SEPTEMBER 2018 | WWW.THE-SCIENTIST.

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 SEPTEMBER 2018

Contents
THE SCIENTIST THE-SCIENTIST.COM VOLUME 32 NUMBER 9
© PATRICK LANDMANN, SCIENCE SOURCE; MODIFIED FROM © ISTOCK.COM, PARA-GRAPH, GREENVECTOR; © ISTOCK.COM, CHRISCHRISW

Features ON THE COVER: © SCIENCE SOURCE, SHEILA TERRY

36
Mending Muscle
Scientists race to develop CRISPR
therapies that could save the lives of kids
with muscle-wasting conditions.
BY SANDEEP RAVINDRAN
42
Muscle’s Timekeepers
Just 20 years ago, scientists didn’t even
realize muscles had their own circadian
clocks. Now researchers are beginning to
appreciate the clocks’ importance in health.
BY DIANA KWON
48
The Elderly Muscle
Researchers untangle the multifarious
nature of muscle aging. So far, the only
reliable treatment is exercise.
BY GILLIAN BUTLER-BROWNE,
VINCENT MOULY, ANNE BIGOT,
AND CAPUCINE TROLLET

09. 201 8 | T H E S C IE N T IST 3

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 SEPTEMBER 2018

Department Contents
13 63
19 FROM THE EDITOR
No Enemy
BIO BUSINESS
A New Dawn for ALS Therapies?
Science journalists are essential After two decades of failure, novel
to advancing the quality of scientific insights and technical
the research enterprise. progress are spurring meaningful
BY BOB GRANT innovation in the field.
BY JENNY ROOD
19 NOTEBOOK
Muscle Vibes; Avian Aromatherapy; 66 READING FRAMES
Ratted Out; Silent Swimmer Repeating the Beat
In this adapted excerpt from Sandeep
35 MODUS OPERANDI Jauhar’s new book, Heart: A History,
Exosome Engineering the author explores the past and
Researchers identify a handy future of artificial hearts.
exosome-binding tool for tinkering BY SANDEEP JAUHAR
with the versatile vesicles.
72
54
BY RUTH WILLIAMS FOUNDATIONS
Homo sapiens Exposed, 1556
54 THE LITERATURE BY SUKANYA CHARUCHANDRA
How the filamentous protein
PAUL MARASCO, LABORATORY FOR BIONIC INTEGRATION; © KIMBERLY BATTISTA; © CORWIN VON KUHWEDE

myosin gives red blood cells their IN EVERY ISSUE

distinctive shape; aging muscle 11 CONTRIBUTORS
experiences mitochondrial redox 14 SPEAKING OF SCIENCE
imbalances, and exercise doesn’t 67 THE GUIDE
help much; a muscle protein 68 RECRUITMENT
can help mice recover from
a restless night
CORRECTIONS:
56 PROFILE In the June article “Incomplete Immunity,” the credit for the accompanying
image was incorrect. The photograph of the bird was taken by Marie Read.
All Muscle
The July article “Needles in Haystacks” stated that Kristin Aquilino works
Having pioneered the study with the California Wildlife Foundation. She works with the California
Department of Fish and Wildlife. The Scientist regrets these errors.
of muscle physiology in
mammals, Angela Dulhunty ANSWER
59 uncovered how ion channels
PUZZLE ON PAGE 14
enable muscle movement.
BY ANNA AZVOLINSKY L UGOS I L A UR E L
A E L T A A
59 SCIENTIST TO WATCH NUC L E U S ORD E R
Avnika Ruparelia: Muscle Mender A K E A L I Y
BY SUKANYA CHARUCHANDRA T ROOP S I L I CON
E E Q A X
60 LAB TOOLS MA R S U P I A L
The Native Niche J A A N F
Stem cell experts share their I NH A B I T V I P E R
hard-won tips on making your G O R C E H E
cells grow faster, healthier, S I N A I HORMON E
and in higher numbers. A I N S T Z
BY AMBER DANCE
WH A L E S P E YO T E

09. 2018 | T H E S C IE N T IST 5

 SEPTEMBER 2018

Online Contents

THIS MONTH AT THE-SCIENTIST.COM:

VIDEO VIDEO VIDEO

Muscle Maven
Profilee Angela Dulhunty describes
her research on the structure
and function of proteins that make up
skeletal and cardiac muscles.
Modern Prosthesis
See amputee Amanda Kitts manipulate
objects using nervous impulses to
control an early prototype of her
advanced artificial limb.
Eel-bot
University of California, San Diego,
graduate student Caleb Christianson
explains the development of his eel-
inspired robot.

AS ALWAYS, FIND BREAKING NEWS EVERY DAY, AND LEAVE YOUR COMMENTS ON INDIVIDUAL STORIES ON OUR WEBSITE.

Coming in October
HERE’S WHAT YOU’LL FIND IN NEXT MONTH’S ISSUE

• Researchers are discovering a plethora of life deep

within the Earth’s crust.
© ISTOCK.COM, ISARESCHEEWIN

• Light pollution is disrupting ecosystems around the globe.

• The surprising immune functions of belly fat

• The latest approaches in single-cell multi-omics

AND MUCH MORE

6 T H E SC I EN TIST | the-scientist.com
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 SEPTEMBER 2018

Contributors
During her early rotations as a medical student in 1973, Gillian Butler-Browne met children
with Duchenne muscular dystrophy who were incurable. She was so drawn to the field of muscle
research that Butler-Browne never completed her medical degree, instead going on to do a
traveling PhD that took her from London to the United States, Germany, and ultimately France,
where she had the opportunity to study muscle at the Pasteur Institute. Not many people were
working on muscle, Butler-Browne says: “There was a niche that nobody was filling in France.”
Currently the Director of the Research Center at the Institute of Myology in Paris, Butler-
Browne will be stepping down at the end of the year to go back to research. She’s interested in
aging muscle and its regeneration, and how exercise can help—she walks at least 10 kilometers
a day. At the end of a long day, Butler-Browne likes to unwind with music and cooking, both
of which she considers intertwined with her science. She was a vocalist when she made the
switch to medicine and for a while played bass guitar in a folk/rock band. Read about
the effect of exercise on aging muscles in her feature article “The Elderly Muscle” on pg. 48.

Born in India, Sandeep Jauhar moved with his family to Southern California at the age of
eight. Questions about the universe, philosophy, and literature piqued his interest. “One thing
I really wasn’t interested in was medicine,” says Jauhar, now a cardiologist. Visits to his grand-
father’s clinic in New Delhi had disillusioned him. Despite his parents’ wishes for him to be
a doctor, Jauhar started his PhD in physics at the University of California, Berkeley, in 1991.
Midway through his graduate studies, a close friend developed lupus. “In the course of try-
ing to help her with her physical illness, I started voraciously reading everything I could about
medicine and about lupus.” Medicine’s many unknowns intrigued Jauhar, who eventually real-
ized he “wanted to become a doctor after all.”
Between getting his PhD and going to medical school, Jauhar completed a fellowship at Time
magazine that heightened his interest in journalism. The connections he formed there guided
him to an internship at the local newspaper while he attended medical school at Washington
University in St. Louis. In the afternoons, he spent time at the St. Louis Post-Dispatch, where
he would write local stories that in turn made him think of bigger stories—such as one about a
leprosy hospital in Louisiana that he pitched to The New York Times. His first week in Manhattan,
where he served as a medical resident at New York Hospital, The Times published his story. He
went on to write “regular dispatches from the hospital” for the outlet, he says, and continues to do
so. Since then, Jauhar has gone on to publish two best-selling memoirs, Intern and Doctored, on
his experiences in the medical field. For Jauhar, who keeps a journal and describes himself as self-
reflective, writing about his time in the hospital was a “natural outcropping of his work.” Read his
essay, adapted from his latest book, Heart: A History, on pg. 66.
ANTON OTTAVI; © MARYANNE RUSSELL; TED ECKELS

Growing up in Chile, Julio Fernandez was captivated by science at an early age. As an

11-year-old, he received his “first diploma” that attested to his doorbell repair skills. By age
15, he was certified in radio and television repair. These early experiences laid the foundation
for his work at Columbia University, where he now not only studies proteins but also builds
some of the instruments needed to explore the molecules. Fernandez says his love of ideas
keeps him going: “The wonder of understanding something new has never left me,” he writes
in an email to The Scientist. He has two dueling and divergent interests. While Fernandez
is “addicted to computer programming,” he is also drawn to the self-sufficiency of people in
the 19th century. With his wife, Fernandez has a ranch nestled in the mountains near Santa
Fe, New Mexico. “When we get here from NYC [New York City], we step back in time.” With
nature (and bears!) keeping them company, they bake fresh bread, plant fruit trees, and so
on. Read his story on pg. 28 about how a giant protein, called titin, is changing the paradigm
of muscle function.

09. 2018 | T H E S C IE N T IST 1 1

Read The Scientist on your iPad!
 FROM THE EDITOR

No Enemy
Science journalists are essential to advancing the quality of the research enterprise.

BY BOB GRANT

I
n this special issue focused on the leading edge of scien-
tific research into all things muscle, one key fact stuck
out as I read through the interesting stories it contains.
As researchers Gillian Butler-Browne, Vincent Mouly,
Anne Bigot, and Capucine Trollet write in “The Elderly
Muscle” on pg. 48, “To you readers over age 30, we’ve got
some bad news for you. Chances are good you’ve already
begun losing muscle.”
This is a reality that my 40+-year-old body registered
long before I read that passage. Nonetheless, seeing it
spelled out in front of my face drove home the depressing
point all the way to the depths of my withering sarcomeres.
Butler-Browne and her coauthors do give us a ray of hope,
detailing exercise’s ability to halt or reverse this trend, even
in people older than 70.
This gradual wearing down of physiology brings to instances of misconduct, waste, shoddy research, and hype
mind another alarming and erosive trend happening in our in the scientific community. Far from being an antagonist,
world. The President of the United States has lately been much less an enemy, of science or researchers, science jour-
stepping up his already disturbing attacks on the media, nalists reporting, writing, and editing such stories seek to
often repeating the phrase: “The press is the enemy of the propel the pursuit of knowledge by holding those at the
American people.” As his rhetoric has become more fre- helm accountable and keeping them honest. Transparency
quent and vitriolic in the past few months, a consortium and accountability make the scientific enterprise stronger,
of more than 300 US newspapers, from Maine to Hawaii, and by extension create a safer, more informed citizenry
recently banded together to simultaneously run editorials protected by sound policy. As journalists covering science,
combating the idea that media outlets seek to harm the it is our duty to shine a light on discoveries and help inform
populace or spread untruths about the unassailable char- the public, but also to bring scientific fact to bear on busi-
acter of a misunderstood leader. ness or political issues, and to expose the darker corners of
I won’t spend too much ink here contravening these the scientific enterprise so that the many can learn from
patently ridiculous assertions. My colleagues at The Boston the mistakes of the few.
Globe, the Honolulu Star-Advertiser, Anchorage Daily News, These precepts of responsible journalism, like aging
and other outlets have done a sufficient job in this regard. muscle, can atrophy if not exercised. And this withering
I can, however, share insights from the perspective of can be accelerated when people in power actively seek to
a science journalist. In our business, we’re no strangers undermine our efforts.
to the post-publication butting of heads that sometimes As you peruse this issue, which includes a plethora of
breaks out among researchers, politicians, industrialists, interesting muscle facts and one or two well-founded mus-
and members of the public. After all, one person’s well- cle opinions, know that we at The Scientist pledge to con-
supported scientific fact can be another’s political or busi- tinue to fulfill our journalistic duty with strength and clar-
ness headache. Exposing the empirical dangers of widely ity, as we have for more than three decades. g
sold chemicals, highlighting the damage wrought by cli-
mate change, tamping down the hype of wonder drugs’
marketing, or correcting mistaken public perceptions
ANDRZEJ KRAUZE

about vaccines is all in a day’s work for a science journalist.

And just like those who make a living reporting on poli-
tics, science journalists must often relate unflattering facts
about the people and institutions they cover. For decades, Editor-in-Chief
The Scientist has been one of the clearest voices calling out eic@the-scientist.com

09. 2018 | T H E S C IE N T IST 1 3

 QUOTES
Speaking of Science
1 2 3 4
8 9
11 12
Note: The answer grid will include every letter of the alphabet.
13
15
17 18
21 22
23
BY EMILY COX AND HENRY RATHVON
ACROSS
1. Cinematic player of a human
hematophage
4. Dicotyledon used to make wreaths
8. 1911 atomic discovery of Ernest
Rutherford
10. Grouping between class and family
11. Murder : crows :: ___ : monkeys
12. Element used to make semiconductors
13. Koala, possum, or wombat
17. Have as a natural environment
19. Venom-injecting snake
21. Peninsula bridging Asia and Africa
22. Secretion of an endocrine gland
23. Mammals with large calves
24. Hallucinogen from a cactus
14 T H E SC I EN TIST | the-scientist.com
5 6 7 Many key positions remain
unfulfilled, divisions are
understaffed, and process
10 has slowed to a crawl.
—An anonymous US Fish and Wildlife Service
staffer answering a survey conducted by the Union of
Concerned Scientists in which several thousand federal
scientists weighed in on the state of science under the
Trump administration (August 14)
14
16 Women have a huge capability,
19 20 sense of problem solving
and reasoning that they can
contribute to science but both
my own experience and general
statistics suggest this capability
is regularly overlooked
24 and we don’t receive equal
opportunity to participate.
—Michaela Kendall, an environmental scientist at
Birmingham City University who made a freedom of
information request to the UK’s Engineering and Physical
Sciences Research Council, revealing that 90 percent of
the government grants awarded for such research over the
DOWN past decade have gone to men (The Guardian, August 10)
1. Wearing a natural coat of wool
2. Lizard that licks its own eyeballs
3. 1973 film of a cryopreservation
subject
5. Bikini, for one
6. In math, the √ sign
7. Where phonatory muscles are housed
ANDRZEJ KRAUZE
9. Hirsute biped of cryptozoology
13. Evergreen shrub with berries and
spiny leaves
14. Division, vis-à-vis multiplication
15. Puzzle solved without pen or pencil
16. Change states of matter, in a way
18. Seawater
20. Prefix on synthesis
Answer key on page 5
Data Reporting Guidelines
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This means you may want to change
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Western blots. Get robust and
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 FREEZE FRAME

Caught on Camera
Selected Images of the Day from the-scientist.com

MUSCLE MOSAIC
»

A newly developed staining technique to characterize rodent

muscle fibers (rat plantaris muscle shown here) paints cells
four different colors.
Posted: November 11, 2016

3-D NANOFIBERS »
Researchers created a 3-D model of
synthetic nanofibers, such as these
fibers (pink) surrounded by cardiac
muscle cells (purple), to learn how
to engineer more efficient scaffolds
for biomedical applications.
Posted: March 7, 2018

16 T H E SC I EN TIST | the-scientist.com
 » BEATING HEART
Scientists developed an open-source stem cell line with
fluorescent tags—these mark troponin l1—for cardiac cells.
Posted: July 2, 2018

» COLD HEARTED
Cardiologists can now cool the human heart
in a localized way to help reduce muscle
damage from heart attacks. The dark spot at
lower left is the cooled portion.
Posted: January 22, 2018

MUSCLE BOUQUET

»
Culture-grown muscle stem cells from mice, which
have fused together to form myotubes (some of
them flurorescently labeled in green), showing cell
nuclei (blue) and distinct striations (red)
Posted: January 16, 2018

Muscle Mosaic: S. Sawano et al., PLOS ONE, doi:10.1371/journal.pone.0166080, 2016.;

3-D Nanofibers: V. Balashov et al., Acta Biomaterialia, doi:10.1016/j.actbio.2017.12.031, 2018;
Beating Heart: Allen Institute; Cold Hearted: Catharina Hospital, Eindhoven;
Muscle Bouquet: Kevin Murach, University of Kentucky College of Health Sciences
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 NEWS AND ANALYSIS
Notebook
Muscle Vibes
W
hen Amanda Kitts’s car was
hit head-on by a Ford F-350
PAUL MARASCO, LABORATORY FOR BIONIC INTEGRATION
truck in 2006, her arm was
damaged beyond repair. “It looked like
minced meat,” Kitts, now 50, recalls. She
was immediately rushed to the hospital,
where doctors amputated what remained
of her mangled limb.
While still in the hospital, Kitts dis-
covered that researchers at the Reha-
bilitation Institute of Chicago (now the
Shirley Ryan AbilityLab) were investi-
gating a new technique called targeted
muscle reinnervation, which would
enable people to control motorized pros-
thetics with their minds. The procedure,
which involves surgically rewiring resid-
ual nerves from an amputated limb into
a nearby muscle, allows movement-
related electrical signals—sent from
the brain to the innervated muscles—to
move a prosthetic device.
Kitts immediately enrolled in the
study and had the reinnervation surgery
around a year after her accident. With
her new prosthetic, Kitts regained a func-
tional limb that she could use with her
thoughts alone. But something impor-
tant was missing. “I was able to move a
prosthetic just by thinking about it, but I
still couldn’t tell if I was holding or letting
go of something,” Kitts says. “Sometimes
my muscle might contract, and whatever
I was holding would drop—so I found
myself [often] looking at my arm when
I was using it.”
SEPTEMBER 2018
I’VE GOT A FEELING: Amanda Kitts takes part
in a test of a prosthetic designed to recreate
a user’s sensation of limb movement.
What Kitts’s prosthetic limb failed to
provide was a sense of kinesthesia—the
awareness of where one’s body parts are
and how they are moving. (Kinesthesia
is a form of proprioception with a more
specific focus on motion than on posi-
tion.) Taken for granted by most people,
kinesthesia is what allows us to uncon-
sciously grab a coffee mug off a desk or
to rapidly catch a falling object before it
hits the ground. “It’s how we make such
nice, elegant, coordinated movements,
but you don’t necessarily think about it
when it happens,” explains Paul Marasco,
a neuroscientist at the Cleveland Clinic in
09. 2018 | T H E S C IE N T IST 1 9
 NOTEBOOK
Ohio. “There’s constant and rapid com-
munication that goes on between the
muscles and the brain.” The brain sends
the intent to move the muscle, the muscle
20 T H E SC I EN TIST | the-scientist.com
moves, and the awareness of that move-
ment is fed back to the brain (see “Pro-
prioception: The Sense Within,” The Sci-
entist, September 2016).
GOOD VIBRATIONS: The prosthetic
makes use of a kinesthetic phenomenon
whereby vibrating a person’s muscle
provides a false sense of movement.
Prosthetic technology has advanced
significantly in recent years, but proprio-
ception is one thing that many of these
modern devices still cannot reproduce,
Marasco says. And it’s clear that this is
something that people find important,
he adds, because many individuals with
upper-limb amputations still prefer old-
school body-powered hook prosthetics.
Despite being low tech—the devices
work using a bicycle brake–like cable
system that’s powered by the body’s own
movements—they provide an inherent
sense of proprioception.
To restore this sense for amputees
who use the more modern prosthetics,
Marasco and his colleagues decided to
create a device based on what’s known
as the kinesthetic illusion: the strange
phenomenon in which vibrating a per-
son’s muscle gives her the false sense of
movement. A buzz to the triceps will
make you think your arm is flexing,
while stimulating the biceps will make
you feel that it’s extending (Exp Brain
Res, 47:177–90, 1982). The best illustra-
tion of this effect is the so-called Pinoc-
chio illusion: holding your nose while
someone applies a vibrating device to
your bicep will confuse your brain into
thinking your nose is growing (Brain,
111:281–97, 1988). “Your brain doesn’t
like conflict,” Marasco explains. So if it
thinks “my arm’s moving and I’m hold-
ing onto my nose, that must mean my
nose is extending.”
To test the device, the team applied
vibrations to the reinnervated muscles on
PAUL MARASCO, LABORATORY FOR BIONIC INTEGRATION
six amputee participants’ chests or upper
arms and asked them to indicate how they
felt their hands were moving. Each ampu-
tee reported feeling various hand, wrist, and
elbow motions, or “percepts,” in their miss-
ing limbs. Kitts, who had met Marasco while
taking part in the studies he was involved in
at the institute in Chicago, was one of the
subjects in the experiment. “The first time
I felt the sense of movement was remark-
able,” she says.
In total, the experimenters docu-
mented 22 different percepts from their
participants. “It’s hard to get this sense
reliably, so I was encouraged to see the
 The first time I felt
the sense of movement
was remarkable.
—Amanda Kitts
capability of several different subjects
to get a reasonable sense of hand posi-
tion from this illusion,” says Dustin Tyler,
a biomedical engineer at Case Western
Reserve University who was not involved
in the work. He adds that while this is a
new, noninvasive approach to proprio-
ception, he and others are also working
on devices that restore this sense by stim-
ulating nerves directly with implanted
devices (Sci Rep, 8:9866, 2018).
Marasco and his colleagues then melded
the vibration with the movement-controlled
prostheses, so that when participants decided
to move their artificial limbs, a vibrating stim-
ulus was applied to the muscles to provide
them with proprioceptive feedback. When
the subjects conducted various movement-
related tasks with this new system, their per-
formance significantly improved (Sci Transl
Med, 10:eaao6990, 2018).
“This was an extremely thorough set of
experiments,” says Marcia O’Malley, a bio-
medical engineer at Rice University who
did not take part in that study. “I think it
is really promising.”
Although the mechanisms behind the
illusion largely remain a mystery, Mar-
asco says, the vibrations may be activat-
ing specific muscle receptors that provide
the body with a sense of movement. Inter-
estingly, he and his colleagues have found
that the “sweet spot” vibration frequency
for movement perception is nearly iden-
tical in humans and rats—about 90 Hz
(PLOS ONE, 12:e0188559, 2017).
For Kitts, a system that provides pro-
prioceptive feedback means being able
to use her prosthetic without constantly
watching it—and feeling it instead. “It’s
whole new level of having a real part of your
body,” she says. —Diana Kwon
Avian
Aromatherapy
Male European starlings—small, green-
ish-black birds with a hint of irides-
cence—may not be much to look at. But
they do like to add a little flourish to their
nests: fresh herbs. Originally considered
a strategy for attracting a mate, the use of
aromatics in nest building is now thought
to have an additional role in keeping baby
starlings healthy.
“People have proposed that the birds
add the herbs to their nests because they
improve the fitness of their offspring,” says
Juan Vicente Gallego Rubalcaba, a biol-
ogist at Rey Juan Carlos University in
Madrid who studies the adaptive strate-
gies of birds. Indeed, studies have shown
that starlings raised in herbed dwellings
tend to fare better—having fewer para-
sites and weighing more—than youngsters
brought up in nonherbed nests.

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09. 2018 | T H E S C IE N T IST 2 1

NOTEBOOK
What researchers haven’t been able
to pinpoint is how exactly the herbs exert
an influence over baby birds’ health. To
answer this question, Helga Gwinner of
the Max Planck Institute for Ornithol-
ogy has been studying a wild European
starling (Sturnus vulgaris) colony near
Ammersee, a lake in Germany. Her past
experiments have shown that the hatch-
lings of starlings that add herbs to their
nests have higher red blood cell counts,
stronger immune function, and fewer
bacteria than hatchlings in nonherbed
nests. Yet she and her colleagues have also
found that herbs don’t appear to directly
reduce the number of nest parasites such
as mites, fleas, and lice—thus ruling out
one leading hypothesis for how herbs
confer benefits upon hatchlings.
To get to the bottom of the mys-
tery, Gwinner recently set up an exper-
iment to test another hypothesis: that
the herbs improve offspring health by
keeping eggs in the nest warm. Herbs
might provide direct thermal benefits
through improved insulating proper-
ties, the team reasoned, or through heat
produced by decomposition of the mate-
rial, keeping the eggs toasty while parent
birds are out of the nest.
During the 2006 and 2007 mating
seasons, Gwinner and her colleagues
attached 53 bird boxes to trees near
Ammersee, watched males build nests,
and waited for the first clutches of eggs
to be laid. Once the eggs in the nests
were ready for incubation, the research-
ers swapped out the original nests with
36 artificial ones. Half of these contained
herbs, such as hogweed, cow parsley, and
goutweed, and half were made of just dry
grass to act as controls. Gwinner’s team
also added a “dummy” egg—a disguised
temperature sensor—to each nest to
record the eggs’ temperatures for roughly
13 days, the length of the incubation
period. From the egg temperatures, the
team could infer when the starling par-
ents left the nest, when they came back,
and how long they sat on their eggs.
The dummy eggs in the herby nests
did log higher average temperatures than
those in the plain nests over the course
of the study. But temperatures fell at the
same rate in the parents’ absence, suggest-
ing that the change was not due to physi-
cal insulation by the nest. Instead, the egg
temperature data indicated that parents
in herbed nests spent more time sitting
on their eggs—at least 15 minutes longer
per day compared with birds in nests lack-
ing the aromatics. They also left the nest
less often, at least early in the incubation
period, and made shorter excursions out-
side (Proc R Soc B, 285:20180376, 2018).
The results show that parental behav-
ior, not insulation, is behind the higher
temperature in nests that incorporate
herby materials. “I think that the volatiles
of the nest herbs acted as a tranquilizer,”
Gwinner says. “Parents sat more quietly
on the eggs, nest temperatures dropped
less often below critical thresholds, so
the eggs and the embryos [within them]
experienced a more-constant and higher
temperature.”
I think that the volatiles of
the nest herbs acted as a
tranquilizer.
—Helga Gwinner
Max Planck Institute for Ornithology
© ANDRZEJ KRAUZE
 800.656.7625 www.rockland-inc.com

Hints from these experiments and others also suggest that

nest herbs affect the internal development of the eggs before lay-
ing, particularly the concentration of androgens in each yolk. In
a previous study, Gwinner and her colleagues collected and cal-
culated the quantity of herbs male birds had added to the nests
and then measured yolk testosterone. “We found a positive cor-
relation of yolk testosterone and the amount of green nest mate-
rial,” Gwinner says, explaining that the presence of herbs in the
nest may somehow induce females to increase yolk testosterone
concentration in their eggs during development. Higher concen-
trations of testosterone, as well as increased temperatures, could
have influenced nestling growth in the experiment.
Rubalcaba, who was not involved in the study, notes that
adding aromatic green plants is clearly good for the offspring.
Gwinner’s finding of improved egg incubation might not be
the only factor to boost the health of the baby birds in herbed
nests, he says.
It’s possible that herbs might also protect the immune sys-
tems of the parent birds, notes Michel Raymond, an evolution-
ary biologist at the Institute of Evolutionary Sciences in Mont-
pellier, France, who has previously studied birds’ herb use but
was not involved in this study. Although Gwinner’s team had
earlier found no evidence that the herbs affected parasitic load,
the authors note in the current paper that the greater heating
of eggs in herbed nests could prevent bacteria from growing
on the parents, keeping them healthier. If so, “the energy saved
(the immune system is costly) could be invested parentally,”
Raymond writes in an email to The Scientist. If the parents are
healthier, they can invest more energy in caring for the chicks,
making them fitter in the long run.
—Ashley Yeager

Rockland Immunochemicals, Inc. manufactures

Ratted Out antibodies to Green Fluorescent Protein (GFP) and
Red Fluorescent Protein (RFP), which are offered
When working on archaeological digs in the Gambier Islands of
as monoclonal and polyclonal. These antibodies
French Polynesia, zooarchaeologist Jillian Swift has her attention
are raised against full-length proteins.
trained on the ground. Nevertheless, she’s often struck by a strange
lack of activity overhead: “It’s kind of eerie, where there’s just not
Rockland offers highly-referenced anti-GFP and
a single bird in the sky.”
anti-RFP signal amplification antibodies that can
This avian silence fell over some of the places where Swift
be used for a variety of applications, which include
works—such as several small islands clustered near Mangareva,
immunofluorescence microscopy, flow cytometry,
the central island of the Gambiers—centuries ago, after humans
and Western blotting.
and their attendant rats arrived, often making quick meals of
local birds’ eggs and young. But now, the bones of these wayfar-
Learn more by visiting rockland-inc.com/gfp-rfp
ing rodents and their descendants offer researchers such as Swift,
who’s currently based at the Max Planck Institute for the Science
of Human History in Germany, a unique insight into the history
of human settlements. That’s because the composition of those
bones—specifically, the ratios of isotopes of elements such as car-
bon and nitrogen—are “really sensitive to environmental change,”
she says. Using those ratios to make inferences about what the
animals would have been eating when they were alive, researchers
 NOTEBOOK
can gain insight into the state of the whole
ecosystem during the rodents’ lifetimes.
To find bones that could retell the tale
of settlement in Oceania, Swift recently
collected specimens on those quiet
islands surrounding Mangareva. She
also borrowed other researchers’ speci-
mens from those same islands and from
a site on one of the Marquesas Islands
and pored over samples from sites on the
island of Tikopia from the Bernice Pau-
24 T H E SC I EN TIST | the-scientist.com
ahi Bishop Museum in Hawaii. The rat
species she focused on, Rattus exulans,
is commensal with humans, and would
have hitched rides in the canoes of set-
tlers from other Polynesian islands. Using
specimens dating back more than 1,000
years, Swift and her collaborators ana-
lyzed the isotopic composition of nitro-
gen and carbon in the bones’ collagen to
look for changes over time in the land-
scapes’ nutrient flows.
The analyses revealed a consistent
pattern following human colonization
of each island, even though these ini-
tial settlements were spread over hun-
dreds of years. “I was doing this island
to island, site to site . . . and then put-
ting it all together I realized there was
this nearly universal decline in [the ratio
of nitrogen-15 to nitrogen-14] through
time across all of these islands,” she says.
“These nutrient flows are changing so
substantially, and it seems to be occur-
ring everywhere and really tied into these
. . . massive landscape changes that occur
as soon as people enter into new regions.”
(PNAS, 115:6392–97, 2018). The carbon
composition changed, too, though not
in such a consistent way. On Agakauitai,
near Mangareva, for example, the ratio of
carbon-13 to carbon-12 isotopes increased
after colonization, but on Tikopia and one
of the Marquesas islands the ratio fell
after settlement.
The results don’t reveal how, exactly,
humans changed the isotopic composi-
tion of the islands as they settled in. Peo-
ple can alter that composition through
many different activities, such as graz-
ing animals or fertilizing crops, and “the
really tricky part is teasing those different
influences apart,” says archaeologist Paul
Szpak of Trent University in Peterbor-
ough, Ontario, who was not involved in
the study. Szpak and colleagues recently
linked increases in relative nitrogen-15
composition to intensifying agricultural
practices in Ireland during the Bronze
Age (Sci Adv, 4:eaas9383, 2018).
Still, Swift has some ideas about how
humans might have wrought the changes.
TOP: JILLIAN A SWIFT; BOTTOM: PATRICK V KIRCH
Other studies on the Polynesian islands
have found charcoal generated when
people cleared forests by burning them,
she notes; that shift in island flora would
have affected the ratios of nitrogen and
ISLAND DIGS: The remains of rats on Polynesian
islands such as Agakauitai, French Polynesia
(left, top panel), provide zooarchaeologists with
a record of environmental changes, and by proxy,
human activity, in the region. One recent study
excavated the animals’ bones (left, lower panel) to
track those changes over more than 1,000 years.
 Rats bones are a unique
archaeological resource.
carbon isotopes in the environment. And
then there’s the matter of those missing
birds—killed off by people and the rats
and other animals they brought with
them—and the absence of their nitro-
gen-filled poop. “The islands lose a key
nutrient input in the seabird guano,”
Swift says, although she adds that that’s
just one of many factors that can change
island landscapes’ nutrient composition.
On Agakauitai, she and her coauthors
suggest, the shift toward carbon-13 indi-
cates that people—and the rats that ate
their scraps—favored a fish-rich diet, while
the opposite trend on some other islands is
consistent with an increasing reliance on
the C3-photosynthesizing crops, such as
taro, that Polynesians tended to cultivate.
Although the isotopes in their bones
may not tell a complete story, rats are
unique archaeological resources in a few
ways, says molecular anthropologist Lisa
Matisoo-Smith of the University of Otago
in New Zealand who was not involved in
the new study. For example, unlike Homo
sapiens settlers, rats have maintained a
constant presence on the islands. “Even
when humans may have abandoned entire
islands or parts of islands or sites, the rats
are still going to be there,” she tells The
Scientist. Analyzing isotopes and changes
in diet in rat remains is “a brilliant idea
. . . and to see it applied through time
and across space in the Pacific gives us
some really valuable insight”—into both
the changes wrought by humans’ arrival
and into the changing activities of those
humans over time.
Swift, a.k.a. @DrRatGirl on Twitter,
plans to continue mining the bones of
rats and other animals that live around
humans for more of this insight. “Com-
mensal animals in general are a really
under-researched and interesting topic,”
she says. “There’s a lot they can tell us
about people and environments.”
—Shawna Williams
Silent Swimmer
A flattened, almost completely transpar-
ent object moves slowly through the water.
In its appearance, the colorless structure
somewhat resembles an eel larva—and
for good reason: this marine robot was
designed with a leptocephalus, the name
of a curious stage in the lifecycle of several
eel species, in mind.
“We want to make a soft robot that can
swim around coral reefs without damaging
them,” says Caleb Christianson, a doctoral
student at the University of California, San
Diego (UCSD), who has been working on a
prototype for more than a year and a half.
Marine robots have various uses. The
oil and gas industries use unmanned vehi-
cles for tasks such as installing under-

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 NOTEBOOK

water structures, constructing pipelines,

and drilling. Scientists, meanwhile, are
increasingly using the technology for envi-
ronmental sensing, biological research,
and finding ways to protect marine
resources.
But most ocean-going robots come
with a catch: they are noisy. The decibel
levels and vibrations of the propellers and
jets scare away fish and other animals.
What’s more, most robots are built with
metallic frames, which can bump into
and damage fragile ocean life. Christian-
son decided to take a different approach,
with a design incorporating “muscles that
don’t have any rigid materials to them,” he
says. “They’re just completely soft.”
In May 2016, Christianson and his
colleagues at UCSD set out to build this
soft robot. They weren’t the first to look to
the natural world for this purpose—other
research groups have used fish and manta
rays as the basis for marine-robot design.
But Christianson decided to focus on eel
larvae, finding the wavelike motion of the
fish to be an ideal model for the technology
they were working on in the lab. What’s
more, replicating the larva’s transparency
would help the team’s creation blend into
the ocean environment.
To create their eel-inspired robot, the
researchers used transparent dielectric elas-

JACOBS SCHOOL OF ENGINEERING, UC SAN DIEGO; © WIKIMEDIA COMMONS, KILS

tomer actuators (DEAs)—“pieces of rubber
that respond to an electrical stimulation,”
Christianson explains. While the rubber
itself doesn’t conduct electricity, a voltage
applied to the upper and lower surfaces of
the elastomer causes it to flatten, making the
material both thinner and longer.
To achieve movement, each individual
DEA, or artificial muscle, required two
electrodes: one, a small pouch of water
inside the rubber, and the other, the water
surrounding the robot in a tank in the lab.
Initially, the researchers started with
two actuators, which, placed back-to-back,
had the ability to bend to the right or to
the left. To achieve more-fluid motion and
improve propulsion, the team next placed
three of these actuator pairs end-to-end,
creating a 22-cm-long, 5-cm-high, and
1.5-mm-thick robot. “We arranged several
of these in such a way that we can get an
undulating motion to mimic the motion
of an eel swimming,” says Christianson.
By applying voltage to the six actuators in
diagonal pairs from the front to the back
of the eelbot via external cables, Christian-
son’s group was able to prod the robot into
eel-like motion around the tank.
It was at this point in the project, around
June 2017, that Dimitri Deheyn, a marine
biologist at UCSD’s Scripps Institution of
Oceanography, became involved in this
research. To see how closely the robot resem-
bled its inspiration, Deheyn used a hyper-
spectral imaging system that gives every
single pixel of the sample its own spectral
identity. The more light going through the
robot, the less conspicuous it would be in the
open ocean. “We were able to have a high
SOFT AND SLEEK: Researchers have
designed a marine robot that blends in with its
environment (above) and moves by contracting
artificial muscles to perform an undulating
motion resembling that of an eel larva (below).
percentage of light going through, close to
85 to 95 percent or more, indicating that it
is truly a transparent robot as opposed to
a translucent robot,” says Deheyn. In areas
with a reduced passage of light, the trans-
parency matched that of the bony bits of an
eel larva (Sci Rob, 3:eaat1893, 2018).
The researchers also found that they
could reproduce another aspect of eel biol-
ogy in their robot—fluorescence—by adding
a dye to the fluid pouch electrode. Certain
species of eel naturally fluoresce, possibly

26 T H E SC I EN TIST | the-scientist.com
 siD ydobitnA tseB ehT The Best Antibody Discovery
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as a form of communication during spawn- Technology Is Now at Your Fingertips
ing events, or as a way to attract prey. But
fluorescence might be useful for under-
water robots, too, where other communica-
tion channels such as radio waves are imprac-
tical, the researchers suggest in their paper.

We want to make a soft robot

that can swim around coral
reefs without damaging them.
—Caleb Christianson
University of California, San Diego

The eel-inspired device isn’t quite ready

for use in the oceans, though. The current
prototype moves forward at 1.9 mm per
second while the electronics package that
applies the voltage floats on the surface
of the water of the tank. “The speed with
which it’s moving is quite slow,” says Stefan
Williams, who works on marine robotics at
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Amino Acid position
who was not involved in the research, says Amino Acid position

these are “early stages” for the use of soft CDR-H3 residue utilization in antibodies derived from human samples and the
noitisop dicA onimA Amino Acid position Amino Acid position
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space. But it’s very exciting to see progress.”
Exceptional Human
—Sukanya Charuchandra Antibody Discovery
Technology
noitpecxE Exceptional Human
09. 2018 | T H E S C IE N T IST 27
ydobitnA Antibody Discovery
olonhceT Technology
 CRITIC AT LARGE
Reimagining Muscle Function
Titin throws a powerful curveball into the muscle physiology field.
Is it time for a paradigm shift?
BY JULIO M. FERNÁNDEZ
W
atching the elaborate motion of a pitcher throw-
ing a baseball at more than 100 miles an hour
illustrates the essential role of stretching prior
to delivering large amounts of mechanical power. Similarly,
practitioners of the millennia-old practice of yoga often seem
to defy gravity with impressive feats of muscle stretching.
By contrast, astronauts returning to Earth from long voy-
ages display a significant loss of muscle tone, as if the pro-
longed absence of gravitational loading had robbed them of
some essential source of mechanical power. Yet much of the
biomechanics at the foundation of these familiar phenom-
ena remains beyond the reach of modern scientific inquiry.
Perhaps these gaps will be filled in when we develop a better
understanding of how muscle tissue works.
According to most physiology textbooks, we already fully
understand muscle contraction at the molecular level, and any
further knowledge would only deal with minor details about
the participating molecules specific to different muscle types.
28 T H E SC I EN TIST | the-scientist.com
Muscle is a highly organized tissue with a crystal-like arrange-
ment of its filaments. The standard model of muscle contrac-
tion focuses mainly on the sliding of myosin motors along
actin filaments, while other muscle proteins play scaffolding
or regulatory roles. But more recently, physiologists have sug-
gested adding the giant elastic protein titin to the model of the
muscle sarcomere, the basic contractile unit of muscle tissues.
© SHUTTERSTOCK.COM, JOSE LUIS CALVO
Despite its being the longest continuous polypeptide in the
human body, some medical textbooks still do not include titin
in their description of the sarcomere.
Titin was discovered in the late 1970s by researchers
Koscak Maruyama (J Biochem, 80:405–07, 1976) and Kuan
Wang et al. (PNAS, 76:3698-702, 1979). By its placement
within the muscle sarcomere, it was readily evident that titin
operated under a stretching force during the muscle elonga-
tion that precedes every muscle contraction. But the standard
bulk biochemistry techniques available at the time couldn’t
interrogate this behavior because they lacked the ability to
apply a uniaxial force. It wasn’t until the advent of single-pro-
tein force spectroscopy towards the end of the 20th century
that researchers were able to study the behavior of individual
titin molecules under physiological conditions.
By the mid-1990s, I had already built a modified atomic
force microscope (AFM) to probe the mechanical forces
involved in exocytosis. Then, while on a sabbatical semester
from the Mayo Clinic in January 1997, I joined the physics
laboratory of Hermann Gaub at Ludwig-Maximilians University
in Munich. My plan was to continue my exocytosis project using
the modified AFMs in Gaub’s laboratory. However, upon my
arrival, Gaub proposed that we start instead with a recently
discovered muscle protein that was huge and presumably easier
to handle. With then–graduate student Matthias Rief, I was
able to observe astonishingly long and detailed sawtooth-pattern
traces of native titin extracted from bovine cardiac tissue as the
AFM cantilever stretched the titin molecule, unfolding protein
domains, which refolded when the force was released. In a few
short weeks we had submitted a paper to Science that was
published that same May (276:1109–12, 1997).

What we now know about titin should be

sufficient to trigger a paradigm shift in our
understanding of muscle contraction.

Upon my return to Mayo in late May 1997, I took a job

as chair of the Department of Physiology and Biophysics,
but I also told my laboratory that I had become mesmer-
ized by titin and that we would switch from efforts to study
the mechanics of exocytosis to developing this new field
of inquiry. We built better AFM instruments, engineered
the first tandem homopolyproteins to provide unambigu-
ous mechanical fingerprints, and submitted grants to the
National Institutes of Health to fund the venture.
We generated some truly novel findings on protein dynamics
under force that are crucial to understanding how titin works in
intact muscle tissue. This was no easy task. Our work on protein
dynamics under force has been frowned upon by bulk-protein
biochemists who use molar concentrations of harsh chemical
denaturants to trigger unfolding, producing simplistic thermo-
dynamic models that fail to explain our observations—such as
the surprisingly large amount of mechanical work done by a
protein folding while under a stretching force. This was simply
due to the fact that the unfolded state configuration of a protein
such as titin depends on the pulling force, requiring incorp-
oration of polymer physics into the traditional models used by
protein biochemists. After more than two decades of research,
it is now clear that protein unfolding and folding under force
is prevalent in biology and plays crucial roles in processes from
protein translation to protein degradation and most things
 Actin Filament
RELAXED
Myosin filament
Z line
H zone Sarcomere
Actin Filament
Titin
Z line
Myosin filament
I band
CONTRACTED
H zone
Titin
Myosin filament
Z line
MUSCULAR DOGMA: The standard textbook model of muscle
contraction includes only two main players, the filaments actin and
myosin, which slide past each other, causing the sarcomere—the basic
unit of muscle tissue—to shorten. Driven by ATP-powered myosin
motors, sarcomere contraction causes whole muscles to shorten. In the
past 40 years, researchers have explored the role of another filament:
titin. Its emerging role in muscle function, unwinding during muscle
relaxation (above) and folding during contraction (below), suggests
that the ATP-driven motors also act as latches allowing titin to fold,
providing a powerful boost to muscle contraction.
in between, as demonstrated by a growing group of scientists
using our techniques.
Our most important observation is that proteins can
readily fold against a pulling force, delivering a large
amount of mechanical work that surpasses that generated
by ATP-fueled motors. From this, it is evident how titin
might operate in intact muscle tissues. Titin modules are
arranged in tandem and can number up to 100 in the elas-
tic I band region, which spans sarcomeres. Passive stretch-
30 T H E SC I EN TIST | the-scientist.com
ing, which occurs when muscle is relaxed and elongated
during activities such as yoga, will unfold and extend titin
modules under a wide range of forces and time scales. Hold-
ing a yoga pose for long periods of time results in storage of
large amounts of potential energy in the stretched muscle
through titin unfolding.
By contrast, titin folding occurs over only a few pico-
newtons in the physiological force range. Strikingly, this range
matches the range of forces produced by active ATP-driven
myosin motors. Given how these molecules are organized in
the sarcomere, it seems likely that the activation of the ATP-
driven motors relieves the force on titin, triggering spontane-
ous titin folding. This partnership suggests that the motors act
as release latches for the elastic energy stored in titin during
animal motion. Our most recent data show that in the physio-
logical force range, the power output of disulfide-bonded titin
domains matches the power output of the myosin thick fila-
ment, suggesting that these two sources of energy combine to
deliver muscle power.
After more than two decades of research,
it is now clear that protein unfolding and
folding under force is prevalent in biology
and plays crucial roles in processes from
protein translation to protein degradation
and most things in between.
For a long time, the classical sliding filament model of
muscle contraction developed in the 1950s seemed unassail-
able, and challenging this concept was deemed a fool’s errand.
Yet this theory was developed without knowledge of the exis-
tence of the giant third filament: titin. Unsurprisingly, all the
data obtained from intact muscle fibers were assumed to orig-
inate solely from the interactions between actin and myosin.
Hence, it is long overdue to consider modifying the sliding
filament theory to incorporate these striking mechanical fea-
tures of titin, and to reexamine muscle mechanics experiments
in the light of this new theory.
It speaks volumes about the state of science today that the
muscle field has been reluctant to consider the large amount
of force-spectroscopy data pointing to a role of titin in muscle
© IKUMI KAYAMA/STUDIO KAYAMA
contraction. Instead, titin has been relegated solely to the
domain of muscle elasticity, with many researchers afraid
to besmirch the purity of the standard model of ATP-driven
muscle contraction. What we now know about titin should be
sufficient to trigger a paradigm shift in our understanding of
muscle contraction. g
Julio M. Fernández is a professor and principal investigator in
the Department of Biological Sciences at Columbia University.
 REGIONAL NATIONAL REGIONAL NATIONAL
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09. 2018 | T H E S C IE N T IST 3 1

 The Brain-Microbiome Axis: Links Between
COMINGSOON Neurological Disease and Microbiota
The human microbiome comprises trillions of commensal microbes. These bacteria, fungi, and viruses, which naturally reside within the human body, have
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brains, as well as how microbiome manipulation may open therapeutic avenues for these disorders, The Scientist is bringing together a panel of experts who
will share their research, summarize the state of the science, and discuss the next steps for those looking to adopt the technique.

JANE FOSTER, PhD WEDNESDAY, SEPTEMBER 26

Associate Professor
Department of Psychiatry & 2:30–4:00 PM, EASTERN TIME
Behavioral Neurosciences
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www.the-scientist.com/BrainMicrobiomeAxis
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• How resident microbiota can influence the
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Researchers have long known that cellular senescence, telomere shortening, and changes in gene expression contribute to aging. Recently, scientists
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 Identifying Novel Biocatlyst Candidates via High-
COMINGSOON throughput Stability Monitoring
The stability of enzymatic biocatalysts is of primary importance in the biotechnology, food, chemical, and pharmaceutical industries, but screening
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 MODUS OPERANDI

Exosome Engineering
Researchers identify a handy exosome-binding tool
for tinkering with the versatile vesicles.

BY RUTH WILLIAMS
Exosome

R
CP05 + M12
eleased from almost all cell types, found in blood and other
bodily fluids, and thought to play a role in cell-to-cell communi-
cation, the tiny vesicles called exosomes are garnering a great CP05 + PMO
deal of research interest both as potential diagnostic tools and as vehi-
cles for drug delivery. Many researchers are developing methods for
capturing, modifying, and tissue-targeting exosomes. One such tool—
the protein CP05—may accomplish all three.
Haifang Yin of Tianjin Medical University in China and colleagues
discovered CP05 in a search for proteins that bind CD63—a trans-
membrane protein abundant on the surface of exosomes. They’ve used
this interaction to not only isolate exosomes from human serum via
CP05-coated magnetic beads, but also to load cargo onto exosomes
and send them to various tissues in mice.
To accomplish the latter, the team created peptide chimeras of CP05
with the tissue-targeting peptides M12, RVG, or SP94 to direct exo- Duchenne muscular dystrophy model Treated mouse
somes to muscle, brain, or subcutaneous tumors, respectively. They also
delivered an oligomer that corrects a splicing error in dystrophin to the TISSUE-TARGETED THERAPEUTIC EXOSOMES: To treat a mouse model
of Duchenne muscular dystrophy, molecules of the exosome-binding pep-
muscles of mdx mice (which display the murine equivalent of Duchenne
tide CP05 are linked either to the muscle-targeting peptide M12 or an
muscular dystrophy symptoms). Both expression of functional dystro- oligomer (PMO) that corrects a splicing error in the gene for dystrophin.
phin protein and the grip strength of the animals increased. These two conjugate peptides paint the surface of exosomes from cultured
The use of CP05 is “an interesting concept,” says Pieter Vader of mouse cells. When injected into the mice, the exosomes home to muscle,
the University Medical Center Utrecht, in the Netherlands, because where they deliver the oligomer, boost functional dystrophin levels,
and improve muscle function.
“it circumvents the whole issue of having to engineer cells.” Producing
exosomes targeted to specific tissues usually involves genetic engi-
neering of the cells from which the exosomes are to be collected, Vader existing methods for isolating exosomes, such as centrifugation, size
explains. But with Yin’s system, “[you] just click something on that exclusion chromatography, and immunoaffinity.
steers exosomes toward certain tissues.” “What we have is a technology that can deliver cargo specifically in
While the results of the targeting experiments are “very impres- vivo,” says Yin. The next step is “to partner with researchers who have
sive,” adds Vader, who was not involved with the research, it’s unclear specific therapeutic cargoes for specific diseases to see if we can help
whether the CP05-coated magnetic beads would be preferable to them.” (Sci Transl Med, 10:eaat0195, 2018) g

AT A GLANCE

EXOSOME TISSUE TARGETING METHOD CARGO LOADING TISSUES TARGETED REFERENCES

MODIFICATION IN VIVO

Genetic Cells are engineered to express a fusion
engineering protein consisting of an exosomal mem-
brane protein and a tissue-targeting pep-
tide. Exosomes displaying the fusion pro-
tein are then collected from the cells.
© GEORGE RETSECK
The cargo molecule is Brain, tumor Nat Biotechnol, 29:341–45, 2011
transfected either into the
engineered cells or into Theranostics, 7:1333–45, 2017
the exosomes directly.

With CP05 A chimeric peptide consisting of
peptide CP05 and a tissue-targeting peptide
is incubated with exosomes and binds
to the membrane protein CD63.
CP05 is conjugated to the Brain, muscle, tumor Sci Transl Med, 10:eaat0195, 2018
cargo molecule and incu-
bated with exosomes at the
same time as the tissue-tar-
geting chimeric peptide.

09. 2018 | T H E S C IE N T IST 3 5

FATTY MUSCLE: In patients with muscular
dystrophy, muscle fibers (purple) are replaced
by adipose tissue (black) as muscles waste away
and lose function. (Mesenchymal tissue shown
in green and yellow.)

© PATRICK LANDMANN,
SCIENCE SOURCE
 Mending
Muscle
Scientists race to develop CRISPR therapies
that could save the lives of kids
with muscle-wasting conditions.

BY SANDEEP RAVINDRAN

I
f Chengzu Long hadn’t been quite so unlucky, he might never
have attempted to study and treat Duchenne muscular dystro-
phy. As a PhD student in Eric Olson’s lab at the University of
Texas Southwestern Medical Center, Long had spent years knock-
ing out genes in mice to try to identify their role in muscle devel-
opment and disease, only to find that each of the resulting knock-
outs had no discernible differences from wildtype individuals.
In the fall of 2013, with only about a year left until his
planned graduation, Long decided to take a different approach:
rather than generate yet another knockout mouse that might
again lack a phenotype, he would use the new CRISPR-Cas9
© PATRICK LANDMANN/SCIENCE SOURCE

gene-editing technique to correct a disease-causing mutation,

such as those that cause muscle wasting in Duchenne muscular
dystrophy. “I thought to myself, I’m really good at making mice
without a phenotype, so maybe we can use CRISPR to cure an
existing mutation,” says Long, now an assistant professor at New
York University.
Like many muscle disorders, or myopathies, Duchenne is
caused by mutations in a single gene—the X chromosome’s DMD
gene, which encodes the protein dystrophin. Without a functional
dystrophin protein, Duchenne patients gradually lose their mobil-
ity as their muscles degenerate. Most die by their early 30s from

09. 2018 | T H E S C IE N T IST 37

 breathing complications or heart failure. “Duchenne is devastat- ing muscle function, Olson adds. “It’s estimated that as little as 15
ing,” says Olson, whose lab has worked on muscle development percent of normal dystrophin levels could be curative, or at least
and disease for 30 years. “Right now there [are] 300,000 boys in highly beneficial.”
the world with Duchenne, so it’s a large patient population, and What’s tricky, though, is restoring the defective parts of the gene
there’s a desperation for a really transformative therapy.” in the first place. Doing so relies on a template-driven DNA repair
Because the genetics of the disease are well understood, process called homology-directed repair (HDR), which occurs
researchers could theoretically replace the mutated version of DMD infrequently in nondividing cells such as those of skeletal and heart
with a healthy copy to cure the disease. Unfortunately, the gene for muscle. “The problem with inserting stuff is that it’s very ineffi-
dystrophin is massive, with 2.6 million base pairs. As a result, it’s cient and makes the drug more complicated,” says Gersbach. “We’re
not feasible to insert the entire gene, or even just the 11,000 coding exploring a number of ways by which we might increase that effi-
base pairs (introns excluded), into a viral vector that could deliver ciency, but for the time being that’s not really an option.”
the therapeutic package to the muscle. “Gene editing therefore was As an alternative, researchers can use CRISPR to initiate a
a great opportunity to correct the endogenous gene rather than try- different DNA repair process called nonhomologous end joining
ing to deliver” a nonmutated version of it, says Charles Gersbach, a (NHEJ), which doesn’t rely on a template and is far more efficient
biomedical engineer at Duke University. than HDR. NHEJ can’t replace a defective gene with a wildtype
By the end of 2013, multiple labs had successfully used
gene editing to rescue the dystrophin protein in vitro, using
cells from patients. So Long decided to try his luck at using
CRISPR-Cas9 to edit the dystrophin gene in vivo. He injected
the CRISPR system into the zygotes of mdx mice, which carry a
single mutation in the gene for dystrophin. He then implanted
the zygotes into female mice, and confirmed in their 10-day-
old progeny that CRISPR-Cas9 had successfully corrected the
mutation that causes Duchenne symptoms. When the cor-
rected mice were a month old, Long tested their muscle func-
tion and found that it had also improved compared with mice
carrying the uncorrected mutation. “We published one of the
first in vivo rescues of phenotype [using CRISPR] in an ani-
mal model,” he says.
Now, just four years later, Olson has improved the gene-
editing strategy to address the majority of human Duchenne muta-
tions, and he and others have successfully tested these techniques
in various mouse models, 3-D–engineered heart muscle, and dogs.
With multiple research groups gearing up for clinical trials over
the next few years, what they learn will have implications beyond
Duchenne, as similar approaches could be applied to treat many
other muscle disorders.

Mini proteins offer partial improvements

After successfully correcting the Dmd mutation in mdx mice dur-
ing embryonic development, Long and Olson decided to use adeno-
associated virus 9 (AAV9) to deliver the CRISPR system into mice
after birth. They were again able to correct the gene mutation that
led to the Duchenne phenotype, resulting in major improvements
in muscle function.2 Gersbach at Duke and Amy Wagers, a stem cell
biologist at Harvard University, simultaneously published similar
results.3,4 “For the very first attempt, I personally thought the effi-
ciencies were remarkably good,” says Wagers. 
There are a few reasons why successful gene edits, once made,
CHENGZU LONG

were so efficient in these examples. Muscle cells are multinucleated,

with each cell having hundreds of nuclei. “If you can correct just
a few of them you can protect the whole muscle fiber,” says Gers- DON’T MISS A BEAT: CRISPR-mediated exon skipping improves the function
bach. And just a little dystrophin can go a long way toward improv- of heart muscle cells taken from Duchenne patients.

38 T H E SC I EN TIST | the-scientist.com
 version, but it can direct the removal of problematic portions of
it. And there is good reason to think that such a strategy might
work for Duchenne.
In Duchenne, DMD mutations disrupt the gene’s reading frame,
causing translation to terminate prematurely and leading to a com-
plete lack of a functional dystrophin protein. In a closely related
disease called Becker muscular dystrophy (BMD), patients carry
mutations in the DMD gene that are in-frame, typically deletions
that result in a smaller but still partially functional dystrophin. As
a result, patients with BMD generally suffer less-severe symptoms
and survive considerably longer than Duchenne muscular dystro-
phy patients. “The dystrophin protein is built like a shock absorber
with a series of redundant coils in the center,” says Olson. “You can
delete several of those coils and still retain function.”
To improve Duchenne patients’ prognoses, then, researchers
can provide them with smaller versions of the dystrophin protein.
One promising approach is to scale down gene therapy so that the
DNA encoding a functional, pared-down protein can fit into a viral
vector. A recent trial testing the implantation of such a “micro-
dystrophin” showed that the therapy increased levels of the small
protein in muscles and reduced levels of a Duchenne-associated
enzyme, called creatine kinase, in three patients. The results are
promising, although it’s still too early to know what their clinical
significance will be. “There are a number of these types of trials that
are ongoing that look really exciting,” says Gersbach.
Alternatively, researchers can omit mutated exons to avoid the
premature termination of translation. A few biotech companies
have been testing antisense oligonucleotides to enact an “exon-
skipping” strategy, obviating the need to edit the genome. The anti-
sense oligonucleotides bind to the mRNA produced from mutated
exons and cause them to be skipped during translation, restoring
the transcript’s reading frame to produce a smaller but functional
protein. The US Food and Drug Administration (FDA) recently
approved a drug, Sarepta Therapeutics’s eteplirsen (Exondys 51),
that uses antisense oligos to skip exon 51.
But the drug would only work on 13 percent of Duchenne
patients—those with mutations in that exon—and so far the clin-
ical benefits appear relatively modest. The drug is also expected
to cost about $300,000 a year. In addi-
tion, the effects of the antisense oligos
are transient, and patients will need
regular injections to maintain the exon
skipping. “There’s a real need for a long-
THE DYSTROPHIN PROTEIN: Dystrophin is part
of a protein complex linking the cytoskeleton
of muscle fibers to the surrounding connective
tissue (basal lamina). It’s a long protein with
© STEVE GRAEPEL
numerous redundant coils (purple balls), and
Cytoskeleton
acts like a shock absorber during contraction.
Without functional dystrophin to support
muscle strength and stability, muscle fibers
are easily damaged.
I think it’s not unreasonable to imagine
that we could get into patients
in a few years.
—Eric Olson, University of Texas Southwestern Medical Center
term therapy that can go at the cause of this disease, which is the
genetic mutation,” Olson says.
That’s where CRISPR-based genome editing may be able to
help. Independent studies by Olson, Gersbach, and Wagers ini-
tially used a double-cut strategy—cutting on each side of an exon
to excise it from the genome and glue the cut ends back together
using NHEJ. But Olson recently developed an approach that uses a
single cut to skip, rather than excise, a defective exon, bringing the
protein back in frame. He used CRISPR-initiated NHEJ to alter a
genomic region before the defective exon, causing the exon to be
skipped during later splicing of the mRNA. “We spent a lot of time
trying to conceive of the simplest possible approach to modify the
genome to correct it, and that led us to single-cut CRISPR,” he says.
Olson and Long used the single-cut strategy to skip exon 51 in a
mouse model of Duchenne and restore up to 90 percent of dystro-
phin protein expression in skeletal muscles and the heart.5 But again,
translating this to humans would only affect about 13 percent of boys
with Duchenne. To test a strategy that could rescue dystrophin func-
tion in a majority of patients, the researchers simultaneously tar-
geted the top 12 exons that are “hotspots” for DMD mutations. By
making a single cut before each of these exons, the team restored
dystrophin protein expression in heart muscle cells, or cardiomyo-
cytes, derived from patient stem cells.6 “We found, quite amazingly,
that with CRISPR-edited cardiomyocytes the force of [heart muscle]
contractions really recovered after the genome editing,” says Long.
Skipping multiple exons in the dystrophin gene—in partic-
ular, those with the highest rates of Duchenne-causing muta-
tions—will be key to developing a broadly applicable ther-
apy, says Olson, who says he believes his team is getting close.
“There are more than 4,000 mutations that have been identi-
Basal lamina
Laminin
Muscle cell membrane
Dystrophin
09. 2018 | T H E S C IE N T IST 3 9
 THE GENETICS OF DUCHENNE
Duchenne muscular dystrophy results from mutations in the DMD
gene that encodes the dystrophin protein. There are many types
of mutations that can cause the disease; each disrupts the reading
frame such that translation terminates prematurely, producing no
functional dystrophin protein. DMD mutations are particularly com-
mon in “hotspot” areas of the gene (exons 45–55 and 2–10).
Because the genetic underpinnings of Duchenne are known,
researchers can devise gene-editing fixes to the problem. Several
potential treatments are now being tested in preclinical and clinical
studies. In some cases, they aim to correct the mutations in the
DMD gene; other times, the goal is simply to restore a shorter
but still partially functional dystrophin protein.

Wildtype DMD gene Example: A mutation in exon 44 disrupts the reading frame and causes
translation to stop prematurely, leading to a dysfunctional protein.
Full protein
translated
STOP
1-79 1-43 44 45-79

Duchenne-causing mutations Strategies for fixing the problem

Point mutation or small deletion or duplication Exon deletion: Making cuts on either side of the mutated exon to remove it can
restore the gene’s reading frame.
STOP STOP
1-34 35 36-79 43 44 45 43 45

Deletion of one or more exons Exon reframing: Creating small deletions to repair mutated exon can also restore
the reading frame.
STOP STOP
1-43 45-79
43 44 45 43 44 45
Duplication of one or more exons
Exon skipping: The mutated exon can be skipped during mRNA processing
STOP by editing the splicing site preceding the exon.
1-2 2 3-79 STOP
43 44 45 43 44 45
Splice-site mutation (in the genomic region preceding an exon)

Exon knock-in: If an exon is missing entirely, it can be knocked back in to restore

1-42 43 44-79 the complete gene sequence.
STOP
Exon 43 not included in mRNA 43 45 46 43 44 45 46

STOP
1-42 44-79 44

fied in Duchenne patients worldwide, so one of the huge chal-
lenges has been to devise a strategy that would allow you to con-
solidate large numbers of patients with different mutations and
correct them with a common method,” he says. “We believe we
can, in principle, correct somewhere between 60 and 80 per-
cent of Duchenne mutations using this single-cut gene-edit-
bach is collaborating with Sarepta Therapeutics to develop
CRISPR gene editing for boys with the condition, while Olson’s
work has led to the creation of Exonics Therapeutics, a biotech
focused on designing gene editing therapies for Duchenne and
other neuromuscular diseases.
Scaling up to accommodate the size of human muscles will
© STEVE GRAEPEL

ing strategy.” be difficult. Skeletal muscle accounts for about 40 percent of

human male body weight (closer to 30 percent in females).
From pipette to patient Treating such a large amount of tissue requires a big dose of the
If all goes well, researchers will soon test Duchenne gene edit- nuclease and delivery vector, which presents both manufactur-
ing in nonhuman primates, and then in human patients. Gers- ing and safety issues. High doses can exacerbate the potential for

40 T H E SC I EN TIST | the-scientist.com
The hope is that technologies
developed for Duchenne can be
applied for other myopathies.
—Charles Gersbach, Duke University
immune reactivity against the bacterial Cas9 protein or the viral
vector, for example. Preexisting immunity can exclude certain
people from the treatment, while patients who develop antibod-
ies after treatment may only have a single shot at the therapy.
The timing of the Duchenne treatment will also be crucial.
“The earlier the intervention, the more effective this therapy can
be,” says Olson. “If one waits till very late in the disease when
there’s a loss of muscle tissue and there’s nothing left to fix, then
that’s going to be a tougher clinical challenge.”
But the biggest challenge is likely to be getting the therapy
into patients, says Long. Treating Duchenne requires correcting
both skeletal muscle—to restore mobility—and cardiac muscle,
to prevent heart failure. This will require systemic delivery of
any therapy through the blood. That’s worked so far in mice, but
it remains to be seen how well it will work in humans. “Delivery
into patients is a huge, huge challenge,” Long says.
Despite the long road ahead, these cutting-edge Duchenne
therapies have advanced at a blistering pace over the past few
years, and the future looks bright. “There’s no question that
there will be challenges coming, but I’ve never been more opti-
mistic about something,” says Olson. “I think it’s not unreason-
able to imagine that we could get into patients in a few years.”
And Duchenne could be just the beginning. There are hun-
dreds of incurable muscle and heart disorders that are caused
by mutations in a single gene. “Duchenne is the obvious test
bed,” says Gersbach, “but the hope is that technologies devel-
oped for Duchenne can be applied for other myopathies.” g
Sandeep Ravindran is a freelance science writer living in New
York City.
References
1. C. Long et al., “Prevention of muscular dystrophy in mice by CRISPR/Cas9–
mediated editing of germline DNA,” Science, 345:1184–88, 2014.
2. C. Long et al., “Postnatal genome editing partially restores dystrophin expression
in a mouse model of muscular dystrophy,” Science, 351:400–403, 2016.
3. C.E. Nelson et al., “In vivo genome editing improves muscle function in a
mouse model of Duchenne muscular dystrophy,” Science, 351:403–407, 2016.
4. M. Tabebordbar et al., “In vivo gene editing in dystrophic mouse muscle and
muscle stem cells,” Science, 351:407–11, 2016.
5. L. Amoasii et al., “Single-cut genome editing restores dystrophin expression in
a new mouse model of muscular dystrophy,” Sci Transl Med, 9:eaan8081, 2017.
6. C. Long et al., “Correction of diverse muscular dystrophy mutations in
human engineered heart muscle by single-site genome editing,” Sci Adv,
4:eaap9004, 2018.
BEYOND DUCHENNE
In 2015, Ronald Cohn used CRISPR to remove duplicated exons,
restoring the full-length dystrophin protein in cells from a Duchenne
patient. Now, the chief pediatrician at the Hospital for Sick Children
in Toronto is testing whether CRISPR can treat another myopathy,
congenital muscular dystrophy type 1A (MDC1A).
MDC1A affects one in 150,000 people worldwide and is caused
by a mutation in a gene called laminin alpha 2. It can lead to severe
muscle wasting, paralysis, and death by the age of 30. Unlike the
gene for dystrophin, laminin alpha 2 lacks redundant regions, so
researchers can’t just excise or skip mutated exons or create a
truncated but functional form of the protein.
In a mouse model of MDC1A, Cohn and his team used AAV9-
delivered CRISPR to make two strategic cuts to repair a mutation
in a splice site, thus restoring a skipped exon and the full-length
protein. The treatment significantly improved the mice’s muscle
strength and function (Nat Med, 23:984–89, 2017). Cohn hopes to
test this treatment in MDC1A patients in the next few years, and
sees even more potential in the future.
“It’s in theory applicable to any disease with a splice-site
mutation, and there are about 50,000 splice-site mutations
associated with diseases,” he says. Cohn is currently testing the
technique on tissues from Duchenne patients with rare splice site
mutations, with promising results.
Other myopathies may also benefit from modern technologies.
Molecular biologist Jocelyn Laporte at the Institute of Genetics
and Molecular and Cellular Biology in France has been working
for decades on rare and severe muscle disorders known as
centronuclear myopathies, which can lead to death in the first
year or two of life. Laporte and his team noticed increased levels
of dynamin 2 in mice lacking the disease-associated gene Mtm1,
and in muscle biopsies from centronuclear myopathy patients.
The researchers used genetic crosses to produce Mtm1 knockout
mice with only one Dnm2 allele (the double knockout is lethal),
reducing dynamin 2 by 50 percent and successfully improving both
the muscle function and longevity of diseased mice (J Clin Invest,
124:1350–63, 2014).
Injecting mice early in life with antisense oligonucleotides or
small hairpin RNAs to reduce dynamin 2 expression prevented
disease development (Nat Commun, 8:15661, 2017). Injecting
them later reversed some symptoms, including decreased muscle
strength and mass (Mol Ther, 26:1082–92, 2018). “Reducing
dynamin 2 really seems to have therapeutic potential,” says Belinda
Cowling, a former postdoc with Laporte and currently head of
research at biotech company Dynacure, launched in 2016 to
translate the work to the clinic.
Laporte and Cowling hope to start clinical trials in 2019
using antisense oligonucleotides to reduce human dynamin
2 expression. “It’s quite an exciting moment to be working on
centronuclear myopathies,” says Cowling. “And given its role
in membrane remodeling, you could imagine that dynamin 2
may play a role in other diseases, so targeting it could also have
broader therapeutic potential.”
09. 2018 | T H E S C IE N T IST 41
 CREDIT LINE

42 T H E SC I EN TIST | the-scientist.com
 Muscle’s
Timekeepers
Just 20 years ago, scientists didn’t even realize muscles had their own circadian clocks.
Now researchers are beginning to appreciate the clocks’ importance in health.
BY DIANA KWON
I
n the early 2000s, Stefano Schiaffino, a muscle physiologist
at the University of Padova in Italy, was faced with puzzling
results: two seemingly identical experiments involving hind leg
muscles in rats had yielded different findings.
MODIFIED FROM © ISTOCK.COM, PARA-GRAPH, GREENVECTOR
Schiaffino and his team were investigating nuclear factor of
activated T cells (NFAT), a transcription factor that responds to
the level of muscle activity. Despite using similar procedures, the
researchers found that in the tissues from one set of animals,
NFAT had moved from the cytoplasm into the nucleus in a large
proportion of cells, while in tissues from another experiment, this
change had not occurred.
The explanation for this difference turned out to be simple:
timing. The researcher responsible for one trial had sacrificed
the nocturnal animals in the evening, while another had con-
ducted the same procedure for the second trial in the morning.
This meant that the first group of animals was more active at
the time of measurement than the second. When the scientists
repeated the second experiment late in the day, when the ani-
mals were more likely to be awake, they observed high levels of
NFAT in the nuclei of the muscle cells, essentially replicating the
first experiment. “At that time, I’d been working for many years
on muscle, but had never thought about the circadian rhythms,”
recalls Schiaffino, whose research now focuses on this aspect of
muscle biology.
Around the same time, on other side of the globe, muscle
physiologist Karyn Esser, then at the University of Illinois at Chi-
cago, also stumbled upon a surprising discovery: genes encoding
essential elements of biological clocks being expressed in rat mus-
cle tissue. Esser had been studying how muscles adapt to physi-
cal activity, but the unexpected finding piqued her interest—so
much so that she decided to take a sabbatical at Northwestern
University to investigate it further with geneticist Joseph Taka-
hashi, a pioneer in the field of circadian rhythms. “It was a sort of
90-degree tangent from what I’d been doing,” recalls Esser, now
at the University of Florida. “But the more I read about circadian
rhythms and clocks, the more [the findings] just made sense. I
couldn’t back away once I started.”
A growing body of evidence now points to these cyclical
dynamics as mediators of metabolism. Disrupting them may have
consequences for health, predisposing individuals to conditions
09. 201 8 | T H E S C IE N T IST 4 3
 such as diabetes. Research also indicates that these clocks may
influence muscle strength and structure, and may even regulate
neurological processes such as sleep.
“Clock systems are a sort of core, primordial part of our
genome that instruct and prepare cells for the work of using
nutrients, moving around, breathing, and [other] fundamental
processes,” says Joseph Bass, a clinical endocrinologist at North-
western University. “This is a story that’s evolving across a lot of
different experimental systems—and muscle is now a new experi-
mental system on the block.”
I think there has evolved a fairly
clear picture that the clock
is segregating . . . aspects of
metabolism to fit with the rest
and activity cycles of the day.
—Karyn Esser, University of Florida
Managing metabolism
The study of circadian rhythms, the daily cycles that regu-
late tissue and cell function, was once focused primarily on
the suprachiasmatic nucleus, the “master clock” in the brain.
Beginning in the late 1990s, scientists began uncovering
44 T H E SC I EN TIST | the-scientist.com
peripheral clocks—timekeepers located throughout the body—
and in 2007, Esser, Takahashi, and their colleagues confirmed
their presence in muscles.
Using microarrays to examine the transcriptomes in mouse
tissue, the researchers found a number of genes expressed in
a rhythmic fashion in muscle.1 These included the clock genes
Bmal1 and Per2, as well as genes involved in a variety of functions,
such as transcription and metabolism.2
By the time Schiaffino pivoted the course of his research to focus
on circadian biology, Esser and her colleagues had published these
results. “We were lucky that some pioneering groups had started
to do circadian transcriptomics on skeletal muscle,” says Kenneth
Dyar, a postdoc at the Institute for Diabetes and Obesity of Helm-
holtz Zentrum München in Germany who joined Schiaffino’s lab as
a graduate student in 2006. “So we had a short list of probable circa-
dian clock–dependent genes because they were cycling over 24 hours.”
Schiaffino and his colleagues decided to knock out Bmal1, a
core clock gene, from the muscles of mice. Upon doing so, they
discovered that the tissue’s ability to take up glucose in response
to insulin was impaired. Further analyses revealed that this was
due to decreased levels of proteins such as GLUT4, an insulin-
dependent glucose transporter, and TBC1D1, a factor involved in
the movement of GLUT4 to the plasma membrane of cells. The
researchers also found reduced activity of pyruvate dehydroge-
nase, an enzyme involved in metabolizing glucose in muscles.3
These findings implied that “the intrinsic muscle clock is
an important controller of glucose metabolism,” Schiaffino
says. This makes sense, he adds, because a muscle can become
a “sponge for glucose” when insulin is released in a healthy ani-
© ISTOCK.COM, VUKOSLAVOVIC
 Insulin
Glucose
ACTIVE MUSCLE
GLUT4
Insulin receptor
Energy
production
ATP
MUSCLE AT REST
Glucose storage
mal—after a meal, for example. In fact, skeletal muscle is the
body’s major glucose storage unit, responsible for around 70
percent of the body’s uptake of the sugar.
Esser posits that this daily cycle helps muscles prepare to tran-
sition from rest (and fasting), when the cells tend to store fuels,
to a wakeful period, when the animal is eating and its cells burn
fuels to generate energy for activity. In a 2015 study, she and her
colleagues discovered that, in mice, genes involved in metabo-
lism were primarily expressed before the rodents entered their
active phase in the evening. Prior to sleep, on the other hand, the
expression of genes involved in storing glucose and lipids peaked.4
Earlier this year, Charna Dibner of the University of Geneva
and her colleagues reported similar outcomes in human mus-
cle cells: disrupting the clock in vitro altered the expression of
a number of genes, including those encoding proteins involved
in glucose transport and lipid metabolism, and impaired the
muscle cells’ ability to take up glucose in response to insulin.5
“It was very consistent with what we see in the mouse, suggest-
ing a very conserved function for the muscle clock,” Esser says.
The muscle clock also appears to regulate the type of fuel
that the cell burns. Although active tissues require more energy,
cells still need some fuel during sleep, but rather than rely pre-
dominately on glucose, which powers contractile activity during
© THOM GRAVES
waking hours, they burn lipids and amino acids while at rest.
By examining mouse tissues at various time points, Schiaffino’s
team observed that Bmal1 and its target gene, REV-ERBα, play
a key role in this fuel selection process.6
MANAGING METABOLISM
One of the most clearly defined roles of a
muscle’s clock is maintaining the tissue’s
ability to take up glucose, a process that
occurs in response to insulin levels and mus-
cle contractions. During an animal’s waking
hours, feeding—which releases insulin from
the pancreas—and physical activity induce
the movement of the glucose transporter
GLUT4 to the cell membrane. Studies show
that disrupting clock genes in the muscle
impairs the transcription of GLUT4 and other
key genes involved in this process.
Proteins required to metabolize sugars
and lipids are also produced in a circadian
manner. Researchers have found that genes
that regulate the storage of these fuels reach
peak expression levels when animals are
preparing for rest, while those involved in
breaking them down for energy production
peak just before the active phase begins.
“I think there has evolved a fairly clear picture that the
clock is segregating . . . aspects of metabolism to fit with the
rest and activity cycles of the day,” says Esser.
Muscle clocks and health
Those who have flown overseas or from one North American
coast to another will likely understand what it feels like when
the body’s rhythms are out of sync. Traveling long distances
across multiple time zones throws off the usual clock-setting
cues, or zeitgebers, such as the daily light-dark cycle. Jet lag can
cause a variety of temporary symptoms, including dizziness, irri-
tability, and indigestion.
Longer-term perturbations of these rhythms can have
lasting effects on the body. Researchers have also found that,
in rodents, mutations in circadian clock genes can cause obe-
sity, metabolic syndrome (a cluster of conditions that includes
high sugar and low insulin levels in the blood), and diabe-
tes.7,8 A number of epidemiological studies have shown that
people who work night shifts are at a higher risk for these
conditions as well.9,10
Together, these findings imply that disrupting the muscle
clock may predispose individuals to metabolism-related ail-
ments. However, the specific mechanisms underlying this con-
nection have yet to be determined. “If we succeed in showing
that the [muscle] clock is involved in this passage from insulin-
sensitive to insulin-resistant muscle, then we can imagine ther-
apeutic directions, like using clock modulators,” Dibner says.
09. 201 8 | T H E S C IE N T IST 4 5
 “But what happens in type 2 diabetes is still a remaining ques-
tion in humans.”
Muscle clocks may affect aspects of health other than metab-
olism as well. Esser and her colleagues have found that knock-
ing out tissue-specific timekeepers leads to weaker muscles in
mice.11 Her team has also observed, in unpublished work, that
perturbing these clocks can influence the physiology of sarco-
meres, the basic units of muscle tissue. “When we disrupt the
clock, we’re starting to see variations in the length of the sarco-
mere along a single fiber,” Esser says. “[This can] affect force
generation, and the prediction is that you might make this mus-
cle more susceptible to injury.”
Muscle clocks can also influence rodents’ slumber. Neu-
roscientist Ketema Paul of the University of California, Los
Angeles, and his colleagues revealed that removing Bmal1
from the muscles of mice increased the amount of time the
rodents spent in non-REM sleep.12 Moreover, the researchers
found that, while knocking out this gene in the brain or mus-
cles impaired the rodents’ ability to regain normal sleep pat-
terns after six hours of forced wakefulness, increasing Bmal1
expression in muscle made them better at bouncing back after
sleep deprivation. (See “Power Nap,” pg. 55.) In addition to pro-
viding a better understanding of the mechanisms that mediate
sleep homeostasis, Paul says, these findings hint at potential
treatments that target nonbrain tissues for sleep disorders or
improve recovery after sleep loss.
Many questions remain about the way muscle keeps time,
such as how external signals are incorporated. Studies, pri-
marily in rodents, suggest that feeding and exercise may serve
as primary environmental cues. Oxygen levels may also play a

Light

MUSCLE TIME
The muscles’ intrinsic timekeepers keep
the body’s metabolic pathways in sync with
activity and rest cycles during the day.
12
The muscle clock is regulated by feeding
and physical activity—behaviors controlled
9 3
in part by the suprachiasmatic nucleus
6
(SCN), the body’s so-called master clock.
Researchers have found that the muscle’s
Muscle
intrinsic rhythms could be tweaked in
clocks
influence mice by changing the timing of feeding
Master clock
(SCN)
sleep (Gene Dev, 14:2950–61, 2000), which is an
important cue for other peripheral clocks as
well. Scheduled exercise can also tune the
Feeding Activity muscle’s clocks, affecting the expression
of circadian genes such as those involved
in maintaining the muscles’ contractile
properties (Med Sci Sports Exerc, 44:1663–70,
2012). Conversely, disrupting muscle clocks
Other
Muscle clock can affect sleep, suggesting that rhythms in
peripheral clocks
the body’s peripheral tissues feedback on
the brain, possibly through circulating
hormones or other chemical messengers.
THE SCIENTIST STAFF

Glucose and lipid

metabolism

46 T H E SC I EN TIST | the-scientist.com

role. A recent study by Bass and his colleagues at Northwest-
ern University revealed that muscle clocks interact with the
hypoxia-inducible factor (HIF) pathway, which responds to the
availability of oxygen.13 When the muscles are rapidly depleted
of oxygen—during exercise, for example—HIF helps the body
transition from metabolizing glucose through mitochondrial
respiration, an aerobic process, to anaerobic glycolysis, which
takes place in the cytoplasm and produces lactic acid.
“The clock seems to be gating the capacity of the skeletal
muscle to activate HIF and HIF-dependent metabolism at dif-
ferent times of day,” says Clara Peek, a biochemist at North-
western University who took part in that work. “I think [this]
new molecular connection in mice possibly explains some of
the connections between metabolism and the clock in muscle.”
MODIFIED FROM © ISTOCK.COM, DARUMO
How the muscle clocks interact with other peripheral clocks
also remains an open question. The muscles do not act alone.
With metabolism, for example, researchers have found that
other peripheral timekeepers, such as those in the liver, pan-
creas, and adipose tissue, also play a part. Studies by Dibner’s
group14 and others15 have shown that the clocks cycling within
the pancreas’ islet cells are crucial for maintaining proper insu-
lin secretion. (See “Out of Sync,” The Scientist, 2013.)
“In animals with a central nervous system, clocks are orga-
nized—to the best of our approximation—in a hierarchical man-
ner,” says Bass. “At the top of the pyramid is the so-called mas-
If we succeed in showing that
the muscle clock is involved
in this passage from insulin-
sensitive to insulin-resistant
muscle, then we can imagine
therapeutic directions, like
using clock modulators.
—Charna Dibner, University of Geneva
ter clock, which is aligned with the light/dark cycle, and through
mechanisms that are still not fully known, it orchestrates the align-
ment of peripheral tissue clocks with the environmental cycle.”
And when it comes to uncovering the molecular path-
ways that keep time in muscles and control that clock’s effects
on the body, Esser says, researchers have only just begun.
“There’s a lot still to learn.” g
References
1. B.H. Miller et al., “Circadian and CLOCK-controlled regulation of the mouse
transcriptome and cell proliferation,” PNAS, 104:3342–47, 2007.
2. J.J. McCarthy et al., “Identification of the circadian transcriptome in adult
mouse skeletal muscle,” Physiol Genomics, 31:86–95, 2007.
3. K.A. Dyar et al., “Muscle insulin sensitivity and glucose metabolism are
controlled by the intrinsic muscle clock,“ Mol Metab, 3:29–41, 2014.
4. B.A. Hodge et al, “The endogenous molecular clock orchestrates the
temporal separation of substrate metabolism in skeletal muscle,” Skeletal
Muscle, 5:17, 2015.
5. L. Perrin et al., “Transcriptomic analyses reveal rhythmic and CLOCK-driven
pathways in human skeletal muscle,” eLife, 7:e34114, 2018.
6. K.A. Dyar et al., “Transcriptional programming of lipid and amino acid
metabolism by the skeletal muscle circadian clock,” PLOS Biol, doi:10.1371/
journal.pbio.2005886, 2018.
7. F.W. Turek et al., “Obesity and metabolic syndrome in circadian clock mutant
mice,” Science, 308:1043–45, 2005.
8. B. Marcheva et al., “Disruption of the clock components CLOCK and BMAL1
leads to hypoinsulinaemia and diabetes,” Nature, 466:627:31, 2010.
9. C. Vetter et al., “Night shift work, genetic risk, and type 2 diabetes in the UK
Biobank,” Diabetes Care, 41:dc171933, 2018.
10. S. Fun et al., “Meta-analysis on shift work and risks of specific obesity types,”
Obes Rev, 19:28–40, 2018.
11. E.A. Schroder et al., “Intrinsic muscle clock is necessary for musculoskeletal
health,” J Physiol, 593:5387–404, 2015.
12. J.C. Ehlen et al., “Bmal1 function in skeletal muscle regulates sleep,” eLife,
6:e26557, 2017.
13. C.B. Peek et al., “Circadian clock interaction with HIF1α mediates oxygenic
metabolism and anaerobic glycolysis in skeletal muscle,” Cell Metab, 25:86–92, 2017.
14. C. Saini et al., “A functional circadian clock is required for proper insulin secretion
by human pancreatic islet cells,” Diabetes Obes Metab, 18:355–65, 2016.
15. M. Perelis et al., “Pancreatic cell enhancers regulate rhythmic transcription of
genes controlling insulin secretion,” Science, 350:aac4250, 2015.
09. 2018 | T H E S C IE N T IST 47
 The Elderly Muscle
Researchers untangle the multifarious nature of muscle aging.
So far, the only reliable treatment is exercise.
BY GILLIAN BUTLER-BROWNE, VINCENT MOULY, ANNE BIGOT, AND CAPUCINE TROLLET
T
o you readers over age 30, we’ve got
some bad news for you. Chances
are good you’ve already begun los-
ing muscle. And it only gets worse. Up to
a quarter of adults over the age of 60 and
half of those over 80 have thinner arms
and legs than they did in their youth.
In 1988, Tufts University’s Irwin
Rosenberg coined the term “sarcopenia”
from Greek roots to describe this age-
related lack (penia) of flesh (sarx). Muscle
aging likely has several underlying factors,
including decreased numbers of muscle
stem cells, mitochondrial dysfunction, a
decline in protein quality and turnover,
and hormonal deregulation. Loss of mus-
cle mass is associated with—and possibly
preceded by—muscle weakness, which can
make carrying out daily activities, such as
climbing stairs or even getting up from a
chair, difficult for many seniors. This can
lead to inactivity, which itself leads to mus-
cle loss at any age. Thus, older people can
48 T H E SC I EN TIST | the-scientist.com
enter a vicious cycle that will eventually
lead to an increased risk of falls, a loss of
independence, and even premature death.
The good news is that exercise can stave
off and even reverse muscle loss and weak-
ness. Recent research has demonstrated
that physical activity can promote mito-
chondrial health, increase protein turnover,
and restore levels of signaling molecules
involved in muscle function. But while sci-
entists know a lot about what goes wrong in
aging, and know that exercise can slow the
inevitable, the details of this relationship
are just starting to come into focus.
The role of muscle stem cells
Skeletal muscle consists of multinucleated
fibers formed by the fusion of muscle pre-
cursor cells, or myoblasts, during embry-
onic and fetal development and postna-
tally until the tissue reaches its adult size.
Mature fibers are post-mitotic, meaning
they do not divide anymore. As a result, in
adulthood both muscle growth and repair
are made possible only by the presence of
muscle stem cells.
In 1961, Rockefeller University bio-
physicist Alexander Mauro, using electron
microscopy, first described muscle stem
cells, calling them “satellite cells” because of
their position at the periphery of the muscle
fiber.1 Subsequently, researchers have dem-
onstrated that satellite cells are the only cells
able to repair muscle—which explains why
recovery from muscle injuries among the
elderly is slow and often incomplete: the
number of satellite cells falls from 8 percent
of total muscle nuclei in young adults to just
0.8 percent after about 70 to 75 years of age.
© ISTOCK.COM, CHRISCHRISW
Of course, a decline of the satellite
cells’ ability to divide and repair could
also be to blame, but research does not
support this idea. In pioneering studies
carried out in 1989, biologists Heather
Carlson and John Faulkner at the Univer-
sity of Michigan showed that muscle iso-
CREDIT LINE

201 8 | T H E S C IE N T IST 49
09. 2018
 lated from a two-year-old rat was repaired
faster and better when grafted into two-
to three-month-old rats.2 More recently,
we isolated these cells from young and
old adults and were surprised to find that
elderly human satellite cells grew in cul-
ture as well as those from young subjects
did.3 So it seems that declining function of
satellite cells is not the problem; there are
just fewer of them in muscle to do the job
of repair and growth.
The elderly human satellite cells we exam-
ined did, however, show dramatic changes in
their epigenetic fingerprint, with the repres-
sion of many genes by DNA methylation.
One gene, called sprouty 1, is known to be
an important regulator of cell quiescence.
Reduced sprouty 1 expression can limit satel-
lite cell self-renewal and may partially explain
the progressive decline in the number of sat-
ellite cells observed in human muscles dur-
ing aging. Indeed, stimulation of sprouty 1
expression prevents age-related loss of satel-
lite cells and counteracts age-related degen-
eration of neuromuscular junctions in mice.4
Mitochondrial contributors
Other likely culprits of muscle aging are
the mitochondria, the powerhouses of
muscle. To work efficiently, skeletal mus-
cle needs a sufficient number of fully func-
tional mitochondria. These organelles
represent around 5 percent to 12 percent
of the volume of human muscle fibers,
depending on activity and muscle special-
ization (fast-twitch versus slow-twitch).
And research suggests that abnormalities
in mitochondrial morphology, number,
and function are closely related to the loss
of muscle mass observed in the elderly.
In 2013, David Glass of Novartis and
colleagues found that markers of mito-
chondrial metabolism pathways were sig-
nificantly downregulated as rats aged, and
this correlated with the onset of sarcope-
nia.5 Although the findings are merely cor-
relative, the timing and near-perfect rela-
tionship between decline in mitochondrial
gene expression and the onset of sarcope-
nia provides strong evidence that mito-
chondrial dysfunction may be driving
sarcopenia. The expression of genes and
production of proteins that regulate mito-
5 0 T H E SC I EN TIST | the-scientist.com
chondrial fission and fusion—processes
that maintain mitochondrial volume and
function—also dropped, suggesting that
mitochondrial dynamics are also per-
turbed during muscle aging.
Up to a quarter of adults over the age of 60
and half of those over 80 have thinner arms
and legs than they did in their youth.
As with muscle stem cell decline, the
underlying cause of poor mitochondrial
health may be gene regulation. In 2016,
Alice Pannérec and her colleagues from
Nestlé Institute of Health Sciences and
Manchester Metropolitan University in
the UK examined the transcriptomes of rat
and human muscle and found that suscep-
tibility to sarcopenia in both species was
closely linked to deregulation of gene net-
works involved in mitochondrial processes,
regulation of the extracellular matrix, and
fibrosis, the formation of excess connective
tissue in a muscle caused by the accumula-
tion of extracellular matrix proteins.6
Protein quality control
Even if they eat plenty of protein, older peo-
ple often cannot maintain muscle mass,
probably because their bodies cannot turn
proteins into muscle fast enough to keep up
with the natural rate of the tissue’s break-
down. Moreover, the muscles of older peo-
ple undergo lower levels of autophagy, a
process that under healthy conditions recy-
cles used and damaged proteins, organelles,
and other cell structures. (See “Eat Your-
self to Live: Autophagy’s Role in Health and
Disease,” The Scientist, March 2018.) This
can result in an imbalance between protein
production and degradation that is likely
linked to muscle aging.
There may also be other ways that
reduced autophagy may contribute to both
muscle loss and muscle weakness during
aging. In order to maintain muscle strength,
muscle cells must get rid of the intracellular
garbage that accumulates over time. In the
case of muscle cells, this garbage includes
old organelles such as mitochondria and
endoplasmic reticuli, clumps of damaged
proteins, and free radicals, all of which can
become cytotoxic over time. By recycling
mitochondria, muscle fibers boost energy
production and preserve muscle function.
If muscle fibers fail to clear these potentially
dangerous entities, they will become smaller
and weaker. Sure enough, in a study from
Marco Sandri’s group at the University of
Padova in Italy, mice whose skeletal mus-
cles lacked one of the main genes that con-
trols autophagy, Atg7, had profound muscle
loss and age-dependant muscle weakness.7
Blood signals
In 2005, Stanford University stem cell biol-
ogist Thomas Rando and colleagues com-
bined the circulation of young and old mice
and found that factors in the blood of young
mice were able to rejuvenate muscle repair
in aged mice. (See “How old cells can regain
youth,” The Scientist, February 17, 2005.) It
is now well known that the levels of circu-
lating hormones and growth factors dras-
tically decrease with age and that this has
an effect on muscle aging. Indeed, hormone
replacement therapy can efficiently reverse
muscle aging, in part by activating path-
ways involved in protein synthesis.
Moreover, the muscle itself is a secre-
tory endocrine organ. Proteins produced
by the muscle when it contracts flow into
the blood, either on their own or encased
in membrane-bound vesicles that pro-
tect them from degradation by circulating
enzymes. Bente Pedersen of the Centre of
Inflammation and Metabolism and Cen-
tre for Physical Activity Research in Den-
mark was the first to use the term myo-
kine to describe these proteins. Secreted
myokines can act locally on muscle cells
or other types of cells such as fibroblasts
and inflammatory cells to coordinate mus-
 HOW MUSCLES AGE
Sarcopenia, the loss of muscle mass with age, can start as early as one’s 30s, and affects a large proportion of the elderly. Fortunately, exercise
can combat muscle aging, likely by reversing many of the age-related physiological changes at the root of this decline.

BLOOD-BORNE FACTORS
Signaling factors known as myokines can be released into the blood directly or
through excreted vesicles, and travel through the circulatory system to coordinate
muscle physiology and repair. For example, apelin, which decreases with age,
YOUNG boosts the formation of new mitochondria, stimulates protein synthesis OLD
MUSCLE and autophagy, and supports the function of muscle stem cells. MUSCLE

MAINTAINING PROTEIN BALANCE

Old muscles undergo lower levels of autophagy. Combined with lower protein
production, this can result in an imbalance of proteins linked to muscle aging.

Myokines
Mitochondrion Autophagosome

Muscle Differentiated
stem cells muscle cells

MUSCLE STEM CELLS

Muscle stem cells, or satellite cells,
decrease in number as we age. In
elderly-human cells DNA methylation
suppresses the expression of some
genes, including sprouty 1, an important
regulator of satellite cell self-renewal.

MITOCHONDRIA
Muscles develop abnormalities in mitochondrial
morphology, number, and function with age.
© SCOTT LEIGHTON

EXERCISE: A sedentary lifestyle can induce molecular processes of muscle aging, such as decreases
in the efficiency and number of mitochondria. Conversely, exercise reverses a gene expression profile
consistent with mitochondrial dysfunction and restores levels of mitochondrial proteins. Exercise
also increases autophagy levels and restores levels of myokines involved in muscle function.

09. 2018 | T H E S C IE N T IST 51

cle physiology and repair, or they can have sity in France recently discovered a novel versity of Birmingham and Steven Har-
effects in distant organs, such as the brain. myokine, which they termed apelin.8 The ridge at King’s College London examined
Although several of these myokines researchers have demonstrated that this the muscles of 125 male and female ama-
have been identified—in culture, human peptide can correct many of the pathways teur cyclists and showed that a lifetime
muscle fibers secrete up to 965 different that are deregulated in aging muscle. When of regular exercise can slow down mus-
proteins—researchers have only just begun injected into old mice, apelin boosted the cle aging: there were no losses in muscle
to understand their role in muscle aging. formation of new mitochondria, stimulated mass or muscle strength among those who
The first myokine to be identified, interleu- protein synthesis, autophagy, and other were older and exercised regularly. More
kin-6 (IL-6), participates in muscle main- key metabolic pathways, and enhanced surprisingly, the immune system had not
tenance by decreasing levels of inflamma- the regenerative capacity of aging muscle aged much either.9
tory cytokines in the muscle environment, by increasing the number and function of Exercise’s influence on muscle health
while increasing insulin-stimulated glucose satellite cells. As with IGF-1, levels of cir- likely acts through as many mechanisms
uptake and fatty-acid oxidation. Elderly culating apelin declined during aging in as those underlying age-related muscle
people with high circulating levels of IL-6 humans, suggesting that restoring apelin loss and weakness. For example, the num-
are more prone to sarcopenia. Another levels to those measured in young adults ber of satellite cells can be increased by
myokine, insulin-like growth factor 1 may ameliorate sarcopenia. exercise, and active elderly people have
(IGF-1), can trigger the swelling of muscle more of these cells than more-sedentary
fibers, including after exercise. IGF-1 levels Exercise to combat muscle aging individuals do. This is the reason why
decrease with age, as do levels of the cell- Although the causes of muscle loss are exercise prior to hip and knee surgery can
surface receptor that IGF-1 binds to, and numerous and complex, there is now speed up recovery in the elderly.
mice that overexpress IGF-1 are resistant copious evidence to suggest that exer- Physical activity also affects the mus-
to age-related sarcopenia. cise may prevent or reverse many of these cle’s mitochondria. A lack of exercise
Nathalie Viguerie and colleagues from age-related changes, whereas inactivity decreases the efficiency and number of
the Institute of Metabolic and Cardiovascu- will accelerate muscle aging. Earlier this mitochondria in skeletal muscle, while
lar Diseases at INSERM-Toulouse Univer- year, for example, Janet Lord of the Uni- exercise promotes mitochondrial health.
Last year, Caterina Tezze in Sandri’s lab
at the University of Padova identified a
strong correlation between a decline in the
levels of OPA1, a protein involved in shap-
AGE-RELATED MUSCLE DISEASES ing the mitochondria, and a decrease in
Sarcopenia is part of the general process of aging, but it can be triggered prematurely in some muscle mass and force in elderly subjects,
late-onset muscle diseases. For example, oculopharyngeal muscular dystrophy (OPMD) while OPA1 levels were maintained in the
is a rare genetic disease that primarily affects the eyelid (oculo) and throat (pharyngeal) muscles of senior athletes who had exer-
muscles. Mutations in the polyadenylate binding protein nuclear 1 (PABPN1) gene lead to the cised regularly throughout their lives.10
production of an abnormal protein that forms aggregates only in nuclei of muscle fibers. The Exercise can even spur muscle cells
late onset of the disease, which generally appears between 50 and 60 years of age, suggests to maintain more-youthful levels of gene
that the affected muscles successfully cope with the abnormal protein for many years. transcripts and proteins. For example,
However, the ability to deal with abnormal proteins decreases with age, and an imbalance Sreekumaran Nair from the Mayo Clinic
between elimination and aggregation could trigger the onset of OPMD. in Rochester, Minnesota, and colleagues
OPMD shows mechanistic similarities with severe degenerative disorders in which found that high-intensity aerobic interval
perturbed RNA metabolism and pathological assemblies of RNA-binding proteins are involved training reversed many age-related differ-
in the formation of cytoplasmic or nuclear aggregates. In patients with spinocerebellar ataxias, ences in muscle composition, including
ALS, Alzheimer’s, Huntington’s, or Parkinson’s diseases, these aggregates form in the neurons. restoring mitochondrial protein levels.11
In the case of myotonic dystrophy and inclusion body myositis, they form in the muscle fibers. And Simon Melov at the Buck Institute
Defining the exact alteration in RNA metabolism is an interesting question facing researchers for Research on Aging and Mark Tarnop-
studying muscle aging. Of note, all of these diseases are also characterized by abnormal olsky of McMaster University in Canada
mitochondria, which are observed in aging muscle. and their colleagues have found that while
Research into these diseases should not only lead to specific treatments, but also healthy older adults (average age 70) had
to interventions for the generally healthy aging population. And the reverse is also true: a gene-expression profile that was consis-
understanding how to stall muscle aging may provide tools to ameliorate pathological tent with mitochondrial dysfunction prior
conditions. Therefore, cooperation between the pathophysiology and aging fields to to a resistance exercise training program,
study these diseases, for which animal and cellular models exist, should be a focus of in just six months this genetic fingerprint
future research. had completely reversed to expression lev-
els comparable to those observed in young
subjects. Additionally, exercise improved
muscle function: the older adults were
59 percent weaker than the younger
adults before training, and only 38 per-
cent weaker afterward.12 Different types
Exercise may prevent or reverse many
of these age-related changes, whereas
inactivity will accelerate muscle aging.
of exercise can trigger variable but spe-
cific responses in the muscle. For exam-
ple, whereas strength training is efficient
at making muscle, high intensity interval
training in aerobic exercises such as biking
and walking had the greatest effect at the
cellular level at combating age-related loss
and weakness, according to Nair’s work.
Exercise also appears to influence
autophagy. In December 2011, Sandri and
his colleagues were the first to report, in
mice, that autophagy activity could be
boosted by voluntary physical activity,
in this case, running on a treadmill.13 In
January 2012, the team of Beth Levine
at the University of Texas Southwestern
Medical Center confirmed that exercise
rapidly increased autophagy activity and
that autophagy is required for exercise
to have its beneficial effects: physically
active mice that were unable to ramp up
autophagy did not show any increase in
muscle mass, mitochondrial content, or
insulin sensitivity after running.14
Finally, exercise can also apparently
restore levels of myokines that decline
with age. For example, when elderly sub-
jects followed a regular program of physi-
cal activity, there was a direct correlation
between the improvement in their phys-
ical performance and the increase in the
level of circulating apelin.15 Similarly, Ivan
Bautmans from Vrije Universiteit Brussel
showed that increased circulating levels of
inflammation markers correlate with mus-
cle fatigue in geriatric patients, and that
resistance training decreased inflamma-
tory myokines in young adults.16
By these mechanisms and others we
have yet to discover, exercise can improve
overall strength in the elderly, and specifi-
cally, the metabolic vigor of skeletal mus-
cle. Being the most abundant tissue in
the average human body, accounting for
30 percent to 40 percent of its total mass,
muscle is not only critical for locomotion
and breathing, but also for glucose, lipid,
and amino-acid homeostasis. The age-
associated loss of muscle mass and quality
thus contributes to the general metabolic
dysfunction commonly seen in elderly
patients. In older women, one hour of
brisk walking produced elevated insulin
sensitivity on the following day. There-
fore, it is never too late to exercise to try to
combat the consequences of muscle aging.
A detailed understanding of the
molecular and cellular pathways
involved in muscle aging could pave the
way for the development of therapeutic
interventions to boost protein synthesis
and increase muscle mass. For now, reg-
ular exercise combined with good nutri-
tion is still the most effective way to fight
sarcopenia, and possibly aging over-
all. In addition to detailing the under-
lying causes of muscle aging, future
research should seek to define optimal
physical exercise and nutritional pro-
grams to combat age-related muscle loss
and weakness. It may not significantly
increase human lifespan, but it will cer-
tainly help people reach the end of their
lifespan in a healthier condition. g
Gillian Butler-Browne studies neuromuscu-
lar diseases and gene therapy at Sorbonne
Université, INSERM, Institut de Myolo-
gie, Centre de Recherche en Myologie, in
Paris, France. At the same institution, Vin-
cent Mouly studies muscle regeneration in
health and disease, Anne Bigot studies mus-
cle aging, and Capucine Trollet studies age-
related muscle disease and gene therapy.
References
1. A. Mauro, “Satellite cell of skeletal muscle fibers,”
J Biophys Biochem Cytol, 9:493–95, 1961.
2. B.M. Carlson, J.A. Faulkner, “Muscle
transplantation between young and old rats:
Age of host determines recovery,” Am J Physiol,
256:C1262–66, 1989.
3. A. Bigot et al., “Age-associated methylation
suppresses SPRY1, leading to a failure of re-
quiescence and loss of the reserve stem cell pool in
elderly muscle,” Cell Rep, 13:1172–82, 2015.
4. W. Liu et al., “Loss of adult skeletal muscle stem
cells drives age-related neuromuscular junction
degeneration,” eLife, 6:e26464, 2017.
5. C. Ibebunjo et al., “Genomic and proteomic profiling
reveals reduced mitochondrial function and
disruption of the neuromuscular junction driving rat
sarcopenia,” Mol Cell Biol, 33:194–212, 2013.
6. A. Pannérec et al., “A robust neuromuscular system
protects rat and human skeletal muscle from
sarcopenia,” Aging, 8:712–28, 2016.
7. E. Masiero et al., “Autophagy is required to maintain
muscle mass,” Cell Metab, 10:507–15, 2009.
8. A. Besse-Patin et al., “Effect of endurance
training on skeletal muscle myokine expression
in obese men: identification of apelin as a novel
myokine,” Int J Obes, 38:707–13, 2014.
9. N.A. Duggal et al., “Major features of
immunesenescence, including reduced thymic
output, are ameliorated by high levels of physical
activity in adulthood,” Aging Cell, 17:e12750, 2018.
10. C. Tezze et al., “Age-associated loss of OPA1
in muscle impacts muscle mass, metabolic
homeostasis, systemic inflammation, and epithelial
senescence,” Cell Metab, 25:1374–89.e6, 2017.
11. R. Sreekumar et al., “Gene expression profile in
skeletal muscle of type 2 diabetes and the effect of
insulin treatment,” Diabetes, 51:1913–20, 2002.
12. S. Melov et al., “Resistance exercise reverses aging in
human skeletal muscle,” PLOS ONE, 2:e465, 2007.
13. F. Lo Verso et al., “Autophagy is not required to
sustain exercise and PRKAA1/AMPK activity but is
important to prevent mitochondrial damage during
physical activity,” Autophagy, 10:1883–94, 2014.
14. C. He et al., “Exercise-induced BCL2-regulated
autophagy is required for muscle glucose
homeostasis,” Nature, 481:511–15, 2012.
15. C. Vinel et al., “The exerkine apelin reverses age-
associated sarcopenia,” Na Med, doi:1010.1038/
s41591-018-0131-6, 2018.
16. P. Arnold et al., “Peripheral muscle fatigue in
hospitalised geriatric patients is associated
with circulating markers of inflammation,” Exp
Gerontol, 95:128–35, 2017.
17. X. Wang et al., “A 60-min brisk walk increases
insulin-stimulated glucose disposal but has
no effect on hepatic and adipose tissue insulin
sensitivity in older women,” J Appl Physiol,
114:1563–68, 2013.
09. 2018 | T H E S C IE N T IST 53
 EDITOR’S CHOICE PAPERS

The Literature
CELL & MOLECULAR BIOLOGY

In the Blood Healthy RBC

THE PAPER
A.S. Smith et al., “Myosin IIA interacts with the spectrin-actin
membrane skeleton to control red blood cell membrane curvature
and deformability,” PNAS, 115:E4377–85, 2018.

Healthy red blood cells are puffy with a dimpled middle. “It’s just a
really cool shape,” says Velia Fowler, a cell biologist at The Scripps
Research Institute in San Diego. For decades, researchers have Spectrin
Myosin
been wondering what gives red blood cells their characteristic Actin

curves, and now Fowler and her colleagues have the answer: myo-
sin proteins tug on the red blood cell’s cytoskeletal membrane, contraction
creating a divot at the center.
Myosin-inhibited RBC
Back in the 1980s, when Fowler started working with red blood
cells, it wasn’t clear whether they even contained myosin. She sus-
pected they might, because the protein appeared to play a role in giv-
ing other cells their shapes. After painstaking experiments, Fowler
finally showed that red blood cells do carry the protein, but exactly
how it influenced erythrocytes’ shape remained a mystery. “We didn’t
have the tools to do those experiments then,” she says. The myosin
filaments in red blood cells are tiny, only around 200 to 450 nano-
meters long, making them extremely challenging to image. And the
first inhibitor of myosin IIA—the specific protein found in red blood
cells—wasn’t developed until the early 2000s, so scientists couldn’t
see what happened when the protein wasn’t functioning in the cells. relaxed
In the new study, the team attached an immunofluorescent tag
IN A PINCH: In healthy red blood cells (RBCs), myosin fibers (blue) contract,
to myosin and a phalloidin tag to actin in human red blood cells
pulling on actin (pink) and spectrin proteins (purple) connected to the cell
and observed the cells with both superresolution and total internal membrane and helping to give the cells their distinct, indented shape (top).
reflection fluorescence microscopy. Filaments of myosin attach to When the myosin fiber is experimentally manipulated so that it slackens or
filaments of actin and the scaffolding protein spectrin that lie just detaches from the cell-membrane proteins, red blood cells instead take on
beneath and parallel to the cell membrane, the team found. When an oval shape (bottom).
myosin and actin interact and myosin contracts, the cell membrane
stiffens, giving the cell a dimple at its center. Inhibiting the motor “They’ve gotten rid of the cell nucleus, mitochondria, and all
activity of the protein causes the myosin filaments to expand so cytoskeletal proteins.” These simplified cells “have been a pow-
they no longer tug on spectrin and actin. That leads to less tension erhouse for generating concepts about how [plasma] mem-
in the membrane and, ultimately, the disappearance of the dimple. branes are organized in other cell types,” Bennett explains.
By expanding and contracting, the myosin filaments likely make Understanding the importance of myosin’s contraction in
it possible for red blood cells to shape-shift as they tumble in the shaping red blood cells, he says, may help in teasing out its
© KIMBERLY BATTISTA

bloodstream’s shear flow and squeeze through microvessels such as
capillaries, then pop back into their dimpled form, Fowler says.
This discovery could also give clues to how myosin works in
other types of cells, Vann Bennett, a biochemist at Duke Uni-
versity who was not involved in the new study, tells The Scien-
tist. “Red blood cells are a true experiment of nature,” he says.
functions in other cell types.
Another recent study supports myosin’s widespread impor-
tance in maintaining cell structure: the research showed the
protein is critical for axons to grow and shape themselves, sug-
gesting it could be involved in brain plasticity (Neuron 97:P555–
70.E6, 2018). —Ashley Yeager

5 4 T H E SC I EN TIST | the-scientist.com
 FEEL THE BURN: While exercise helps build muscle even as we age, it
does little for cells’ mitochondrial redox imbalance.
CELL & MOLECULAR BIOLOGY
Old and Stressed Out
THE PAPER
G.P. Holloway et al., “Age-associated impairments in mitochondrial
ADP sensitivity contribute to redox stress in senescent human skele-
tal muscle,” Cell Rep, 22:2837–48, 2018.
TOO MUCH OF A GOOD THING
The electron transport chain within mitochondria helps produce energy
in the form of ATP through redox reactions, generating reactive oxygen
species (ROS) in the process. In rodents, rising levels of ROS have been
linked to aging in mice, Graham Holloway of the University of Guelph in
Canada notes. In a recent study, his team used biopsies from a quadriceps
muscle to test whether the association held up in humans.
LATERAL THINKING
The researchers grew human muscle fibers from biopsies in culture
in the presence of varying amounts of the ATP precursor ADP and
measured the amounts of oxygen used up and hydrogen peroxide
(an ROS) released in mitochondria.
GROWING OLD
In healthy young adults, optimally functioning ADP ferries electrons
quickly through the final step of cellular respiration, meaning fewer
electrons can slip away to form ROS. Holloway’s group recorded
higher amounts of hydrogen peroxide in the muscle fibers from older
people (age ~70) at lower levels of ADP, indicating that the fibers had
© ISTOCK.COM, ALVAREZ; © ISTOCK.COM, UNOL
become less sensitive to the molecule. “The paper has systemati-
cally shown how mitochondrial function and bioenergetics is greatly
affected during aging,” writes Jin Han, who studies energy metab-
olism at Inje University in South Korea but was not involved in this
study, in an email to The Scientist.
LITTLE HELP
When Holloway and his colleagues tried the same experiment in
biopsies from older individuals undergoing resistance training at
the gym, the muscle fibers were in better health—stronger, leaner,
and more sensitive to ADP. But the skewed levels of peroxide
emission remained unchanged. —Sukanya Charuchandra
MUSCLED UP: In mice, it’s not just the brain that influences recovery from
a night of poor sleep.
CELL & MOLECULAR BIOLOGY
Power Nap
THE PAPER
J.C. Ehlen et al., “Bmal1 function in skeletal muscle regulates sleep,”
 eLife, 6:e26557, 2017.
UPSIDE DOWN
The protein Bmal1, which helps regulate the body’s internal clock, is
found in especially high levels in the brain and in skeletal muscles.
Mice completely deficient in Bmal1 were known to suffer from sleep
impairments, but the specifics at play weren’t clear. At the Univer-
sity of California, Los Angeles, Ketema Paul and colleagues looked to
these mice for clues about the role Bmal1 plays in sleep regulation.
MUSCLE PLAY
When Paul’s team restored levels of the Bmal1 protein in the
mice’s brains, their ability to rebound from a night of bad sleep
remained poor. However, turning on production in skeletal muscles
alone enabled mice to sleep longer and more deeply to recover
after sleep loss.
SWEET DREAMS
For decades, scientists have thought sleep was controlled purely
by the brain. But the new study indicates the ability to catch up on
one’s sleep after a bout of sleeplessness is locked away in skeletal
muscles, not the brain—at least for mice. “I think it’s a real paradigm
shift for how we think about sleep,” says John Hogenesch, a chrono-
biologist at Cincinnati Children’s Hospital Medical Center who dis-
covered the Bmal1 gene but was not involved in this study.
TARGET LOCKED
Paul’s group also found that having too much of the Bmal1 protein
in their muscles not only made mice vigilant but also invulnerable
to the effects of sleep loss, so that they remained alert even when
sleep-deprived and slept fewer hours to regain lost sleep. “To me,
that presents a potential target where you could treat sleep disor-
ders,” says Paul, noting that an inability to recover from sleep loss
can make us more susceptible to diseases.
—Sukanya Charuchandra
09. 2018 | T H E S C IE N T IST 5 5
 PROFILE
All Muscle
Having pioneered the study of muscle physiology in mammals,
Angela Dulhunty uncovered how ion channels enable muscle movement.
BY ANNA AZVOLINSKY
F
or Angela Dulhunty, the main draw of studying cells’ electri-
cal properties was the reward of instantaneous data. Rather
than having to wait sometimes days to get the results of a
biochemistry experiment, with electrophysiology “you see what
is happening in an individual cell in the moment,” says the mus-
cle biology researcher and now emeritus professor at Australian
National University in Canberra.
Dulhunty was attracted to learning how muscle works as an
undergraduate student studying physiology and biochemistry at
the University of Sydney. “Biochemistry in those days was a lot
of learning metabolic cycles, which was not as intuitive for me as
understanding how the micro-components of tissues and organs
inform their functions,” she says.
Dulhunty’s first real lab experience was during her final year
as an undergraduate. She was completing her honors thesis,
studying how hearing is registered by the ear and translated into
electrical signals through the nervous system.
She liked the hands-on lab work as much as the intellectual
exercise of cogitating on a biological problem. “I don’t think you can
separate the lab work from the thinking part of research. I’ve always
enjoyed putting the two together,” she says. This was among the
reasons Dulhunty continued to do her own hands-on experiments
in the lab, long after she started her own laboratory back in 1975.
In 2000, she made what she calls a “completely unexpected
and serendipitous discovery.” Dulhunty and her colleagues were
studying how the ryanodine receptor, a type of protein receptor,
functions in muscle cells. Ryanodine is an ion channel, embedded
in an internal membrane within the muscle cell, that surrounds a
pocket of calcium ions. The channel regulates the changes in cal-
cium ion concentration that control the muscle contractile appa-
ratus and, in turn, muscle movement.
I don’t think you can separate the lab work
from the thinking part of research. I’ve always
enjoyed putting the two together.
Dulhunty had set up electrophysiology experiments on a
receptor from mammalian cardiac muscle fiber to measure its
activity, and her initial measurements on the receptor’s activity
were going nicely. On a whim, she decided to add the enzyme
glutathione transferase to the muscle cells’ medium, just because
the chemical was sitting on the lab bench next to her. “I had an
extra 20 minutes to play, and I thought, ‘What would happen if I
5 6 T H E SC I EN TIST | the-scientist.com
added the enzyme? Would it interact with the ryanodine recep-
tor?’” Dulhunty recalls.
She did not expect anything to happen, but the addition of
the enzyme blocked the cardiac ryanodine receptor’s function.
“I could see immediately that the glutathione transferase began
to inhibit the cardiac muscle receptor’s activity,” she recalls.
Because the effect was unexpected, she didn’t believe it initially
and repeated the experiment several more times to make sure
the results were real. Within a few months, Dulhunty and her
colleagues published their first paper on the role of the omega
class glutathione S-transferase, GSTO1-1, in inhibiting the ryano-
dine receptor in cardiac muscle and in increasing the activity of
the skeletal muscle ryanodine receptor. The team continued the
work: finding the part of the GSTO1-1 molecule responsible for
inhibiting the ryanodine receptor, they modified the molecule for
maximum efficacy. They also explored its potential as a therapeu-
tic drug for use in preventing cardiac arrhythmias. Three years
after the initial discovery, they found that another protein struc-
turally related to glutathione transferases, a chloride intracellular
ion channel, CLIC-2, could also dampen the activity of the ryano-
dine receptor in the heart. This was the start of Dulhunty’s work
to discover the significance of these proteins in muscle.
Throughout her career, Dulhunty has been driven by her curi-
osity to know how the underlying physiology of the body works,
and, as a result, has made important discoveries about how skel-
etal and heart muscle contractions are generated and regulated.
AN EARLY START
Dulhunty was born in 1946 in Sydney, Australia, an only child.
Her father was a geologist at the University of Sydney, and her
mother had been a sheep farmer and breeder prior to getting
married. Dulhunty was a “tomboy,” who couldn’t wait to get home
from school, change into shorts, and run around and climb trees
in the Australian bush with her friends, she says. Thanks to her
father, Dulhunty was also interested in science from an early age,
which led her to study biochemistry when she entered the Univer-
sity of Sydney in 1965. She also began riding horses as a young-
ster, and every summer during university she was a counselor and
instructor at a horse riding camp in the Snowy Mountains, close
CHRIS THEKKEDAM
to where she now lives just outside of Canberra.
When wrapping up her undergraduate studies, Dulhunty
thought she wanted to go to medical school so that she could do
medical research, but a professor of biochemistry advised her to
attend graduate school instead if research was her main interest.

ANGELA DULHUNTY
Emeritus Professor, John Curtin School of Medical Research,
Australian National University, Canberra, Australia
Honorary Member, Australian Physiological Society
President of the Australian Society for Biophysics (ASB, 2010 – 2012)
Founding director of Biotron Pty Ltd, a biotechnology company
Head of the John Curtin School of Medical Research Department of
Molecular Bioscience (previously Division of Biochemistry and
Molecular Biology, 2007-2014)
Greatest Hits
• Along with Clara Franzini-Armstrong, uncovered that
caveolae—tiny pocket structures in the surface membrane
of individual muscle fibers whose function was previously
a mystery—allowed muscle to stretch to more than double
its length without incurring damage
• By developing techniques of muscle electrophysiology,
pioneered the study of mammalian muscle biology
• Found that potassium and chloride ion activity in muscle
contraction differs drastically between amphibian and
mammalian muscle
• Established that specific members of the glutathione
transferase structural family differentially modulate the
activity of calcium-dependent ryanodine receptors in
cardiac and skeletal muscle
• Found that a mutation in the type 2 chloride intracellular
ion channel (CLIC-2) in humans can result in an X-linked
human disorder
After seeking advice from other professors, she ended up in Peter
Gage’s membrane physiology and neuroscience lab at the Univer-
sity of New South Wales in Sydney. “He was exciting to talk to and
an inspiring scientist,” Dulhunty says.
BASIC QUESTIONS
Dulhunty began her PhD in 1969, getting straight to work on
her thesis, as no coursework was required for a graduate degree
in Australia at the time. She initially worked on characterizing
a deadly toxin secreted by the Southern blue-ringed octopus
(Hapalochlaena maculosa), which lives in tidal rock pools along
the southern coast in Australia. Using live octopuses, Dulhunty
found that the water-soluble maculotoxin inhibits action poten-
tials in motor nerves and muscle fibers, preventing the transmis-
sion of neuromuscular signals.
Dulhunty also began her initial work on the biophysical prop-
erties of muscle fibers, which contract in milliseconds based on
the translation of signals from the brain to the muscle. “The basic
question,” she says, “was, how does the electrical signal on the sur-
face of the muscle fiber get translated into a muscle contraction?
And that is still what I am working on today!”
In the 1970s and 1980s, Dulhunty—and most labs studying
muscle—used individual toad or frog muscle fibers for electrophysi-
ology experiments because the animals have robust muscle fibers
that can be dissected and separated relatively easily. “Mammalian
fibers are much harder to work with. It is very difficult to separate
out an individual fiber because they are much longer, and there is
so much surrounding connective tissue,” Dulhunty explains.
BEYOND THE LIMIT
After completing her PhD in 1973, Dulhunty applied for and was
awarded a postdoctoral fellowship from the Muscular Dystrophy
Association of America and had her first overseas adventure. Dul-
hunty worked at the University of Rochester in New York in the
lab of Clara Franzini-Armstrong, who is now an emeritus pro-
fessor at the University of Pennsylvania. At the time, Armstrong
had already made her name in science by demonstrating how the
structure of the muscle fiber membrane facilitates its function.
Continuing to work on frog skeletal muscles, Dulhunty com-
bined her expertise in electrophysiology with Armstrong’s skills
in electron microscopy. “This in itself was remarkable at the time,
as the two vastly different techniques were generally not com-
bined into a single study,” she says. In 1975, Armstrong and Dul-
hunty uncovered a key function of caveolae, Latin for “little caves,”
09. 2018 | T H E S C IE N T IST 57
 PROFILE
tiny indented structures in the surface membrane of individual
muscles that are also found in fat tissue and in blood vessels. The
pair found that the thousands of caveolae in frog muscle open,
enabling the muscle to survive “enormous stretches to more than
double its rest length and without significant damage to the cells,”
says Dulhunty. Their findings revealed just how well-equipped
muscle is to adapt to stress and resist cell death.
Dulhunty stayed in the US for a year and a half. When she wasn’t in
the lab, she took time to travel, visiting Woods Hole in the summer, and
taking a three-week road trip from the East Coast to the West Coast and
back to Rochester in a “little Mustang car.” Still, she wanted to go back
to Australia. And, she wanted to find out more about how mammalian
muscles worked, which other researchers were not yet attempting.
After leaving Rochester in 1974, Dulhunty started her own lab
at the University of Sydney. Her goal was to better understand how
the electrical signal on the surface membrane of a muscle cell is
translated into the release of calcium ions that initiates muscle
contraction. Dulhunty homed in on how ion channels in the mus-
cle affect membrane potential—the difference in electric poten-
tial between the interior and the exterior of a living cell. Her first
achievement on this front, in 1977, was authoring a Nature paper
that revealed details of mammalian muscle contraction. She found
that the threshold of response to potassium ions is higher in rat
muscle than in amphibian muscle. Dulhunty then went on to dem-
onstrate the importance of chloride ions and membrane-bound
chloride transporter proteins in setting the electrical membrane
potential in mammalian skeletal muscle.
“It was assumed that chloride functioned the same way in
amphibians and in mammals, but we found chloride ion func-
tion was very different from that in amphibian muscle,” Dulhunty
explains. The studies helped uncover what goes wrong in several
human childhood-onset genetic neuromuscular disorders, includ-
ing myotonia congenita and myotonic dystrophy. Both occur when
chloride channel presence is altered in muscle. Largely following
from Dulhunty’s work, researchers have shown that the removal of
chloride ions from the muscle membrane results in overexcitable
muscle fibers that contract involuntarily.
BUILDING A LAB
In 1984, Dulhunty moved her laboratory to Australian National
University in Canberra. By then she and Gage were a couple, and
he also moved his lab to the university, where the two continued to
collaborate until his death in 2005.
Once her lab was established, Dulhunty continued to develop
techniques to study mammalian muscle. There was a gradual tran-
sition in the field from examining the biophysical properties of
amphibian muscles—which are much easier to study—to mamma-
lian muscle, in large part led by Dulhunty.
Her group was the first to measure the “asymmetrical charge
movement” in mammalian muscle fibers. The phenomenon—ini-
tially identified in nerve and then muscle cells—arises when a charge
moves about a millionth of a millimeter inside the cell membrane,
which is a critical part of normal muscle fiber contraction. To make
5 8 T H E SC I EN TIST | the-scientist.com
this exquisite measurement in the mammalian system, Dulhunty’s
and Gage’s labs first had to design and build the equipment for doing
so, putting three tiny, glass pipette electrodes into the end of an indi-
vidual rat muscle fiber and painstakingly dissecting the minute elec-
trical signals of asymmetrical charge movement from much larger
signals and noise in the system. The signals had been predicted in the
1950s by the 1963 Nobel Prize winners Alan Hodgkin and Andrew
Huxley, but it had taken more than 20 years to develop the technology
required to measure so tiny a charge movement. “It was very exciting
to be a part of that development,” Dulhunty says.
Researchers in Dulhunty’s lab also began to study the ryanodine
receptor ion channel. Among the first to characterize the receptor,
Dulhunty’s lab extracted the essential protein from muscle fibers,
embedding it into an artificial lipid bilayer to study its function.
The receptor works to initiate the final step of muscle contraction.
In 1994 and 1995, the team found that a small, multifunctional
protein that binds an immunosuppressant drug, FK506, was criti-
cal for normal ryanodine receptor function. They also discovered
unsuspected effects of high calcium ion concentrations and oxi-
dizing reagents on the activity of the receptor in cardiac muscle.
“Everything that the lab has done since then has been partly focused
on this ion channel and the way in which it operates, including its
role in human muscle diseases,” Dulhunty says.
The study of the transferases and related proteins is an exam-
ple. In 2012, she and her colleagues found that a mutation in the
gene for human CLIC-2, the type 2 chloride intracellular ion chan-
nel, could result in X-linked intellectual disability, congestive heart
failure, and seizures. “The mutation altered the effect of CLIC-2
on the ryanodine receptor, so altering normal ryanodine receptor
activity could explain the characteristics of individuals carrying this
mutation,” Dulhunty says. More recently, in still unpublished work,
the lab identified additional disease-associated CLIC-2 mutations.
An emeritus professor since 2016, Dulhunty is wrapping up
a few key projects, including identifying additional proteins that
interact with the ryanodine receptor. She also has been working
with a foundation in the US to develop animal models for ryanodine
receptor–associated muscle diseases. The lab, which comprises one
technician and one graduate student, contributes by exploring how
the activity of the ryanodine receptor is affected by genetic altera-
tions. “It’s important to study these human mutations in animal
models, not just in cell culture, because there are major differences
of how muscle proteins are modified in a whole animal compared
to in isolated cell cultures,” Dulhunty says.
She is optimistic about further advances in the field. “When I
started my PhD, muscle contraction was a black box, and now we
generally know how it works, although not all of the most impor-
tant details,” she says. The next generation of scientists, she says, will
have to develop new techniques to reveal these details.
Dulhunty says that in retirement she hopes to help researchers
in her lab and to set aside time to tend her horses. “As soon as I had
enough money, I bought my own horse,” Dulhunty says. That was
30 years ago. She now lives on farm with two Jack Russell terriers
and two horses, which she tries to ride five times a week. g
 SCIENTIST TO WATCH
Avnika Ruparelia: Muscle Mender
Research Fellow, Australian Regenerative Medicine Institute, Monash University, Age: 30
IBY SUKANYA CHARUCHANDRA
B
orn and raised in Kenya, Avnika
Ruparelia moved to Australia with
the hope of becoming a doctor.
When her application to medical school
was denied, she switched her focus to bio-
medical science. As someone who hates
the sight of blood, the career diversion
suited her. Ruparelia, now a research fel-
low at Monash University in Melbourne,
revels in “the feeling of being the only per-
son who knows that one thing,” she says.
“It’s absolutely fantastic.”
© CORWIN VON KUHWEDE
Ruparelia got her start in research as an
undergraduate in the lab of Monash muscle
biologist Robert Bryson-Richardson. In 2009,
she worked with him to map a gene that even-
tually was linked to myofibrillar myopathies
(MFM), a group of diseases resulting in pro-
gressive muscle weakness that is character-
ized by protein clumping and structural failure
within muscle cells. As Bryson-Richardson and
Ruparelia began studying MFM, they and col-
leagues developed a zebrafish mutant model
for one type of the disease, filamin-related
MFM.1 This model allowed researchers to
study not only filamin-related MFM, but also
the normal function of filamin C, a protein
found within skeletal muscles.
A year after developing the model,
Ruparelia focused on a different form of MFM
called BAG3-related MFM. Unlike other forms
of the condition, this one is caused by muta-
tions in a gene that plays no obvious role in
muscles. The protein encoded by BAG3 nor-
mally helps dispose of malformed proteins. By
expressing mutant and normal forms of BAG3
in zebrafish, Ruparelia and her colleagues
found that the mutant form of the protein
trapped and sequestered the functional
form, eventually leading to muscle
damage.2 Ruparelia, who finished
her PhD at Monash in 2014,
also found healthy BAG3
ensnared within aggre-
gates of mutant filamin
protein. BAG3, how-
ever, wasn’t working
to clear the prob-
lematic proteins from
the muscle cells. In fact,
its presence within such clumps
seemed to inhibit other cell-cleansing
pathways as well.3
“It’s important to question the results
you get and look for other explanations,
not just the one you set out to test,” says
Bryson-Richardson. Ruparelia, he adds,
is thorough in questioning her results.
Their work allowed the pair to use a
zebrafish model of BAG3-related MFM to
run a drug screen to test potential therapies
for the disease. Some of the drugs the
researchers have identified helped zebrafish
models with not only the pathology but also
the muscle weakness associated with the
disease. One of the drugs they’re testing is
already FDA-approved to treat other con-
ditions, which might mean it could move
through clinical trials much more quickly
than entirely new medicines.
“I’ve been impressed by Avnika because
she’s had a very focused line of research
in terms of the way that she’s approached
problems and built up a very strong body
of evidence to support that direction,”
says her mentor for the past two years
Gordon Lynch, a muscle biologist at the
University of Melbourne.
Ruparelia is now setting up the first lab
in Australia to use African killifish as a model
organism. She spent the summer in geneticist
Christoph Englert’s lab at the Leibniz Institute
on Aging in Germany to learn how to use the
model and to gather some preliminary data.
The killifish’s short lifespan, Englert writes in
an email to The Scientist, will help Ruparelia
study the effect of aging on the regenerative
capacity of muscle. g
REFERENCES
1. A.A. Ruparelia et al., “Characterization
and investigation of zebrafish models of
filamin-related myofibrillar myopathy,”
Hum Mol Genet, 21:4073–83, 2012.
(Cited 20 times)
2. A.A. Ruparelia et al., “Zebrafish models of
BAG3 myofibrillar myopathy suggest a toxic
gain of function leading to BAG3 insuffi-
ciency,” Acta Neuropathol, 128:821–33, 2014.
(Cited 27 times)
3. A.A. Ruparelia et al., “FLNC myofibrillar
myopathy results from impaired autoph-
agy and protein insufficiency,” Hum Mol
Genet, 25:2131–42, 2016. (Cited 15 times)
09. 2018 | T H E S C IE N T IST 59
 LAB TOOLS

The Native Niche

Stem cell experts share their hard-won tips on making your cells grow faster,
healthier, and in higher numbers.

BY AMBER DANCE

C
ell biologist Alicia Lyle hoped to
use mouse mesenchymal stem cells
to deliver molecular cargos to tis-
sues, and she also wanted to study how
MSCs from different lines of knockout
mice assemble into blood vessels. But Lyle’s
group, at Emory University in Atlanta, soon
hit a snag: growing the cells took ages.
“Even with the tenderest of care, it was
taking somewhere close to eight to twelve
weeks to even reach a point . . . to passage
them,” recalls Lyle, referring to the point
when cells crowd a dish and need to be split
between multiple culture flasks. And by
passage seven or eight, Lyle’s cells began to
senesce, losing their ability to either main-
tain pluripotency or differentiate. and for research into future treatments. THEY’VE GOT THE BEAT: Heart muscle cells
While mouse MSCs are particularly dif- To screen 1 million drugs would require derived from human iPSCs with NME7AB in
ficult to work with, Lyle’s complaints echo on the order of 100 billion stem cells, as the media form much more organized myofibrils,
needed for contraction, compared to those raised
those heard across the stem cell field. To would building a human heart or liver in fibroblast growth factor 2 (FGF2).
both understand stem cells and use them to from scratch.
treat diseases, efficiency will be crucial. “We
want to harness that power of stem cells, we techniques, such as a new approach that
want to use it to treat patients, but then you We want to harness the power encases cells in long, sausage-like tubes.
are limited by how many you can make,” of stem cells . . . but you are And experimentalists can also improve
laments Abba Zubair, medical director for limited by how many you their stem cell quality and yields by com-
transfusion medicine and the Human Cell can make. bining more than one method.
Therapy Laboratory at the Mayo Clinic in Overall, what seems to work is to pro-
—Abba Zubair, Mayo Clinic
Jacksonville, Florida. No one wants to wait vide a cell culture setting that mimics the
around for months while stem cells grow, or natural stem-cell niche as closely as pos-
discover that only a few have the potential How can scientists speed things up? sible, which minimizes cell stress, advises

ANDREW STEWART, MINERVA BIOTECHNOLOGIES

to differentiate as desired.
And stem cell treatments will only
become affordable and commonplace if
the technical processes used to develop
them are efficient. For now, another type
of cell therapy, the first approved CAR
T-cell treatment for cancer, Novartis’s
Kymriah, costs $475,000 for a single dose,
suggesting that stem cell–based treat-
ments using today’s protocols will be simi-
larly complicated and expensive. Still, the
potential demand for stem cells could be
quite high, both to directly treat patients
Every stem cell type, from embryonic to
induced pluripotent to tissue-specific,
has its own particular requirements for
healthy, optimal growth and differen-
tiation. But there are a few general tips
that all stem cell researchers can employ.
Some scientists obtain good results by try-
ing different growth factors. One particu-
lar recombinant growth factor, NME7AB,
which supports cells in their most naïve,
pluripotent state, can streamline experi-
ments. Another tactic is to develop stem
cell-nurturing, three-dimensional culture
Yuguo Lei, a biomedical engineer at the
University of Nebraska–Lincoln.
EMBRACE NAÏVETÉ
Induced pluripotent stem cells (iPSCs),
made from a person’s blood or skin cells,
have become tremendously popular. But
Cynthia Bamdad, CEO of Minerva Bio-
technologies in Waltham, Massachusetts,
says most iPSC cultures are not as “stem”
as they could be.
The stem cells in the inner cell mass
of an embryo, known as naïve stem cells,

6 0 T H E SC I EN TIST | the-scientist.com
 are what many scientists would like to
imitate with iPSCs. They lack epigene-
tic marks that program cell fates, mak-
ing them fully pluripotent. In contrast,
most iPSC cultures are primed stem
cells. They represent a more mature
embryonic tissue, the epiblast that forms
the external layer of an embryo. Primed
iPSCs often contain epigenetic marks,
though the genes turned on and off are
fairly random, says Bamdad.
That means not all cells in an iPSC
culture are truly pluripotent; many are
already on their way to one cell fate or
another. “It is difficult to make primed
stem cells differentiate into mature, func-
tional cell types that we need for therapeu-
tics,” says Bamdad.
The reason iPSCs tend to turn out
primed instead of naïve, she adds, is that
most protocols use media containing fibro-
blast growth factor (FGF). “FGF, which is
used to grow these cells during reprogram-
ming and after, prevents them from going
all the way back to the earliest state that we
call naïve.” Only about 15 percent of iPSCs
generated with FGF turn out to be “bona
fide” iPSCs, says Bamdad.
Minerva’s AlphaSTEM cell culture
products provide a more native environ-
ment, she says, because they use a dif-
LI ET AL., BIOFABRICATION, 10:025006, 2018, DOI:10.1088/1758-5090/AA6B5, CC BY 3.0
ferent growth factor, NME7 (Stem Cells,
34:847–59, 2016). NME7 is typically
expressed in very early embryos, so it
better mimics the naïve stem cell niche.
Using the recombinant NME7AB, Bam-
dad says, yields better iPSCs. “By ‘bet-
ter,’ we mean works every time . . . bet-
ter functional profile, higher yields,” she
says. “They all can become any cell type.”
With AlphaSTEM Naïve HPSC Medium
($295/500 mL), researchers can repro-
gram the majority of cells in their cul-
tures to a pluripotent state, she adds.
Other pro-naïve solutions exist, such
as 2i and 5i media formulations. These
rely on leukemia inhibitory factor (LIF)
from mice and other biochemical inhib-
itors. While these media do yield cells
that are more naïve than primed cells,
Bamdad notes they can create instability
in chromosome numbers, and she’s not
sure how well they mimic true embry-
onic stem cells.
Minerva researchers say naïve iPS cells
could streamline cell production, improve
gene-editing efficiency, and boost the over-
all quality of therapeutic stem cells. “If you
start with better cells, everything’s going to
be easier,” Bamdad says.
ADD DIMENSIONS
Another way to improve the stem cell cul-
ture environment is to consider its topo-
graphy. Cells in the lab often grow in
monolayers on flat glass or plastic. All
those plates require a lot of space, media,
and time to care for them; it’s highly inef-
ficient. One solution is to grow cells in sus-
pension, in large flasks or bioreactors. (See
“Easier Ways to Grow Stem Cells,” Septem-
ber 2017, The Scientist.)
But big bioreactors create problems, too,
says Lei. At first, the stem cells begin to clus-
ter together into little spheroids. That’s good;
stem cells don’t typically grow alone in situ.
But in bioreactors, those spheroids eventu-
ally grow and coalesce into big aggregates.
Nutrients and oxygen can’t reach the cells in
the center of the aggregate, and wastes don’t
easily diffuse out of the blob. “That makes
the cell culture very inhomogeneous,” says
If you start with better cells,
everything’s going to be easier.
—Cynthia Bamdad, Minerva Biotechnologies
Lei. Aggregation can slow growth, induce
apoptosis, or alter differentiation patterns.
Stirring the tank minimizes aggre-
gation, but such agitation creates a shear
force that can kill cells, especially sensi-
tive human stem cells. Lei’s lab has found
that up to 40 percent of stem cells grown in
spinning suspension die every day.
Lei sought a way to manage stem cell
cluster size while protecting the frag-
ile cells from shear forces. He developed
a hydrogel tube in which stem cells can
grow (Biofabrication, 10:025006, 2018).
The lab uses a custom micro-extruder to
make the tubes, which are up to 400 μm
in diameter. As they extrude the alginate
for the tube through a cylindrical channel
(shell flow), they simultaneously pump a
cell suspension through its center (core
flow). These materials land in a calcium
chloride buffer, which makes the alginate
solidify into tubes around a cell suspension
core. With the AlgTubes, his cultures look
like vats of udon noodle soup, Lei says.
The tubes constrain the size of the cell
aggregates so that nutrients and oxygen can
still diffuse in, and wastes can filter out. The
hydrogel barrier insulates cells from shear
forces in moving media.
Lei’s results indicate stem cells are much
more comfortable in the tubes. Fewer than
TOTALLY TUBULAR: Growing human embryonic
stem cells in hydrogel tubes protects them
from moving media and promotes health
and higher yields.
09. 2018 | T H E S C IE N T IST 61
 LAB TOOLS
10 percent die every day. Yields are higher,
with up to 500 million stem cells growing
in every milliliter of media within the tubes,
compared to 2 million to 5 million per mil-
liliter in suspension culture. Lei expects it
will be possible to scale that up. For exam-
ple, one could make 10 billion cells with just
two milliliters of space contained in tubes.
The cells in AlgTubes can be cultured for
more than 50 days, can expand 1,000-fold
in just 10 days, and can be differentiated
into a variety of cell types.
AlgTubes can last for months, and
when the researchers want to get the cells
back out, it’s easy. They use EDTA to che-
late the calcium ions in the hydrogel, and
the tubes fall apart.
Lei can provide scientists with detailed
instructions for making their own AlgTube
extruder, or his group can produce them
for collaborators. He’s started a company,
CellGro Technologies, to commercialize
the extruder.
SYNERGIZE
If one method improves stem cell yields
and quality, wouldn’t two be better?
That’s what Lyle tested with the mouse
MSCs she isolated from bone marrow.
6 2 T H E SC I EN TIST | the-scientist.com
A good protocol to grow and main-
tain mouse MSCs would be a boon for
research, she says, because scientists
already have so many genetically engi-
neered mouse lines. Researchers could
isolate MSCs from those lines, and
then determine how different genes are
involved in differentiation, she says.
Lyle read that basic fibroblast growth
factor (bFGF) worked well for MSCs, likely
due to its pro-growth and pro-survival sig-
naling. (FGF wouldn’t be a bad choice in
Lyle’s case, because she wasn’t trying to
grow naïve cells.) She’d also seen reports
that hypoxia would help. That’s probably
because oxygen levels in the bone marrow,
where MSCs naturally exist, are around 5
percent, much less than the atmospheric 21
percent that most stem cells are cultured in.
The research team tested four treat-
ments: normoxia and hypoxia, each with
or without bFGF. Then, when the cells
covered much of the dish surface and were
ready for their first passage, the scientists
measured the concentration of cells per
square centimeter. With atmospheric oxy-
gen and no bFGF, the cells were ready to
passage in 39 days, and there were about
6,600 cells per square centimeter. With
It doesn’t have to be a one-
pronged approach. It can be
multifactorial.
—Alicia Lyle, Emory University
bFGF, the cells achieved the required con-
fluence within 27 days, and they crowded
together at 43,000 per square centimeter.
With hypoxia and no bFGF, the cells were
ready after 20 days, and their numbers rose
to 99,000 per square centimeter. But with
both hypoxia and bFGF, the cells were con-
fluent at 14 days, and the concentration was
the highest—220,000 per square centime-
ter (Sci Rep, 7:13334, 2017).
Overall, the MSCs in low-oxygen,
bFGF conditions grew 2.8 times faster
than under standard protocols, and
reached passage 3, when cells are typi-
cally ready to test, within one month
instead of the usual two or three. They
were less likely to senesce, and retained
their ability to multiply or differentiate
until at least passage 11.
The results of the dual treatment are
“crazy good,” says Lyle, “I think because it’s
closer to what their native niche is.”
Lyle has already used her protocol to
generate MSCs from transgenic mice that
make luciferase or GFP. Scientists could
then engineer these MSCs and implant
them into mice without the luminescent
or fluorescent markers, and track where
the glowing cells end up and how long
they survive.
And she’d be happy to add other treat-
ments to improve yields and efficiency. “It
doesn’t have to be a one-pronged approach,”
says Lyle. “It can be multifactorial.”
Indeed, Lyle adds, current stem cell cul-
ture protocols probably haven’t reached any-
CAROTI ET AL., SCI REP, 7:13334, 2017.
where near the optimal conditions for effi-
cient growth and division. “There is still a lot
of area for improvement,” she says. g
TWO ARE BETTER THAN ONE: While both low
oxygen (right column) and basic FGF (bottom row)
improve growth of mouse mesenchymal stem cells,
the combination of the two (bottom right) works
the best.
 BIO BUSINESS

A New Dawn for ALS Therapies?

After two decades of failure, novel scientific insights and technical
progress are spurring meaningful innovation in the field.

BY JENNY ROOD

I
n 1999, a paper in Nature Medicine
reported that mouse models of the
fatal neurodegenerative disorder amyo-
trophic lateral sclerosis fared better with
a simple treatment: a diet supplemented
with creatine, a compound that helps reg-
ulate energy levels in the brain and mus-
cles (5:347–50). That promising, albeit
preliminary, result soon launched not one
but three clinical trials, with a total of 386
patients in the US and Europe. Disappoint-
ingly, the trials revealed that creatine had
no effect in people. It was a familiar out-
come: more than 50 other clinical trials
of potential amyotrophic lateral sclerosis
(ALS) drugs, ranging from lithium to cele-
coxib (Celebrex), have failed.
Also known as Lou Gehrig’s dis-
ease, ALS results from the degeneration
and death of motor neurons, and affects
approximately two to five of every 100,000 by the US Food and Drug Administration In the past few years, the ALS research
people worldwide. ALS’s devastating (FDA), small-molecule drugs riluzole and community has gained traction in meet-
symptoms—including progressively wors- edaravone, have largely mysterious mecha- ing these challenges. Genetic discoveries
ening muscle weakness and spasming, and nisms of action and targets, and only mod- have revealed new pathways involved in
difficulties with speech, swallowing, and estly improve survival and quality of life. the disease. Enhanced technologies gen-
breathing, leading ultimately to paralysis Second, ALS is a highly heterogeneous erate more-realistic models and hypoth-
and death—have led to an intense hunt for disease in terms of origin (90 percent to 95 eses. And new therapeutic approaches,
treatments to halt its progression. percent of cases are sporadic rather than such as oligonucleotide therapeutics,
Unfortunately, the desire to give inherited), initial symptoms (patients may have begun to enter the clinic. Together,
patients hope has often outstripped good report limb weakness or difficulty in speak- these advances have made many research-
scientific sense. “Many drugs that have ing or swallowing), and speed of progres- ers optimistic that effective treatments are
gone into ALS clinical trials shouldn’t have, sion (some patients live months, others finally on the way.
because the preclinical data package didn’t decades, after diagnosis). This has made it
support it,” says Steve Perrin, CEO and CSO tricky to model the disease. For many years, Shedding light on ALS biology
of the nonprofit ALS Therapy Development the only available mouse models were those In addition to riluzole, on the market
© ISTOCK.COM, WAVEBREAKMEDIA

Institute (TDI) based in Cambridge, Mas-
sachusetts. Only five of the 420 ALS ther-
apy candidates that his center has retested
in mouse and cellular models have shown
a therapeutic effect.
Progress has been hindered by three
main challenges. First, the disease’s causal
mechanisms are poorly understood. Even
the two ALS treatments currently approved
carrying mutations in SOD1—the first gene
linked to the disorder—which affect only 2
percent of ALS patients.
Finally, there are no quantitative bio-
markers to track disease progression or serve
as clinical endpoints for trials. Currently, phy-
sicians use the subjective ALS Functional
Rating Scale–Revised, which measures 12
motor skills on a five-point scale.
since 1995, and edaravone, which the FDA
approved in May 2017, treatment cur-
rently focuses on managing ALS symp-
toms through multidisciplinary care to
increase weight and aid breathing, com-
munication, and mobility, says Merit Cud-
kowicz, director of the ALS Clinic and
chief of neurology at Massachusetts Gen-
eral Hospital (MGH).

09. 2018 | T H E S C IE N T IST 63

BIO BUSINESS

Although it’s still not clear what trig-
gers onset of the disease, research over the
past decade—in particular, the cumula-
tive identification of roughly 50 potential
disease-linked genes by multiple research
groups—has unveiled numerous relevant
mechanisms, most of which act in the motor
neurons affected by ALS. These new insights
are allowing drug developers to shift from
downstream pathways, such as inflamma-
tion or muscle weakness, to upstream mech-
anisms more likely to drive the disease.
Many identified mutations occur in
genes coding for RNA-binding proteins,
such as TDP-43 and FUS, involved in
splicing, transcriptional regulation, or
other aspects of RNA metabolism. These
genetic changes can lead to cytoplasmic
aggregation of these proteins and dys-
functional mRNA metabolism. Intrigu-
ingly, most ALS patients, even those with-
out these mutations, have abnormally high
TDP-43 levels, which can lead to RNA
misprocessing, says neuroscientist Clo-
tilde Lagier-Tourenne of Harvard Medical
School. This suggests that targeting these
pathways could one day result in broadly
applicable ALS drugs.
An emerging player in both dysfunc-
tional RNA biology and other mechanisms
of ALS is the gene C9orf72, thought to
encode a protein involved in cell signal-
ing. Mutated forms of this gene account
for 25 percent to 30 percent of familial
ALS cases and up to 5 percent of sporadic
cases, making it the largest known genetic
driver of the disease. Its 2011 discovery
was “a key breakthrough in terms of ther-
apeutic development,” says Laura Ferrai-
uolo, a translational neurobiologist at the
University of Sheffield in the UK. Multiple
companies have begun to target the gene
with therapeutics.
Smaller RNAs might also have a role
to play in ALS. The microRNA miR-155 is
upregulated in the spinal cord of ALS patients,
and reducing its levels in SOD1 mutant
mice improved survival and motor func-
tion, says Bill Marshall, CEO of miRagen,
a Colorado-based company developing
the anti-miR-155 compound MRG-107.
He thinks that miR-155 may additionally
be acting in neural support cells, such as
microglia or astrocytes, which also seem to
play a role in the disease.
Proteins misbehave in ALS, too, mis-
folding and aggregating in the cytoplasm
and thereby triggering cellular stress
pathways. The actin regulator profilin 1
clumps in this manner, and the mutated
profilin found in some patients exacer-
bates TDP-43 aggregation, too. That’s
led researchers such as pharmacolo-
gist Mahmoud Kiaei at the University
of Arkansas for Medical Sciences to
search for compounds to prevent profilin
clumping. Other drug strategies focus on
broader causes of aggregation, such as
nuclear pore defects that result in cyto-
plasmic protein accumulation. Massa-
chusetts-based Karyopharm’s preclinical
compound KPT-350 aims to inhibit the
nuclear pore protein exportin-1 (XPO1)
in the hopes of alleviating this accumu-
lation. “If it works, it will have a huge
impact in showing how basic biology can
be translated to the clinic,” says Chris
Henderson, vice president and head of
neuromuscular and movement disorders
research at pharmaceutical giant Bio-
gen, which has acquired rights to fur-
ther developing the compound.
Such discoveries have aided preclini-
cal research, via the development of new
rodent models such as TDP-43 and pro-
filin-1 mutant mice. And the recent rise
of cellular reprogramming technology
has made it possible to work directly
with patients’ own cells. Researchers can
convert individuals’ skin cells into neu-
rons that recapitulate their specific spi-
nal cord features, such as protein aggre-
gation and RNA metabolism defects,
says Ferraiuolo. The cells have also been
a boon for therapeutic testing, allowing
her and other researchers to screen 300
drugs per patient per day.

Beyond small molecules

MULTIPLE TARGETS: Companies are trialing a Rather than develop new versions of tra-
number of new therapies for ALS, from traditional ditional compounds, some companies are
small-molecule drugs to oligonucleotide
therapeutics and stem-cell treatments. These
exploring entirely novel kinds of drugs.
therapies may target one of several processes Antisense oligonucleotides (ASOs), for
linked to ALS, including inflammation of the cell example, are designed to bind to the RNA
body  1 , aggregation of proteins in the cell transcript of a specific gene and trigger its
cytoplasm  2 , defects in nuclear pore complexes destruction before translation (see “Wait-
3 , dysregulation of mRNA processing  4,
2
 ing for Oligonucleotide Therapeutics,” The
and dysregulation of microRNAs  5.
Scientist, December 2016). For years, ASOs
disappointed in clinical trials, often failing
to produce convincing efficacy data. That
3
 5

changed with Biogen’s recently approved
ASO nusinersen (Sprinraza), which treats
THE SCIENTIST STAFF

1
 the childhood motor neuron disease spinal
4

muscular atrophy. Although the disease
and population are different from ALS, that
success has boosted optimism for Biogen’s
continued partnership with Ionis, Spinra-
za’s original developer, to test an ASO tar-
geting SOD1 in ALS.
The SOD1 ASO avoids many of the
pitfalls of previously proposed ALS treat-
ments: “You know your target, you know
your mutation, you have a drug that works
at your mutation, and you actually have
readouts,” explains Jeffrey Rothstein,
director of the Robert Packard Center
for ALS Research at Johns Hopkins Uni-
versity. In mutant SOD1 mice, the ASO
knocks down overall protein levels and
improves survival and muscle strength;
currently, a Phase 1 multiple dosing trial
seeks to discern a similar effect in people.
“It could be the first domino to fall,” says
ALS TDI’s Perrin, potentially making way
for the development of ASOs for other ALS
targets. Biogen and WAVE Life Sciences
are developing C9orf72-targeting antisense
oligonucleotides that are expected to enter
clinical trials soon.
Another promising approach, gene
therapy, also received a boost in the ther-
apeutics industry, following the 2009 dis-
covery that the viral vector adeno-associated
virus 9 (AAV9), unlike most vehicles, could
cross the blood-brain barrier (Nat Biotech,
27:59–65). Gene-therapy company AveXis,
recently acquired by Novartis, pairs AAV9
with short hairpin RNAs targeting SOD1 in
its ALS therapy AVXS-301. Like the SOD1
ASO, AVXS-301 extends life and improves
motor function in SOD1 mice. It’s cur-
rently in preclinical safety testing, and
AveXis plans to file an investigational new
drug application (IND) by early 2019, says
CSO Brian Kaspar. He says he hopes that
it might even be useful in patients without
SOD1 mutations who nevertheless have
abnormal levels of the protein, and that, if
successful, AAV9 could become the basis
for ALS therapies targeting other genes.
Rather than altering genes, the cell ther-
apy NurOwn, developed by New York City–
based BrainStorm Cell Therapeutics, consists
of cells differentiated from patients’ own bone
marrow-derived mesenchymal stem cells
(MSCs). These MSCs are thought to produce
protective neurotrophic factors and immuno-
modulatory molecules that both help main-
tain motor neuron function and reduce
inflammation, explains Chief Medical Offi-
cer and COO Ralph Kern. A Phase 3 trial
aims to enroll 200 patients, half of whom will
receive three spinal injections of these cells
at two-month intervals; results are expected
in early 2020. However, despite positive
reported results to date, some remain skep-
tical. “The hype about stem cells is masking
the unbelievable ignorance about the biol-
ogy of these cells,” says Rothstein, who argues
that it would be better to administer specific
growth factors. Kern counters that the mix of
factors provided by the cells is critical for tar-
geting different types of neurons and differ-
ent patient groups.
I think it will be curable with
the right strategy.
—Mahmoud Kiaei, University of Arkansas
Designing better trials
Both Kern and Jean Hubble, VP of medical
affairs at Mitsubishi Tanabe Pharma Amer-
ica, the maker of the recently approved eda-
ravone, point to a new strategy to develop
promising ALS therapies: restricting clini-
cal trials to patients with rapidly progress-
ing disease, which allows scientists to more
easily determine the therapy’s impact. That
approach might lead to disappointment for
some patients in the short term, but MGH’s
Cudkowicz thinks that more-carefully tar-
geted trials are ultimately more likely to
identify effective drugs for a broader swath
of patients (see “Clinical Matchmaker,” The
Scientist, June 2015).
To get there, some ALS researchers are
pursuing large-scale studies to develop bet-
ter ways to stratify their patients and quan-
titatively track disease progress. For exam-
ple, through its precision medicine program,
which has enrolled 700 patients, ALS TDI
uses data from accelerometers strapped to
patients’ limbs to monitor disease progres-
sion, says Perrin. The initiative also col-
lects standard Functional Rating Scale–
Revised results, voice recordings, full genome
sequences, and RNA and protein biomark-
ers, which are made available to each patient
through a secure online portal. A separate,
multi-institution effort, Answer ALS, is
beginning to spot key differences between
patient groups based on the 8 billion data
points (on motor function, breathing, speech,
speed of thought, and molecular profiles of
individualized iPS cells) collected from each
of the 780 patients enrolled so far, says Roth-
stein, who is involved in the program.
Both efforts rely on partnerships
with big-data experts: ALS TDI is work-
ing with Google and the Broad Institute,
while Answer ALS links multiple medi-
cal centers, IBM Watson, and research-
ers at MIT. The breadth and scale of
these alliances highlight another new
trend: the rise of large collaborative
teams in ALS spanning basic research,
the clinic, and nonprofits. “There’s much
more cohesion, and the academic-industry
partnerships are extremely vibrant,” says
Lucie Bruijn, chief scientist of the ALS
Association, a nonprofit aimed at eradi-
cating the disease.
That’s not to say there won’t be fail-
ures. In the past year, those include Cyto-
kinetics’s tirasemtiv, a small-molecule
no longer in development after a Phase 3
trial failed due to tolerability issues and lack
of efficacy; Neuraltus’s NP001, a proprie-
tary formulation of sodium chlorite, which
missed clinical endpoints for efficacy in
Phase 2 trials; and AB Science’s masitinib,
an anti-inflammatory, small-molecule tyro-
sine kinase inhibitor, which was rejected by
European regulators on the basis of uncon-
vincing trial data.
Overall, however, the explosion of
biological and technical advances in
the ALS therapy field—as well as grow-
ing connections between the players—
have led to general optimism that future
drug development might finally be able
to avoid past pitfalls.
“ALS has been labeled incurable, but
I think it will be curable with the right
strategy,” says the University of Arkan-
sas’s Kiaei. Steve Perrin agrees: “Many of
the things that are in clinical development
today are better shots on goal than they
were a decade ago.” g
Jenny Rood is a freelance writer in Cam-
bridge, Massachusetts, and the senior
development writer at the Broad Institute
of MIT and Harvard.
09. 2018 | T H E S C IE N T IST 65
 READING FRAMES
Repeating the Beat
In this adapted excerpt from Sandeep Jauhar’s new book, Heart:
A History, the author explores the past and future of artificial hearts.
BY SANDEEP JAUHAR
T
he heart is the first organ to form—
starting to throb approximately
three weeks into fetal development,
even before there is blood to pump—and
life ends when its beat ceases for good.
From birth until death in humans, the
heart beats approximately three billion
times. The amount of work it performs is
mind-boggling. Each heartbeat generates
enough force to circulate blood through
approximately 100,000 miles of vessels.
The amount of blood that passes through
an average adult heart in a week could fill
a backyard swimming pool.
So when the heart fails, the body fails.
Every year more than half a million Amer-
icans develop congestive heart failure,
in which the heart weakens or stiffens to
the point where it cannot properly pump
blood to meet the metabolic demands of
the body. The symptoms of end-stage heart
failure—shortness of breath, fatigue, nau-
sea—are some of the most torturous and
feared in the human experience.
Replacing the failing heart with an
off-the-shelf mechanical device has been
the great ambition of cardiologists and
cardiac surgeons for the past half century.
At first glance, the technological obsta-
cles seem insurmountable. Blood quickly
coagulates when it encounters plastic
or metal. Without adequate blood thin-
ning, clots can be expelled from an arti-
ficial heart and course through the body,
blocking arteries and causing strokes or
other damage. An artificial heart also can
never stop pumping, even temporarily,
so power lines must travel into the body,
posing the risk of infection.
But tremendous progress has been
made on this front. The first artificial
heart replacement in an animal was per-
formed at the Cleveland Clinic in 1957.
The organ, built by Dutch physician Wil-
6 6 T H E SC I EN TIST | the-scientist.com
lem Kolff, held two balloon-like sacs filled
with blood inside its plastic ventricles.
Pressurized air filled the ventricles and
compressed the balloons, thus forcing
blood out in much the same way as from a
beating heart. Kolff ’s subject, a dog, sur-
vived for about ninety minutes.
Twelve years later, on April 4, 1969,
Denton Cooley, a surgeon at St. Luke’s
Episcopal Hospital in Houston, implanted
the first artificial heart, made of polyester
and plastic and powered by compressed
air, into Haskell Karp, a forty-seven-year-
old Illinois man suffering from end-stage
heart failure. After the implant, which
was intended to provide only a few days
of support, a frantic search for a donor
heart commenced. A compatible organ
was identified three days later in Boston.
It was transplanted successfully, but Karp
died thirty-two hours after the operation.
In the ensuing years many refine-
ments have been made to artificial-heart
design, including changing the shape of
the organ and developing more blood-
compatible building materials. Still, cul-
tural prohibitions have remained, slow-
ing progress. Many people continue to
be repulsed by the idea that the human
heart could be replaced by a machine
made of metal and plastic. For them, the
heart still carries special spiritual and
emotional meanings that make it impos-
sible to replace with a man-made device.
Research on artificial hearts contin-
ues today. The long-term support record
is held by an Italian patient who sur-
vived for 1,373 days before a successful
heart transplant. But significant obsta-
cles to a durable artificial heart persist,
including infection, bleeding, clotting,
and strokes. Today’s devices produce
continuous blood flow, so patients
emerge from the operating room with-
Farrar, Straus and Giroux, September 2018
out a pulse. The devices still pump
blood, of course, but the flow is con-
stant, not periodic. Incredibly, it’s now
clear that humans can survive for long
periods without pulsatile blood flow.
The workhorse of mechanical support
for heart-failure patients today is actu-
ally not the artificial heart but the left
ventricular assist device (LVAD), which
attaches to the native heart, pumping
blood directly out of the left ventricle and
into the aorta, thus essentially bypassing
the failing organ. LVADs have become a
lifesaving option for end-stage heart-failure
patients. Unfortunately, they are not a
viable option for patients with severe
failure of both right and left sides of the
heart. For such patients, a permanent
artificial heart may still be the best hope.
It remains a dream, but not quite the
pipe dream it was fifty years ago. g
Sandeep Jauhar is director of the Heart
Failure Program at Long Island Jewish
Medical Center. He is the author of Doc-
tored and Intern and writes regularly
for The New York Times. Read an excerpt
of Heart at www.the-scientist.com.
 The Guide
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 2018–2019
October 7–11 Drivers of Type 2 Diabetes: From Genes to Environment (S1) Seoul | South Korea
14–18
17–20
Framing the Response to Emerging Virus Infections (S2) Pok Fu Lam | Hong Kong
21st-Century Drug Discovery and Development For Global Health (S3)iBerlin | Germany the
Keystone
November 11–14 From Rare to Care: Discovery, Modeling and Translation of Rare Diseases (S4) Vienna | Austria
25–29 Leveraging Genomic Diversity to Promote Animal and Human Health (S5)iKampala | Uganda
December 11–15 Role of the Genital Tract Microbiome in Sexual and Reproductive Health (S6)iCape Town, Western Cape | South Africa
January 13–17 DNA Replication and Genome Instability: From Mechanism to Disease (A1) Snowbird, Utah | USA

Symposia
13–17 Host and the Environment in IBD: Scientific Advances Leading to New Therapeutics (A2) Taos, New Mexico | USA
13–17 Mitochondrial Biology in Heart and Skeletal Muscle (J1)
joint with Mitochondria in Aging and Age-Related Disease (J2) Keystone, Colorado | USA
13–17 Single Cell Biology (L1) Breckenridge, Colorado | USA
17–21
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joint with Signal Dynamics and Signal Integration in Development and Disease (J4) Keystone, Colorado | USA
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Antibody Engineering December 9-13, 2018

Manchester Grand Hyatt
& Therapeutics San Diego, CA

The Most Innovative Science Presented by Leading Industry and Academic Experts
The Largest Exhibition Devoted to Antibody Engineering
The Leading Forum to Partner with Global Antibody Innovators and Suppliers

KEYNOTE SPEAKERS SHARE STRATEGIES TO ACCELERATE YOUR ANTIBODY MOLECULES

TCR Sequencing: Transforming the Diagnosis Bioinformatics and Computational
and Care of Cutaneous T cell Lymphomas Approaches to Protein Engineering
Rachael A. Clark, M.D., Ph.D. David Baker, Ph.D.
Shing-Yiu Yip and Cecilia M. Hepp Associate Professor Professor of Biochemistry,
of Dermatology, HARVARD MEDICAL SCHOOL UNIVERSITY OF WASHINGTON

Benralizumab, A Monoclonal Antibody Exploiting the Diverse Functions of

Engineered for Enhanced NK-cell Mediated FcRn to Generate Therapeutics
Eosinophil Depletion
Sally Ward, Ph.D.
Bahija Jallal, Ph.D. Professor, Department of Molecular
and Cellular Medicine, TEXAS A&M
President, MedImmune and Executive Vice President,
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FOUNDATIONS

Homo sapiens Exposed, 1556

BY SUKANYA CHARUCHANDRA

I
t wasn’t until the latter half of the 13th century that human
dissections became acceptable in Italy. Previously, both the
Roman Empire and Islamic law had prevented the dissection
of humans and its depiction. While the Greek surgeon Galen’s
anatomical drawings from the second century had been pre-
served and studied until the Renaissance, they were largely
based on dissections of animals, such as apes.
In the mid-16th century, however, famed Flemish anato-
mist Andreas Vesalius dissected the bodies of executed crimi-
nals—not an uncommon practice in that period—while study-
ing in Paris. He realized that Galen had been “misled” by apes,
whose anatomy was not exactly like that of humans.
“The challenge of anatomy is rendering the 3-D experi-
ence of opening bodies onto a 2-D page,” writes Hannah Mar-
cus, a science historian at Harvard University, in an email to
The Scientist. Lack of refrigeration also presented a challenge.
In overcoming those hurdles to produce the first realistic
depictions of internal human biology, Vesalius’s De Humani
Corporis Fabrica, published in Basel, Switzerland, in 1543,
galvanized the study of anatomy.
Meanwhile, Spanish-born Juan Valverde de Amusco was
learning anatomy under the guidance of Roman surgeon Realdo
Colombo, and possibly of Vesalius himself, at the University of
Padua in Italy. Valverde observed and participated in many dis-
sections under Colombo’s guidance, and pored over old books
on the subject. He later moved to Rome and was welcomed into
the home of Spanish Cardinal Juan Álvarez de Toledo.
In 1555, Valverde served as a doctor at the foremost con-
temporaneous Roman hospital, Santo Spirito, where many
luminaries of anatomy worked during that period, includ-
ing Bartolomeo Eustachi, under whom Valverde studied for
a time. The following year, Valverde crafted the Spanish-lan- SKIN DEEP: This drawing from Valverde’s Historia de la Composicion del Cuerpo
guage anatomical text Historia de la Composicion del Cuerpo Humano is attributed to Gaspar Becerra.
Humano, or Account of the Composition of the Human Body.
In seven parts, the book covered topics such as “bone and car-
tilage,” “ligaments and bandaging,” and “instruments of sen- “Valverde who never put his hand to a dissection and is ignorant
sation and external motion.” Largely copied from the 1543 of medicine as well as of the primary disciplines, undertook to
and 1555 editions of Vesalius’s tome, it included 15 new illus- expound our art in the Spanish language only for the sake of
trations in four copper plates. Valverde’s book also included shameful profit.” Valverde conceded his borrowing, explaining
US NATIONAL LIBRARY OF MEDICINE

more than 60 corrections to Vesalius’s text, which enhanced
the contemporary understanding of the intracranial passage
of carotid arteries, the extraocular muscles, the stapes bone
of the middle ear, and how blood moves through the septum.
Historians attribute the few original illustrations to Spanish-
born Gaspar Becerra.
“Vesalius was angry about Amusco’s work and accused him of
plagiarism,” Marcus writes. In 1564, Vesalius wrote in his book
Anatomicarum Gabrielis Fallopii Observationum Examen that
that Vesalius’s drawings were so thorough that “it would look
like envy or malignity not to take advantage of them.”
Valverde simplified Vesalius’s Latin text considerably,
however, as he considered it difficult to understand. His more
concise (and thus cheaper) text had more than a dozen edi-
tions published in Italian, Latin, Dutch, and Greek, in addi-
tion to Spanish, and facilitated the spread of scientific ideas
and Vesalius’s modern anatomy throughout Europe and the
Spanish Americas. g
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