupil reactivity while viewing ated tremor/ataxia syndrome (FXTAS), a neurodegenerative disorder that develops
photographs of faces in fragile X syndrome,”
in more than 40 percent of males and 16 percent of females carrying the premuta-
PLOS ONE, 14:e0209984, 2019.
9. L. Greiss Hess et al., “A randomized, double-
tion.16 FXTAS typically presents in patients’ 60s as tremors followed by balance
blind, placebo-controlled trial of low-dose problems. In men, and very rarely in women, it can also be associated with dementia
sertraline in young children with fragile X as it progresses. Patients with FXTAS show areas on an MRI scan that look extra-
syndrome,” J Dev Behav Pediatr, 37:619–28, white in several brain regions, indicating damage to the white matter. When a
2016.
sample of the brain is viewed under the microscope, the white matter looks moth-
10. K.H. Yoo et al., “Low-dose sertraline improves
receptive language in children with fragile X
eaten. In addition, these patients develop spherical structures within the nucleus,
syndrome when eye tracking methodology is used called inclusions, of excess FMR1 mRNA and the proteins that it sequesters. These
to measure treatment outcome,” J Psychol Clin inclusions are a pathological marker of FXTAS.
Psychiatry, 7:00465, 2017. In the Ricaurte, Colombia, FXS hotspot, we see more, and more-severely affected,
11. R.E. Monyak et al., “Insulin signaling
premutation carriers than in other regions of the world. The more severe involvement
misregulation underlies circadian and cognitive
deficits in a Drosophila fragile X model,” Mol
may be related to exposure to environmental toxins, perhaps the pesticides used in
Psychiatry, 22:1140–48, 2017. agriculture around the village. Premutation carriers tend to be more sensitive to envi-
12. I. Gantois et al., “Metformin ameliorates core ronmental toxins because the health of their neurons is already threatened by RNA
MODIFIED FROM © ISTOCK.COM, PHOTO5963

deficits in a mouse model of fragile X syndrome,” toxicity. In addition, there may be effects of poverty or drug use. Research now aims to
Nat Med, 23:674–77, 2017.
tease apart the genetic and environmental factors that play a role in outcomes for both
13. A.B.C. Dy et al., “Metformin as targeted
treatment in fragile X syndrome,” Clin Genet,
premutation carriers and those with full-blown FXS.
93:216–22, 2018.
14. S. Nelson et al., “Inferential language use by
school-aged boys with fragile X syndrome:
Effects of a parent-implemented spoken language
intervention,” J Commun Disord, 72:64–76, 2018.
15. R.J. Hagerman et al., “Fragile X-associated
neuropsychiatric disorders (FXAND),” Front
Psychiatry, 9:564, 2018.
16. S. Jacquemont et al., “Penetrance of the
fragile X-associated tremor/ataxia syndrome
in a premutation carrier population,” JAMA,
291:460–69, 2004.
Read The Scientist on your iPad!

EDITOR’S CHOICE PAPERS
The Literature
CELL & MOLECULAR BIOLOGY
Speedy Stomata Guard Cells
THE PAPER
M. Papanatsiou et al., “Optogenetic manip-
K+
ulation of stomatal kinetics improves car-
BLINK1
bon assimilation, water use, and growth,”
Science, 363:1456–59, 2019.
In plants, guard cells control the stoma-

tal opening by expanding and contract-
ing through ion flux. When a plant senses
light, a signal is sent to the guard cell to
increase its ion content, which causes the
cell to take in water and swell in size. In
this state, the stoma is open so that it can
start taking in carbon dioxide for photo-
synthesis. When there’s no light available
for photosynthesis, the plant closes its
stomata so it doesn’t lose water through
evaporation. Biologists suspected that OPEN AND SHUT: To test whether the speed of stomata opening and closing can increase productivity and water
the speed at which the guard cells react use efficiency, researchers added the synthetic light-activated BLINK1 ion channel to guard cells in Arabidopsis  1. .
to changing conditions affects plants’ pro- The channel allowed potassium ions to move into a cell within two minutes of activation by blue light  2.. The
increase in ions inside the cell caused the guard cells to take in water and swell in size, opening the stomata to allow
ductivity and water use efficiency.
entry of carbon dioxide  3.. Within 8 to 10 minutes of being in darkness, the BLINK1 channel’s activity decreased
To test this idea, a team of research- and the guard cells began to shrink, closing the stomata and preventing water from leaving the cell  4..
ers borrowed a tool from neurobiology:
optogenetics, which enables scientists to
use light to control membrane voltage Plants with the BLINK1 channels were in light that demonstrate the effect of the
and thus activate neurons experimen- able to open the stomata more quickly channel, says Leonhardt. But she adds
tally. Changing the voltage of a cell is rel- than control plants, presumably allow- that those conditions are “not very real-
atively easy, says study coauthor Michael ing them to get carbon dioxide sooner. istic,” and that natural light levels might
Blatt of the University of Glasgow, “but it’s The modified plants were also able to not fluctuate as quickly. Thus, BLINK1’s
much more of a challenge to move enough close their stomata more quickly than the speed may not offer much of an advantage
ions to change the total ion content of the controls when put in darkness, prevent- in the field.
cell.” The researchers used genetic engi- ing them from losing water unnecessarily. Blatt notes that this study is a proof
neering and a cell-specific promoter to Likely due to their increased efficiency, the of concept, and the first demonstration
add BLINK1, a light-controlled synthetic plants with BLINK1 grew 2.2 times faster that it is possible to engineer crops to
potassium channel, to guard cells in Ara- than control plants kept under the same improve water use efficiency without
bidopsis thaliana to see if they could move conditions, the authors report. sacrificing growth by controlling the
enough ions for the cells to take in water “It’s quite interesting and very inno- speed of the opening and closing of the
and increase in size. vative,” says plant electrophysiology stomata. The next step, he says, is to look
Within two minutes of exposure to researcher Nathalie Leonhardt of France’s into other ways to change that speed
© KELLY FINAN
blue light, the BLINK1 channel opened, Biosciences and Biotechnologies Institute without the use of the BLINK1 channel,
allowing potassium ions to move into the of Aix-Marseille who was not involved in such as by studying genes that affect
cell. The guard cells swelled as a result, the study. The study’s setup exposes the guard cells’ capacity to change size.
making the stomata bigger. plants to fluctuations and quick changes —Chia-Yi Hou
A BEVY OF VIRUSES: Scientists continue to uncover new branches of the SCANNING FOR CLUES: Researchers link levels of a receptor in the brain
viral family tree. to suicidal ideation in people with post-traumatic stress disorder.
MICROBIOLOGY DISEASE & MEDICINE
Family Discovery A Suicide Biomarker?

THE PAPER THE PAPER
A.A. Abbas et al., “Redondoviridae, a family of small, circular M.T. Davis et al., “In vivo evidence for dysregulation of mGluR5
DNA viruses of the human oro-respiratory tract associated as a biomarker of suicidal ideation,” PNAS, 116:11490–95, 2019.
with periodontitis and critical illness,” Cell Host & Microbe,
25:P719–29.E4, 2019. People with major depressive disorder often have other mental
health problems, such as post-traumatic stress disorder. But while
A few years ago, while using DNA sequencing to study the studying depression a few years ago, Yale University psychiatrist
lung microbiomes of people who had received lung transplants, Irina Esterlis noticed that the brains of people with PTSD
researchers at the University of Pennsylvania stumbled across some looked different from those in people with depression alone. In
unexpected sequences. “We saw a little bit of alignment from some particular, people with PTSD had higher levels of a protein called
of our reads to a pig virus that hadn’t been studied very much,” metabotropic glutamate receptor 5 (mGluR5) on the surface of
says microbiologist Frederic Bushman of the university’s Perelman their brain cells, she says. “That was really odd to us.”
School of Medicine. Esterlis and her colleagues wondered whether the receptor,
The scientists assembled the sequences and discovered two which had recently been linked to suicidal behavior, might
completely novel, circular viral genomes unlike anything they’d seen tell them something about suicidal thinking, or ideation, in
before in humans. Suspecting there might be more of these viruses PTSD patients. Using brain scans, the team found that in
out there, the team searched metagenomic databases of other lung 29 people with PTSD (some of whom also had depression),
and mouth samples and identified 17 genomes that closely resembled “the upregulation of mGluR5 availability was specific to
the first two. The researchers dubbed this new family redondoviruses people who were thinking suicidal thoughts,” as measured by
(“redondo” means “round” in Spanish). “Any time we find a new questionnaires on the day of the scan, Esterlis says. No such
family of viruses, it’s always exciting, because it fills holes in our relationship was found in people with depression alone, or in
understanding of viral taxonomy,” says Columbia University virologist healthy controls.
Ian Lipkin, who was not involved in the work. That mGluR5 levels only predicted suicidal ideation in people
Redondoviruses were particularly abundant in samples from with PTSD is remarkable, says Greg Ordway, a neurobiologist at
people with periodontal disease and from critically ill patients in East Tennessee State University who studies suicide but was not
intensive care. However, disease links remain unclear, says Lipkin. involved in this work. “That would imply that there’s a different
© ISTOCKPHOTO.COM, DEDMITYAY; RALWEL
“I’d like to know what cells [these viruses are] growing in, and I’d biology to suicidal ideation in some disorders than others.”
like to see whether or not there’s an antibody response indicative of However, he cautions that the data show substantial overlap
infection.” The UPenn team is working to answer these questions, in mGluR5 levels between PTSD patients who were having
first by trying to grow redondoviruses in culture, says Bushman. suicidal thoughts and those who weren’t, limiting the receptor’s
This approach is as important as the discovery of the new virus usefulness as a biomarker of suicidal ideation on its own.
family, notes virologist David Wang of Washington University in Esterlis’s team is conducting further research on mGluR5’s
St Louis, who was not involved in the work. “Expanding the [viral] biological role in PTSD, and whether the findings could help
catalog is useful,” he says. “But the catalog is already huge. What we identify people who might be prone to developing the condition.
really need is to find associations, set up culture systems, and set up Additionally, she says, “we are trying to evaluate whether we can
animal models so these viruses can actually be characterized.” modulate mGluR5 . . . so that we can potentially treat PTSD.”
—Catherine Offord —Catherine Offord
09. 201 9 | T H E S C IE N T IST 53

PROFILE
Defining Rare Disorders

Judith Hall was instrumental in incorporating genetics into medical
practice, describing previously unknown disorders.
BY ANNA AZVOLINSKY
A
t a 1978 genetics conference in Montana, Philip Pal- found that the mortician had wrapped the baby in a formal-
lister, then the clinical director of the Boulder River dehyde-soaked cloth and because the baby had never eaten,
School and Hospital, asked medical geneticist Judith it was not decomposed at all. “It’s the anaerobic bacteria in
Hall and a group of other researchers a rather startling ques- the gut that decompose bodies, and this infant turned out to
tion: Should we dig up a decomposing baby? be fully preserved,” Hall says. Also fully preserved: a brain
“This story is ghoulish,” Hall tells The Scientist. tumor close to the pituitary gland.
The baby had been born with extra fingers, had issues Over the next six months, Pallister, Hall, and their collab-
with its anal opening, and hadn’t survived more than a few orators identified an additional four cases of the mysterious
hours after birth. The child’s father didn’t even know that syndrome, all seemingly without a genetic cause. The team
his baby had died when the medical team asked him if they published their findings in 1980. It took another 16 years
could perform an autopsy on the child. “He tore up the form before medical geneticist Leslie Biesecker and colleagues
and went to grieve with his wife,” Hall recalls. Three months connected autosomal dominant mutations in the GLI3 gene,
later, the couple asked for the results of the autopsy, but with- which plays a role in embryo development, including for-
out permission, the medical team hadn’t done one, and the mation of the digits and the brain, to Pallister and Hall’s
baby had been buried. cases. The research would come to characterize Hall’s career,
At the conference, Pallister hypothesized that the baby and similar approaches to mysterious cases would help her
might have had dwarfism, and that’s when he proposed dig- describe other novel disorders.
ging up the body for further analysis. However, Hall recalls,
X-rays taken of the baby after birth had not shown an abnor-
mal pelvic shape, a common characteristic seen in most forms
of dwarfism. As a result, the researchers at the conference My medical training was always tied to
decided that they wouldn’t stand to gain any new information genetics. I grew up with genetics, you
by analyzing the baby’s now decomposing body. could say.
Hall, then a researcher at the University of Washington,
flew back home and returned to her work at Seattle Children’s
Hospital. Her first day back, she saw an infant with the same
features as the ones Pallister had described—an extra fin- “It was an incredible saga. I mean, who digs up a baby that
ger, a missing anal opening, and a similar-looking X-ray. Hall has been buried?” says Hall, now a professor emerita of pedi-
recalls wondering whether there was some sort of epidemic. atrics and medical genetics at the University of British Colum-
The baby went into surgery to correct the anus but died on bia in Canada. “But, if you are curious and want to get to the
the operating table. This time, however, the medical team was bottom of things and want to do that for the families that are
allowed to perform an autopsy, and it revealed a huge tumor affected, sometimes you go to rather unusual lengths.”
in the baby’s brain, pressing on the pituitary gland.
“The pituitary gland responds to stress and is partly respon- RAISED WITH GENETICS
sible for production of cortisol. The cortisol literally helps keep Hall was born in Boston in 1939. She was part of a family of
you alive, and the lack of the hormone likely contributed to the seven—including an older and younger brother and two younger
baby’s inability to get through the surgery,” Hall says. sisters—and remembers making soap, rationing food, and eat-
Despite the tragic outcome, the baby’s autopsy led Hall to ing meat on Sundays only during World War II. Later, the fam-
guess that the other child that didn’t have the autopsy might ily moved to Manchester, New Hampshire, where Hall says she
also have had a similar tumor in the brain. She called Pallister played outdoors, built forts, and collected flowers and bugs.
and told him that she thought they should dig up and exam- Hall’s mother, Louise, had infused Hall with this interest in
KIMI TANAKA
ine the baby that he had described at the conference. After the natural world, encouraging Judith and her siblings to study
receiving permission from the child’s parents and the police, plants, insects, and birds. “There was total wilderness around.
the baby was exhumed. During the examination, the team We would roam and collect specimens outside,” Hall recalls.
Hall did well in her high school classes and entered

Wellesley College in Massachusetts in 1957, choosing a sci-
ence-heavy curriculum. Genetics was still relatively new at
the time—many scientists still presumed that there were
48, not 46, human chromosomes—but Hall’s zoology pro-
fessor was tracking developments in modern biology and
taught Hall and other students about new discoveries as they
emerged, including the confirmation that the human genome
did in fact have 46 chromosomes. The professor also intro-
duced Hall to medical genetics, which Hall chose to focus on
when she entered medical school at the University of Wash-
JUDITH HALL ington in 1961.
Professor Emerita, Departments of Pediatrics and Medical Genetics, There, Arno Motulsky had recently established the school’s
Faculty of Medicine, University of British Columbia, Canada Division of Medical Genetics—one of the first such depart-
Investigator Emerita, British Columbia Children’s Hospital, Canada ments in the country—and was offering a course on the topic.
David L. Rimoin Lifetime Achievement Award from the Hall was the only student who signed up, so she received
American College of Medical Genetics and Genomics Foundation personal tutorials from Motulsky. “My medical training was
for Genetic and Genomic Medicine (2018) always tied to genetics,” Hall says. “I grew up with genetics,
Canadian Medical Hall of Fame (2015) you could say.”
Canadian College of Medical Geneticists—Founders Award (2013)
Royal Society of Canada, Fellow (2011) INTRIGUING CASES
Canadian Paediatric Society, Lifetime Member (2008) After completing her degree, Hall stayed an extra year in
Best Doctors, “Best Doctor in Canada” 2003, 2007–2008 Motulsky’s lab so that she could continue her experiments
American Pediatric Society President (2001–2002) growing adult bone marrow cells in culture under low-oxygen
Ross Award, Canadian Paediatric Society (2002) conditions. She wanted to test whether such hypoxic envi-
Order of Canada (1998) ronments could cause the cells to express fetal hemoglobin in
March of Dimes Colonel Sanders Award for Lifetime Achievements addition to or instead of adult hemoglobin. The project was
in the Genetic Sciences (1994) an attempt to understand the switch from fetal to adult hemo-
globin, which occurs around the time of birth, and whether
Greatest Hits the wild type fetal gene copy could be turned on in individuals
• Showed that expression of fetal hemoglobin can be increased in with mutated adult hemoglobin, including those with sickle
adult bone marrow cells in culture under low-oxygen conditions. cell anemia. (See “In Our Blood,” The Scientist, July/August
• Created a handbook of normal physical measurements to help 2019.) In her first publication, which appeared in Nature in
identify syndromes in the clinic. 1968, Hall found that under low-oxygen conditions, more
• Was instrumental in developing the protocols for documenting fetal hemoglobin was indeed expressed in cell cultures.
abnormal physical features through photographs and videos and The result got Hall thinking about the multitude of
for developing natural history studies of disorders. changes that occur during development, as well as the impor-
• With colleagues, described a novel genetic syndrome, Pallister-Hall tance of tracking the course of a disorder from its onset—what
syndrome, which affects many parts of the body and includes extra medical geneticists call a disease’s natural history. “Medical
digits on the hands and/or feet. geneticists are usually doing three things at once: trying to
• Published more than 100 articles on arthrogryposis—a variety of make the very specific genetic diagnosis, thinking about pos-
disorders characterized by contracted joints—and helped to map sible prevention and therapies, and at the same time trying to
the chromosomal loci and biochemical pathways dysregulated in find the basic mechanism and learn about the natural history
the group of disorders. of the disorder so they can share all of that with the family,”
09. 201 9 | T H E S C IE N T IST 5 5

PROFILE
Hall says. “This last part is the clinician-scientist part that is ries. She credits the families she has worked with for helping
so interesting to me.” her answer questions about their children’s disorders. “I have
After medical school, Hall moved with her husband, Phil- never known a family that didn’t want to help understand their
lip, to Maryland to begin a one-year internship in pediatrics child’s condition and was not curious to meet other families
at Baltimore City Hospital in 1966. Around the same time, the with similar experiences,” she says. “They ask the best ques-
couple’s first daughter was born, and Hall would go on to have tions and challenge you because they are the ones living with
another daughter and a son. “I learned so much by becoming the disorders on a daily basis.”
a mother,” she says. “I began to understand how parents feel In 1981, Hall moved to the University of British Columbia,
about their children, the hormonal responses during preg- and over the next three decades, she developed and published
nancy and when you’re a new mother, and how to talk to fam- on the tools, such as photography, necessary to document
ilies, and also experiencing your child go[ing] through their abnormal physical features. She described how to character-
developmental stages. It was a real insight into pregnancy ize behaviors associated with genetic disorders and explained
and the maternal experience.” how to document the natural history of genetic disorders that
included congenital, physical features. In the clinic, Hall iden-
COLLECTING RARITIES tified families affected by genetic disorders, then collected
After her internship at the hospital ended, Hall stayed in Bal- and shared data with laboratory geneticists, who would some-
timore, first doing an internship in medicine and pediatrics, times succeed in identifying the genes responsible. In 1989,
followed by a fellowship in medical genetics, a residency in she and colleagues also published a clinical handbook on nor-
pediatrics, and then a fellowship in pediatric endocrinology, mal physical measurements to facilitate clinicians’ diagnoses
all at Johns Hopkins University. She chose Hopkins specifi- of patients with abnormal features.
cally to train with Victor McKusick, who established the first “[The crux of my work] is meticulously identifying the
medical genetics clinic at the university. He was involved features of a disorder and thinking about how the body
in defining birth defects, and during her fellowship, Hall works and how these features will evolve as the child
focused on dwarfism. She began to collect familial cases that grows,” says Hall.
helped sort out common forms of dwarfism and whether these Disorders that Hall played a leading role in identifying
disorders are inherited in a dominant, recessive, or X-linked and characterizing include achondroplasia, a bone growth
fashion. During that time she also described a new disorder disorder that causes disproportionate short stature; Turner
in which patients had no radius—a bone in the forearm—and syndrome, a female-specific short stature syndrome that is
a blood platelet deficiency. After observing 13 cases in the accompanied by early loss of ovarian function; and Noonan
clinic and identifying 27 additional cases in the literature, syndrome, which includes symptoms such as unusual facial
Hall and her colleagues named the disorder thrombocytope- features, short stature, and heart defects. However, the group
nia with absent radius. of disorders that Hall has devoted the most time to—what she
calls her “favorite”—are collectively known as arthrogryposis
and characterized by some joints being contracted, result-
ing in stiffness and immobilization of the joints and overall
[The crux of my work] is meticulously muscle weakness. She has published more than 100 articles
on arthrogryposis and helped to map the chromosomal loci
identifying the features of a disorder
and biochemical pathways that are dysregulated in this group
and thinking about how the body works of disorders. Five years ago, she put together two reviews
and how these features will evolve as the describing arthrogryposis cases accumulated over 40 years.
child grows. The goal of the articles was to provide information to better
diagnose the disorders.
Hall, now 80, is still hard at work. She no longer frequents
the clinic, nor is she a salaried employee at the university. But
In 1972, Hall accepted an assistant professor position in the she continues to pore over data that she has collected and to
division of medical genetics at the University of Washington write research articles. Currently, she is working on a paper
School of Medicine. She began to document a wide spectrum that tabulates all of the known genes (about 410 so far) that
of genetic disorders. This was the time when she worked on the can cause arthrogryposis.
cases of the babies with the brain tumors, extra fingers, and “I won’t stop being interested in genetics. It’s such an
affected anuses, a condition that became known as Pallister-Hall exciting time with new studies on transgenerational effects
syndrome. The clinic, Hall says, was her laboratory, where she of genetics and epigenetics and new ways to identify genes
would document cases of rare diseases and identify patterns involved in disorders,” she says. “I can’t imagine not being
that helped identify new syndromes or syndrome subcatego- involved.” g
SCIENTIST TO WATCH
Cigall Kadoch: Unraveling Cancer

BY CHIA-YI HOU
W
hen Cigall Kadoch was a She decided to study the complex she could cofound Foghorn Therapeutics,
teenager, a close family friend more carefully, and a few months after hoping to drive the development of
was diagnosed with late-stage her papers with Crabtree were published, treatments for cancer and other diseases
breast cancer. As Kadoch processed the she was invited to join the Dana-Farber influenced by changes in chromatin. So
news, she became deeply frustrated. “I Cancer Institute. She started in January far she has helped to raise $50 million in
didn’t understand what cancer was, why 2014, at 28 years old—one of the youngest venture capital funding for the company.
it could possibly take someone’s life so assistant professors hired there to date. “Her career is already flying,” says
quickly,” she says. That experience led That same year she was named one of Crabtree, a cofounder of Foghorn Therapeutics.
her to learn more about cancer, starting Forbes Magazine’s 30 Under 30 in Science “I think Cigall will be able to develop into a
in high school just north of San Francisco & Healthcare. Since then, Kadoch’s work major leader in cancer biology.” g
and throughout her undergraduate has focused on how the BAF complex may
studies in molecular and cell biology at suppress tumor formation (Nat Genet,
the University of California, Berkeley. Her 49:213–22, 2017), and how changes to the
interest only grew, and she went on to complex can activate different genes that
study cancer biology as a graduate student lead to cancer (Cell, 171,1:163–78.e19, 2017).
at Stanford University in 2009. “She is a, if not the, world leader in
“She walked into my lab one day,” this particular complex,” says Dana-
says Gerald Crabtree, a pathologist at Farber’s Scott Armstrong, chair of the
Stanford who became Kadoch’s PhD pediatric oncology department where
and postdoc advisor, and “she knew so Kadoch works. He adds that she
much and was energetic . . . I was hugely is passionate about her research:
impressed by her.” Crabtree studied “Her most defining characteristic is
vertebrate development and chromatin, enthusiasm for science.”
the collection of DNA and proteins that Crabtree agrees. “We called
comprise chromosomes. Chromatin is her ‘science in heels’ because
constantly being restructured, and how she always wore heels,” he says,
it’s regulated can affect gene expression, explaining that he could always
in some cases leading to cancer. This idea tell how good the results of her
captivated Kadoch, and she immediately experiments would be by the
started studying the connection between sound and energy of her
chromatin and cancer. walk down the hallway
Kadoch published two of her first to his office.
big papers with Crabtree in 2013, Staying true to her
as a postdoc in his lab. She and her original motivation
collaborators found that, for human for pursuing a career
synovial sarcoma, a rare form of soft- in cancer research,
tissue cancer in children, translocation Kadoch says she’s
© KEN RICHARDSON PHOTOGRAPHY
of 78 amino acids to one subunit of a also determined

chromatin-remodeling complex, the BAF to see her science
complex, leads to the loss of a tumor turned into therapies
suppressor, causing the cells to become to help patients.
cancerous (Cell, 153, 1:71–85, 2013; Nat Over the past few
Genet, 45: 592–601, 2013). That a change years, she has
in a single subunit of the complex could turned down money
be a driving factor for some types of from pharmaceutical
cancer was fascinating, Kadoch says. companies so that
09. 201 9 | T H E S C IE N T IST 57

LAB TOOLS
Genetics’ Next Top Models

You’re well-acquainted with Drosophila, C. elegans, and the humble lab
mouse, but lesser-known model systems are poised to make their mark.
BY AMBER DANCE
G
eneticists tend to crowd around a few favorite organ-
isms that have long histories in research and a wealth
of practiced protocols for manipulating their genes. But
those organisms aren’t always the best choice to answer a scien-
tific question, leading some researchers to use other, less pop-
ular models. Organisms from elsewhere across the tree of life
might offer unique genetic structures, physiology closer to that
of humans, or faster generation times to accelerate investigations
into gene regulation, evolution, and development.
Historically, given the lack of detailed gene-linkage maps
and extensive mutant libraries for such organisms, progress with
little-used models was frustratingly slow. But the advent of
genomics and transcriptomics has created a “democratization” of
model systems, says John (Jack) Werren, an evolutionary geneti-
cist at the University of Rochester in New York.
“Once you have the genomic information, then you can target genes
much more efficiently,” says Werren. Researchers can match phenotype
to genotype with ease, and design CRISPR sequences to edit genomes.
“Lots of people now are developing their individual systems.” Oxytricha
Working with up-and-coming models still has its downsides. trifallax
Some are used only by a dozen or so labs, which means it takes
longer to adapt new technologies such as CRISPR editing. None-
theless, many scientists are happy with model organisms that GOOD FOR STUDYING
strut the runway less traveled. Here, The Scientist profiles four • Chromosome biology—specifically, studies of chromatin dynam-
such models and their potential for discovery. ics and organization, genome rearrangement, and transposons.
• RNA-DNA interactions and epigenetics, because the organism
GENOME REMIX: OXYTRICHA TRIFALLAX uses parental RNAs to guide chromosome reassembly follow-
ing sexual reproduction.
CLAIM TO FAME: MULTIPLE NUCLEI
O. trifallax, like other ciliated protozoans, has more than one GENOME
nucleus, and it organizes its multiple genomes in an extraordi- Each archival nucleus contains about 120 chromosomes in a dip-
nary way. The macronucleus holds the working genome that car- loid arrangement, says Laura Landweber, a molecular evolutionary
ries the genes the organism needs for everyday living and keeps biologist at Columbia University who, with colleagues, assembled
them ready for transcription. The micronuclei, of which O. tri- O. trifallax’s genome sequence in 2014 (Cell, 158:P1187–98). The
fallax has two, act as silent archives, storing the entire genome. working nucleus, on the other hand, contains about 18,000 distinct
The catch is that the archival genomes are messy, probably types of chromosomes, at Landweber’s latest estimate, up from the
because active transposons leap about, fragmenting and scat- 16,000 she reported in 2013 (PLOS Biol, 11:e1001473). With an aver-
tering genes as they go. No matter, because O. trifallax uses age copy number of 2,000 per chromosome type, that’s 36 million
RNA guides to put those genes back together into a new work- chromosomes total. Each macronucleus chromosome typically holds
GRAHAM MATTHEWS
ing nucleus for each daughter cell after sexual reproduction. In one gene, with lengths ranging from 469 base pairs to 66 kilobases.
a way, O. trifallax is like a multicellular organism within one
membrane: the archival nuclei, accessed during sexual repro- REPRODUCTION
duction, mirror the germline, while the working nucleus acts When foods such as algae are plentiful, O. trifallax reproduces
like the nucleus of somatic cells. asexually. The archival nuclei divide by mitosis, while the work-
09. 201 9 | T H E S C IE N T IST 59

LAB TOOLS
ing nucleus divides its chromosome complement in half. In the

lab, the organism can double twice a day. Schizosaccharomyces
Under stressful conditions, such as starvation, O. trifallax gets japonicus
frisky. The organism comes in a handful of mating types—the exact
number isn’t known—and must find a compatible type for conjuga-
tion. Two complementary cells fuse, swap genetic material from their
archival nuclei, then split again. The old working nucleus in each cell
dissolves, and a new one is assembled from the archival genomes.
GENETIC ENGINEERING
Researchers can create artificial chromosomes to add genes to the
working nucleus and study chromosome dynamics. RNAi works too,
with injections of short interfering RNAs reducing the production of
corresponding proteins. O. trifallax has its own CRISPR-like genome-
editing system that scientists can used to knock out or modify genes.
Inserting nucleic acids to make these genomic changes is easily accom-
plished by microinjection, because the organisms are relatively large mitosis. And its centromeres are more like those of humans than
at 100 microns across. S. pombe’s are.
• Oxygen-free metabolism, because S. japonicus doesn’t respire
STRAIN COLLECTIONS oxygen.
Labs share O. trifallax strains with one another; Landweber’s
group possesses a handful of wild-type strains, and dozens of GENOME
genetically engineered lab strains. S. japonicus has a haploid genome of three chromosomes; the
sequence was published in 2011 (Science, 332:930–36).
DOWNSIDE
Landweber longs to do large-scale genetic screens, to transform REPRODUCTION
O. trifallax cultures with an entire library of genes, for example. S. japonicus can reproduce asexually, doubling every two hours,
However, to do so requires protocols that would make it easy to or sexually. To control remixing of genes, researchers in Japan
generate many transformants at once. With microinjection, such isolated mutants of two different mating types (Yeast, 26:221–
large screens are currently impractical. 33, 2009). By mating those types together and dissecting the
resulting spores, researchers can obtain the genotypes they desire
SISTER SPECIES: SCHIZOSACCHAROMYCES within about five days.
JAPONICUS
GENETIC ENGINEERING
CLAIM TO FAME: A POPULAR COUSIN Researchers use electroporation to transform S. japonicus and
S. japonicus is related to one of geneticists’ favorite organisms, the knock genes in or out (Cold Spring Harbor Protoc, doi:10.1101/
yeast S. pombe. “There’s a huge discovery potential in pairing the spe- pdb.prot091850, 2017). Scientists can also overexpress genes or
cies,” says Snezhana Oliferenko, a cell biologist at King’s College Lon- use homologous recombination for gene editing.
don and the Francis Crick Institute. For example, if Oliferenko thinks
she understands some pathway in one yeast species, she reconstitutes STRAIN COLLECTIONS
that pathway in the other species. If it works, she’s on the right track. Oliferenko’s lab is one of the best sources; it has about 4,000
Differences between the two yeast species has also led to new strains on hand.
insights. While S. pombe keeps its nuclear envelope during mito-
sis, S. japonicus breaks it open. That means the S. pombe envelope DOWNSIDE
must grow. By comparing the membrane synthesis pathways in So far, Oliferenko says she hasn’t had much success forcing S.
each organism, Oliferenko determined a key phosphorylation event japonicus into a diploid state—something researchers reported
that only S. pombe can perform (Curr Biol, 26:P237–43, 2016). doing back in 2011 (Yeast, 28:747–54). In other yeasts where
this approach is well established, geneticists can use this strat-
GOOD FOR STUDYING egy to identify essential genes: scientists knock out one copy of
• Mitosis and cytokinesis, which S. japonicus does more similarly a gene from a diploid organism, and if half the haploid spores
GULINE
to animals than S. pombe does. don’t survive, then that gene was essential. If researchers try to
CREDIT
• Chromosomes and chromatin. Thanks to S. japonicus’s large, 21 knock out an essential gene in haploid-only S. japonicus, they
YING
micron by 6.5 micron size, chromosomes are easily visible during simply won’t get any surviving transformants—and they won’t
know whether the gene was required, or whether the transfor- REPRODUCTION
mation just failed. Jewel wasps are easy to grow and mate; one mating yields hun-
dreds of offspring within two weeks. The different species will
HAPLODIPLOIDY: JEWEL WASPS interbreed so long as researchers use antibiotics to cure them of
a Wolbachia infection that prevents hybridization.
CLAIM TO FAME: HAPLODIPLOIDY
In the tiny jewel wasps, genus Nasonia, haploid males hatch from GENETIC ENGINEERING
unfertilized eggs and diploid females from fertilized ones. That Nasonia can be transformed by injection of genetic material, and
means it’s an animal model with whole-organism haploid genet- it’s easy to create knockdowns via RNAi, says Werren. CRISPR
ics, which can simplify analyses. For example, researchers can has also been done.
mutagenize males and easily identify recessive mutations, given
that there’s no homologous gene to mask a phenotype of interest. STRAIN COLLECTIONS
Because the species has evolved to allow somatic-cell hap- Researchers have collected wildtype strains from diverse parts
loidy, there are few deleterious recessive genes lurking in the of Europe and North America. There are also strains in which a
genome. That makes it easier to create inbred lines, which sim- wasp has a large fragment of the genome from another species.
plifies genome assembly. Some basic strains are sold by Carolina Biological Supply and
N. vitripennis is the most popular species, but there are three Ward’s Science. Werren and others distribute more-specialized
others under study. They can be crossbred to study the genes that lines. He estimates that about 100 strains are available.
make each species unique. All are about two millimeters long, Researchers will also need host insects, such as blowflies, which
don’t sting people, and rarely fly, making them easy to handle. can be bought from biological suppliers or, in Europe, bait shops.
GOOD FOR STUDYING DOWNSIDE

• How genes interact to produce complex traits. Both transformation and CRISPR still need to be optimized in
• Evolution, by comparing the species. Nasonia for better efficiency, says Werren.
• Epigenetics because about one-third of Nasonia genes are
methylated. EFFICIENT EGG-LAYER: JAPANESE QUAIL
• Genetic conflict, because the genome contains selfish elements,
such as a chromosome that eliminates paternal DNA after fer- CLAIM TO FAME: SMALL SIZE, FAST DEVELOPMENT
tilization of an egg. Birds provide a convenient model for developmental studies. They
• Cytogenetics because the eggs and embryos are clear. have four limbs, four-chambered hearts, and other characteristics
they share with mammals. But the embryos—being separate from
GENOME the mother’s body—are much more accessible. While chickens are
Males have five chromosomes, females 10. Four species have been popular, they take months to mature. The Japanese quail, Coturnix
sequenced; three are published—N. vitripennis, N. giraulti, N. coturnix japonica, weighs in at 5–9 ounces, matures faster, and is
longicornis (Science, 327:343–48, 2010)—and the N. oneida “basically a mouse with wings,” says Rusty Lansford, a developmen-
sequence is forthcoming. tal biologist at the University of Southern California and Children’s
Hospital Los Angeles.
Nasonia GOOD FOR STUDYING

vitripennis • Early development of the embryos, which can be observed by
direct imaging within the egg or when removed from it. The
species also offers researchers the opportunity to manipulate
development. For example, scientists can transplant tissues
between chickens and quail to see what the implanted cells
become. French scientists even transplanted a bit of quail hind-
brain into chickens to make them trill like quail rather than
squeak like baby chickens (Science, 241:1339–42, 1988).
GENOME
PETER KOOMEN
Quail (and chickens) have a diploid set of 39 chromosome pairs.

Eight pairs and one sex chromosome are large, ranging in size
from 27 to 176 kilobases; the rest are much smaller. Until recently,
quail aficionados had to rely on the chicken genome, which shares
09. 201 9 | T H E S C IE N T IST 61

LAB TOOLS
PEGSummitEurope.com - RAKESH D., PHD, VP, ASTRAZENECA
meeting in Europe.
99 percent of the quail’s genome. Now, a quail sequence is avai The best biologics technology
able (Genomics, 101:345–53, 2013; GigaScience, 7:giy049, 2018)
This has facilitated precision both in labeling genetic elements for b
imaging and in genetic engineering, says Lansford. gonads for later thawing and production of living birds. Lans-
ford is collaborating with the Frozen Zoo at the San Diego Zoo’s
University of California, San Francisco
REPRODUCTION Institute for Conservation
Chemistry andResearch to figure Pharmacology,
Cellular & Molecular out the process.
Qu CEOtwo
within and months,
Director ofmuch
the Board, Immunocore
faster than the five Professor, Departments of Pharmaceutical
to Bahija
takes Jallal, PhD
chickens. While artificial insemination James A. Wells, PhD
is p uralNext
matingTherapeutic Platform
is more effective. Females can lay with Recombinant Antibodies
Coturnix coturnix
Bispecific, Soluble TCR as the days. Attacking Cancer Cell Surfaceomes
DAVID HUSS, UNIVERSITY OF SOUTHERN CALIFORNIA

ab per year, and the eggs hatch in 16–17 japonica embryo
Qu bred for too long; after about five generations,
they start to sicken.
2019 PLENARY KEYNOTES
GENETIC ENGINEERING
Researchers can transform
18-22 embryonic
NOVEMBER cells with
2019 DNA or RNA by
LISBON, PORTUGAL  LISBON CONGRESS CENTER
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electroporation (JoVE, 148:59664, 2019). CRISPR editing, RNA
interference, and morpholinos all work.
Keycode O75
11 October with
Register by
STRAIN COLLECTIONS
to €300
Lansford estimates that there are about a dozen transgenic lines
that carry specific genetic markers. They can be shipped as eggs
Save up
commercial quail farmers.
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from research labs. Scientists can also obtain wildtype eggs from
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2019 PLENARY KEYNOTES

Bispecific, Soluble TCR as the Attacking Cancer Cell Surfaceomes
Next Therapeutic Platform with Recombinant Antibodies
Bahija Jallal, PhD James A. Wells, PhD
CEO and Director of the Board, Immunocore Professor, Departments of Pharmaceutical
Chemistry and Cellular & Molecular Pharmacology,
University of California, San Francisco
The best biologics technology

meeting in Europe.
PEGSummitEurope.com - RAKESH D., PHD, VP, ASTRAZENECA
CAREERS
Fixing Animal Research

Many in vivo preclinical studies are poorly designed and generate
irreproducible data, but efforts to address the problem are on the rise.
BY DIANA KWON
A
few years ago, officials at Swit-
zerland’s Federal Food Safety and
Veterinary Office approached
Hanno Würbel, the head of the animal
welfare division at the University of Bern,
with the task of examining the quality of
experimental design in the country’s ani-
mal research. Growing public awareness
of the reproducibility crisis in science—
which has emerged as researchers discover
that a large proportion of scientific results
cannot be replicated in subsequent exper-
iments—had put pressure on the gov-
ernment authority to examine this issue,
Würbel says. “They wanted to know, what
is the situation in Switzerland . . . and is
there anything that we need to improve?”
To address this question, Würbel and
his colleagues examined scientific protocols
in 1,277 applications for licenses to conduct
animal research that were submitted to and
approved by the Swiss Food Safety and Vet-
erinary Office (FSVO). Their analysis, pub-
lished in PLOS Biology in 2016, concluded
that most of the experiments described
in approved applications lacked scien-
tific rigor. Only a fraction of the protocols
included important measures against bias, clinical research is not reproducible. The to scientific advances, projects that gener-
such as blinding, randomization, or a clear latter study’s authors highlighted poor ate irreproducible data on account of poor
plan for statistical analysis. experimental design as one of the main design create far more unease. According
It’s now one of several studies that causes of the problem and estimated that, to toxicologist Thomas Hartung, director
have pointed to critical flaws in the way in the United States alone, approximately of the Center for Alternatives to Animal
animal experiments are designed—and $28 billion is spent each year on preclini- Testing, a group dedicated to promoting
many researchers argue that these flaws cal experiments that cannot be replicated. and improving the welfare of research
are major contributors to the reproduc- Poorly designed animal studies raise animals, at the Johns Hopkins Bloomberg
ibility crisis plaguing published pre- ethical concerns in addition to financial School of Public Health and the University
clinical research. In 2011, for example, and scientific ones. Preclinical experi- of Konstanz in Germany, “Research that is
scientists at the pharmaceutical company ments, which often involve modeling not quality-controlled is unethical.”
© ISTOCK.COM, JXFZSY
Bayer reported that they were unable aspects of human diseases in animals, can These issues have led numerous mem-
to reproduce the findings from 43 of include procedures that may cause pain or bers of the scientific community to express
67 projects on potential drug targets in otherwise inflict harm on the organisms the urgent need to expose and address the
oncology, cardiology, and women’s health. under investigation. While most scientists flaws of animal studies, with many actively
Meanwhile, a 2015 PLOS Biology paper may consider that harm to be justified in working on ways to improve experimental
reported that more than 50 percent of pre- cases where well-conducted research leads design and tighten regulatory oversight.
09. 201 9 | T H E S C IE N T IST 63

CAREERS
Finding the flaws drug that showed promise in animal mod- Laboratory animal Experimentation)
More than 50 years ago, zoologist els of stroke but failed in later clinical tri- provides tools, guidelines, and support
William Russell and microbiologist Rex als. The team’s investigation revealed for researchers to conduct reviews of
Burch established the 3Rs, core princi- that, although nine papers reported that animal studies in their own fields.
ples that governments in many countries, the drug successfully reduced the size of From the perspective of NC3Rs, “if an
including the US, China, and the member infarcts in animals, only two included experiment is not designed to yield robust
states of the European Union, have since randomization and blinding procedures. results or is not reported in enough detail
woven into legislation and guidelines reg- Furthermore, those two studies reported so that other people can actually use those
ulating the use of animals in research. The lower estimates of efficacy than the other results, then it’s a complete waste” of ani-
3Rs refer to replacement of animal studies seven did. mals and research resources, Percie du
with other methods, reduction of the num- The group has since made similar Sert tells The Scientist. “You might as well
ber of animals used in experiments, and findings for other neurological condi- not do the experiment.”
refinement of experimental techniques to tions, such as multiple sclerosis, Alzheim-
reduce pain and improve welfare—a peren- er’s, and Parkinson’s. For example, a 2016 Improving design
nially controversial topic, as quantifying review of interventions for Alzheimer’s In 2015, NC3Rs released the Experimen-
animal suffering is notoriously difficult. disease reported that of 427 published tal Design Assistant (EDA), a free web tool
in vivo studies, fewer than one in four developed to help researchers reduce the
Research that is not quality- reported blinding and randomization, and flaws in their methodologies and produce
none reported sample size calculations. robust designs for animal experiments.
controlled is unethical.
“Essentially, every review we did said the “It’s basically like having your own statis-
—Thomas Hartung, Johns Hopkins same thing,” says the University of Edin- tical assistant with you when you design
Bloomberg School of Public Health
burgh’s Emily Sena, a leader of the CAMA- your experiment,” Percie du Sert explains.
RADES group that continues to carry out More than 6,000 researchers worldwide
During the approval process for ani- these reviews. “Very few studies took sim- are using the EDA, she says, and the tool
mal experiments, regulatory bodies use ple measures to reduce bias.” On top of is recommended by several funders of ani-
the latest welfare research to weigh poten- that, Sena adds, she and her colleagues mal research, such as the Wellcome Trust
tial harm to animals against possible soci- consistently found that studies that did and the Medical Research Council (MRC)
etal benefits, such as advancements in not incorporate those measures tended to in the UK and the National Institutes of
medicine or new scientific knowledge. report larger treatment effects. Health in the US.
But as investigations such as Würbel’s “[CAMARADES’s] studies have been It’s just one of the ways that NC3Rs
have shown, many poorly designed animal really useful for highlighting the problem,” is working to improve the rigor of animal
studies still seem to be falling through the says Nathalie Percie du Sert, the head of experiments. In 2010, the organization
cracks, accruing the associated costs with- experimental design and reporting at the published its ARRIVE (Animal Research:
out providing the potential benefits. National Centre for the Replacement, Reporting In Vivo Experiments) guide-
One of the groups trying to get a Refinement, & Reduction of Animals in lines, a checklist of items for researchers
handle on the problem is the UK-based Research (NC3Rs), a UK-based scientific to include when describing animal exper-
Collaborative Approach to Meta-Analysis organization dedicated to advancing the iments, such as sample sizes, full descrip-
and Review of Animal Data from Exper- 3Rs. “What they’re measuring is the qual- tions of the organisms under investigation,
imental Studies (CAMARADES) team, ity of reporting in published papers—and and explanations of the measures taken to
which has carried out multiple systematic that highlights that there’s not enough reduce bias. Although the guidelines have
reviews—assessments of all available liter- information in published papers to know been endorsed by numerous journals and
ature pertaining to a given research ques- that the studies are robust.” funders, a 2019 paper by Sena, MacLeod,
tion—of preclinical animal studies. CAMA- Researchers in the US and in Europe and colleagues revealed that many manu-
RADES was founded in the early 2000s by are working to implement these types of scripts did not live up to its standards. A
University of Edinburgh neurologist Mal- critical assessments for animal experi- new version of ARRIVE, which has been
colm MacLeod and his colleagues, who ments more broadly. For example, the updated with changes meant to improve
decided to look into why so many of the Evidence-Based Toxicology Collabora- compliance, was posted as a preprint on
drugs coming out of laboratories working tion that Hartung chairs at the Johns bioRxiv in July and is currently undergo-
on stroke failed in clinical trials. Hopkins Bloomberg School of Pub- ing peer review.
In one of their first reviews, carried out lic Health has started using systematic NC3Rs has also spearheaded efforts
more than a decade ago, CAMARADES reviews to evaluate toxicology studies, to curtail the approval of poorly designed
researchers assessed preclinical publi- and the Netherlands-based organization projects. In the UK, where the organi-
cations for NXY-059, a small-molecule SYRCLE (SYstematic Review Center for zation provides funding for 3R-related
research, NC3Rs collaborates with other minimizes bias by using measures such If an experiment is not
funders such as the Wellcome Trust and as randomization and blinding; “exter- designed to yield robust
MRC to offer training to funding panel nal validity,” how generalizable findings
results or is not reported in
members on how to recognize charac- are beyond a single laboratory; and “con-
teristics of high-quality experimental struct validity,” the extent to which a test enough detail so that other
design. These workshops have been run- measures what it claims to measure. people can actually use those
ning for four years and have included 257 results, then it’s a complete
attendees. A sense of urgency waste.
Similar developments are underway Many of the problems plaguing experimen- —Nathalie Percie du Sert, National Centre for
in other countries. In Switzerland, the vet- tal design in animal research are not unique. the Replacement, Refinement, & Reduction of
ting process for approving animal research Preliminary work by Sena’s group, for exam- Animals in Research
licenses has become more rigorous in recent ple, has found that in vitro preclinical stud-
years, as authorities scrutinize applications ies often actually suffer more than animal
closely for both animal welfare and scien- studies from poor experimental design. “I’ve such a vote, launched by a group of Swiss
tific issues. Kaspar Jörger, the head of the had an undergrad student working with me citizens, gained more than 100,000 sig-
animal protection department at the Swiss who did a pilot project looking at in vitro natories. The move has drawn criticism
FSVO, told The Scientist in January that study design, and none of the studies she from the Swiss National Science Foun-
Würbel’s 2016 study was one of the driv- looked at in her sample were randomized, dation and swissuniversities, a group of
ing forces behind this shift. The FSVO also and none of them were blinded,” Sena says, higher education institutions, which say
now requires that all research institutions noting that the lower level of scrutiny and that the passing of such a bill would hin-
and pharmaceutical companies hire ani- lower cost associated with in vitro research der research. The vote is expected to take
mal welfare officers to help scientists pre- might be contributing factors. place around 2022.
pare their applications for animal research However, the costs associated with The debate should act as a wake-up
permits, and launched the Swiss equivalent poorly designed studies are magnified for call for researchers working with animals,
to NC3Rs, the Swiss 3R Competence Cen- animal research due to the difficult ethi- Würbel says. “[Scientists] need to con-
tre (3RCC), in March 2018. cal questions they raise, and they’re begin- vince people that what [they] are doing
Sena suggests that, in addition to ning to have real consequences for the sci- is useful, valid, and they take ethical con-
such measures, the scientific community entific community as public awareness of cerns seriously,” he explains. “But you can
should develop a formal framework to them rises. In Switzerland, for example, only do this convincingly if you make sure
quantify the scientific rigor of a proposed along with more-rigorous animal license that the science is rigorous and that you
experiment as part of the assessment of procedures, political pressure is building have done all you could do to make sure
that experiment’s potential benefits— to reduce or eliminate animal experimen- that the harm imposed on the animals is
much as it uses the 3Rs to judge potential tation. This April, the federal government as minimal as possible.” g
harm. One way to do this, she says, would announced that it would hold a national
be to establish the 3Vs, which she defines referendum on banning animal experi- Diana Kwon is a Berlin-based freelance
as “internal validity,” how well a study ments altogether after a campaign for science journalist.
ALTERNATIVES TO ANIMALS
Researchers are actively searching for alternatives to animal testing in preclinical research, and in recent years there have been significant
advancements in both computational and in vitro replacements. For example, researchers have used in vitro techniques such as organoids
and organs-on-chips to model a various aspects of human biology. As these technologies become increasingly sophisticated, scientists
have exploited these approaches to make brain organoids capable of generating electrical patterns akin to those found in premature
human babies, and even “bodies-on-chips”—interconnected systems of mini-organs.
Substitutes for animal models will likely have the most immediate effect in the area of toxicity testing, because toxicity studies have very
clearly defined endpoints, says Anthony Holmes, director of science and technology at the National Centre for the Replacement, Refinement, &
Reduction of Animals in Research. There is already some evidence that in silico methods might be superior in this field. Thomas Hartung of Johns
Hopkins University and his colleagues recently developed an artificial intelligence–based chemical screen that they demonstrated to be more
effective at identifying toxic chemicals than animal tests. (See Hartung’s opinion article, “AI Versus Animal Testing,” The Scientist, May 2019.)
While many of these approaches are promising, it is unlikely that they will ever completely replace animal research. “We can model cer-
tain elements of this in in vitro conditions, and we’re getting better at it,” says Ulrich Dirnagl, a neurologist at the Charité University Hospital
Berlin. “But there will be a stage when where we will need to see what’s going on in a functioning organism.”
READING FRAMES
Meeting Our Maker

Humans are the first species to decipher the genetic code that underlies
our biology, but does it hold all the answers?
BY BILL SULLIVAN
A
s author George R.R. Martin would alcoholism, liberalism, adultery, and—well,
attest, good writing takes time. you name it. One study claims to have found
For eons, DNA has been writing genes that influence when a person loses
genetic scripts for “survival machines,” their virginity! DNA screening services
evolutionary biologist Richard Dawkins’s promise to illuminate ancestry, predisposi-
term for living organisms—their primary tions for disease, even certain behavioral
purpose being to live long enough to prop- tendencies. But is gazing at one’s DNA
agate their DNA. As author Samuel Butler sequence a robust method for predicting
recognized in 1877, “A hen is only an egg’s future outcomes, or is it a modern form of
way of making another egg.” phrenology? This is one of the key questions
But our planet has limited resources, that prompted me to write Pleased to Meet
so survival machines that had a leg up on Me, a book that describes how genes work
the competition won the DNA replication with other factors to make us who we are.
relay. Selfish genes were locked in an arms Developmental biologists have long National Geographic, August 2019
race to craft survival machines that were suspected that there must be more to sur-
better, stronger, faster. About 600 million vival machines than their genes, vaguely
years ago, an ancestral neuron emerged calling it epigenetics (“beyond genes”). If like Toxoplasma gondii (which dwells in the
that heralded a new weapon: intelligence. all cells in the body possess the same DNA brains of billions of people) produce factors
It took nearly 4 billion years, but DNA has sequence, why do some become brain cells that may alter gene expression in the host.
finally built a survival machine intelligent and others heart cells? DNA sequences do More recent studies show that mRNA can
enough to expose DNA’s game. We are the not change over our lifetimes, so what also be modified in ways that affect protein
first species to meet our maker. brings about the dramatic transformations synthesis, a process called epitranscriptomics
The realization that we’re an appa- of puberty? Long story short, the expres- that adds yet another layer of complexity to
ratus for the dissemination of genes is sion of the genome is just as important as the prediction of phenotypes from genotypes.
quite different from traditional creationist the genome itself. Of course, much of this For some, the realization that biological
narratives. It is even more humbling to is determined by genes encoding transcrip- forces shape who we are is disconcerting, but
reflect on the power of a related revela- tion factors that regulate gene expression. this knowledge is power. By understanding
tion: instead of passively watching genetic But remarkably, increasing evidence sug- how genes, epigenetics, and epitranscrip-
stories unfold, we can now become the gests that our environment also affects the tomics function in the context of our micro-
authors. Are we ready for this awesome expressed genome through epigenetics, by biome and environment, we will be in a
responsibility? In just a half century, we chemically altering DNA itself or the pro- better position to develop new approaches to
resolved the structure of DNA, made teins associated with it. Genetic analysis treat undesirable behaviors. We’ve met our
genome sequencing easy, and discovered of children who suffered abuse and later maker, but with the advent of gene editing,
ways to edit genes. Although we don’t became suicidal, for example, showed the development of epigenetic drugs, and the
fully understand its language, some increased DNA methylation at their glu- ability to remodel our microbiome, we’re on
are now eager to take a red pen to the cocorticoid receptor gene, which compro- course to take control of our own destiny. g
genome. With the help of the first human mises the ability to manage stress.
genome, published in 2003, researchers Epigenetics demonstrates that nature Bill Sullivan is a professor of pharmacol-
have revealed genes involved in certain and nurture are two sides of the same coin. ogy and microbiology at the Indiana Uni-
diseases, and this knowledge is guiding The phenotypes arising from our genes are versity School of Medicine in Indianapolis,
the discovery of novel therapeutics. highly contextual, and the “you” that exists where he studies infectious disease and
But what about more-complex pheno- today might have been very different had genetics. Read an excerpt of Pleased to
types like personality and behavior? We you been conceived or raised in a different Meet Me: Genes, Germs, and the Curious
regularly hear news about the identification environment. Studies have also found that Forces That Make Us Who We Are at
of a gene for procrastination, extraversion, our microbiomes and nefarious parasites the-scientist.com.
09. 201 9 | T H E S C IE N T IST 67

The Human Microbiome:

COMINGSOON Beyond the Gut
The human microbiome has been linked to health and disease. Recent improvements and better accessibility to high-throughput screening have
transformed scientists’ ability to perform research in this area. This has resulted in a tremendous increase in our knowledge of microbial diversity. The
gastrointestinal tract is just one of many microbial homes in the human body. Other parts of the body that contain a hotbed of microbial communities
include the skin, lungs, reproductive tract, mouth, and breasts. Microbiota in these areas could affect various aspects of our health, including our
ability to accept transplants, our susceptibility to certain diseases, and the health of our offspring. To examine the human microbiome beyond the
gut, The Scientist is bringing together experts from the field to share their research and answer questions from webinar attendees.
GRACE ALDROVANDI, MD THURSDAY, SEPTEMBER 19

Professor, Pediatrics 2:30 - 4:00PM EASTERN TIME
Ronald Reagan UCLA Medical Center
UCLA Mattel Children’s Hospital REGISTER NOW!
UCLA Medical Center, Santa Monica www.the-scientist.com/microbiome-beyondthegut
The webinar video will also be available at this link.
BRENDA ANNE WILSON, PhD TOPICS TO BE COVERED:

Professor of Microbiology, Associate Director
• The microbiome of breast milk
of the School of Molecular and Cellular Biology,
• Vaginal microbiome and reproductive health and
College of Liberal Arts and Sciences
disease
Professor of Pathobiology,
College of Veterinary Medicine
Inaugural Professor,
Carle Illinois College of Medicine WEBINAR SPONSORED BY:
University of Illinois at Urbana-Champaign
Off-target Effects in CRISPR-Cas9 Genome Editing:

ONDEMAND Securing Specificity
A cause for concern regarding the popular CRISPR-Cas9 genome editing technology is the potential occurrence of off-target effects. RNA-guided
Cas9 may cleave DNA sequences that are not exact complements of the guide strand when either strand harbors bulges due to insertions or
deletions. Undoubtedly, a better understanding of the specificity of CRISPR-Cas9 editing can help develop strategies to minimize off-target cleavage.
To take a closer look at the specificity and possible causes of off-target effects in the CRISPR-Cas9 system, The Scientist is bringing together
researchers who will summarize their work on off-target effects limiting the applications of Cas9-mediated genome modification and discuss
strategies for predicting and preventing such effects. Attendees will have the unique opportunity to ask experts about their experience with off-target
effects of the CRISPR-Cas9 system.
TUESDAY, MAY 7
LAURYL NUTTER, PhD 2:30 – 4:00PM EASTERN TIME
Associate Director, Model Production and
Cryopreservation & Recovery WATCH NOW!
The Centre for Pharmacogenomics www.the-scientist.com/crisprcas9offtarget
The Hospital for Sick Children
TOPICS TO BE COVERED:
• Whole genome sequencing to assess Cas9 off-target
DAVID J. SEGAL, PhD effects in genetically engineered mice
Professor, Dept. of Biochemistry
• Will elimination of off-target effects be good enough?
and Molecular Medicine
UC Davis School of Medicine
WEBINAR SPONSORED BY:
Professor, Dept. of Pharmacology
UC Davis MIND Institute
Sample Preparation for Single Cell Analysis:

ONDEMAND Tips and Tricks
Single cell analysis is a powerful technique for characterizing complex tissue types, identifying rare cell populations, uncovering regulatory
relationships between genes, and tracking cell trajectories. From sample handling to cell dissociation, there are a number of technical considerations
that influence cell viability and data quality of single cell genomics, single cell epigenomics, and single cell gene expression experiments. In this
webinar, sponsored by 10X Genomics, Jill Herschleb explores critical sample prep decisions and considerations for assay design and optimization.
THURSDAY, AUGUST 8
JILL HERSCHLEB, PhD 2:30 – 4:00PM EASTERN TIME
Director, Sample Preparation
10x Genomics WATCH NOW!
www.the-scientist.com/singlecellanalysis-sampleprep
• Approaches for optimizing sample preparation for single
cell RNA-seq, single cell ATAC-seq, and single cell CNV
• Differences in sample preparation for single cell
genomics, transcriptomics, or epigenomics applications
• Insights into nuclei versus whole cell preparation uses
• Methods to separate cells or nuclei from contaminating
material
• Sample preparation workflows for large-scale antigen
profiling applications
Immuno-Oncology for Solid Tumors:

ONDEMAND Overcoming Barriers in the Tumor Microenvironment
The solid tumor microenvironment is immunosuppressive and poses a significant obstacle to achieving clinical success with various immunotherapies. Most
notably, immune checkpoint pathways, including PD-1, PD-L1, and CTLA-4, allow tumors to evade the body’s usual immune reaction, leading to disease
progression. Checkpoint blockade via monoclonal antibodies results in augmented T-cell activation and proliferation to enhance the anti-tumor immune response
and has revolutionized the field. But not all patients are candidates, and severe adverse effects persist. Therefore, the development of other approaches, such
as antibody-drug conjugates, vaccines, and adoptive cell-based therapies, has surged. Recent advances in immunotherapy have certainly produced enhanced
treatment options for solid tumors, but obstacles to their mainstream use remain. To explore novel strategies in immuno-oncology for solid tumors, The Scientist is
bringing together a panel of experts to share their research and insights into what it may take to overcome the barriers in the tumor microenvironment.
GRACE DY, MD TUESDAY, JUNE 18

Chief, Thoracic Oncology 2:30 – 4:00PM EASTERN TIME
Associate Professor of Oncology
Department of Medicine Roswell Park WATCH NOW!
Comprehensive Cancer Center www.the-scientist.com/immuno-oncology-solid-tumors
CHRYSTAL PAULOS, PhD • Immunotherapy in lung cancer
Associate Professor of Microbiology
• Adoptive immunotherapy: overcoming barriers
and Immunology
against solid tumors with potent T cells
Co-Leader of the Cancer Immunology Program
at the Hollings Cancer Center
Endowed Peng Chair of Melanoma in Department
of Dermatology and Dermatological Surgery
Medical University of South Carolina,
College of Medicine
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09.2019 | T H E S C IE N T IST 7 1
FOUNDATIONS
Modern Synthesis, 1937

BY CHIA-YI HOU
W
hen Charles Darwin published
On the Origin of Species in
1859, he suggested that traits
could be inherited, and that natural selec-
tion could affect which traits were passed
down. Around the same time, Gregor
Mendel was conducting his pea plant
experiments, which he published in 1866
Mendel gave a few lectures on his findings
about genetics in pea plants, but no one
seemed to grasp the importance for under-
standing how traits are inherited.
Meanwhile, based on Darwin’s writ-
ings, biologists thought natural selection
happened in populations, but didn’t have
an idea how it worked at the level of organ-
isms. There wasn’t an understanding of
genetics and how traits could be passed on
to offspring until Mendel’s work was redis-
covered around 1900 and independently
reproduced by multiple researchers.
After that, biologists started work-
ing on how to fit genetics and natura
selection together in a theory of evolu-
tion. Genetics gave scientists a mathe-
matical framework for exploring natural
selection and opened up questions about TREE OF LIFE: Theodosius Dobzhansky experimented with Drosophila to study the effects of
how reproduction affects which genes get mutations at the population level. He found the flies had high genetic variability and that mutations
passed on to offspring. The experimen- were the source of variation.
tal and theoretical work that effectively
combined Darwin’s theory of evolution Dobzhansky joined labs at Columbia Uni- In his book Genetics and the Origin of
and Mendel’s work on heredity came to versity and then Caltech. He investigated Species, published in 1937, Dobzhan-
be known as the Modern Synthesis, a population genetics in Drosophila pseu- sky argued that genetic mutations were
term coined by Julian Huxley in his 1942 doobscura and gathered empirical evi- sources of variability that, through natu-
book Evolution: The Modern Synthesis. dence for the genetics underlying natural ral selection, could lead to evolutionary
The goal was to “understand the genetic selection and speciation. “He was a real change, and he suggested that these pro-
basis of evolutionary change at the pop- naturalist, but he also knew that genetics cesses could lead to speciation of popula-
ulational level,” science historian Vassi- was crucial to understanding evolution,” tions that are isolated long enough.
liki Betty Smocovitis of the University of says Smocovitis. Dobzhansky published updated ver-
AMERICAN PHILOSOPHICAL SOCIETY
Florida tells The Scientist. Dobzhansky wanted to find out how sions of his book for decades; the last
Theodosius Dobzhansky, born in 1900 genetic variation could lead to shifts version, released in 1970, bore the title
in Nemyriv, Ukraine (then part of the at the population level and potentially Genetics of the Evolutionary Process. By
Russian Empire), to a Russian-speaking to evolutionary changes. To that end, then the structure of DNA had been dis-
family, would become one of the fore- he tracked mutations and differences covered, and he was able to revise his
most architects of the Modern Synthesis. between fly populations in the lab. ideas to include the molecular basis of
After he emigrated from the Soviet Union Dobzhansky noted the arrangements of genetic mutations. “[He] intellectually
to the United States in 1927 on a schol- chromosomes in different Drosophila contributes the most to the Modern Syn-
arship from the Rockefeller Foundation, species as well as their mutation rates. thesis,” says Smocovitis. g
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