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of ineffectiveness. Because the repurposing recommen-
dations were based on weak evidence, it is unlikely that
the World Health Organization experts could have made
better recommendations in March 2020.
The disappointing results of these remdesivir trials
were partly explained by the focus on hospitalized patients
because antivirals are expected to be more effective as
treatment in earlier stages of the infection (when viral repli-
cation, not inﬂammation, is the predominant pathologic
process) or prophylaxis. In fact, remdesivir had a clinical
beneﬁt in monkeys treated within hours of SARS-CoV-2
infection (6), and a randomized trial in nonhospitalized
high-risk humans with COVID-19 published in December
2021 found an 87% lower risk for hospitalization for remde-
sivir versus placebo (7). Nevertheless, remdesivir did not
become the treatment of choice in ambulatory settings
because of the availability of oral alternatives that were per-
ceived to be more effective.
WHY DID MOLNUPIRAVIR MAKE INTO
RANDOMIZED TRIALS?
Molnupiravir was developed at Emory University with
funding from the National Institute of Allergy and Infectious
Diseases and Defense Threat Reduction Agency. While in
preclinical development for inﬂuenza treatment, molnupira-
vir was reported to inhibit SARS-CoV-2 in vitro (8) and block
SARS-CoV-2 transmission in ferrets (9). On 23 March 2020,
Emory University sold an exclusive license to Ridgeback
Biotherapeutics to conduct randomized trials of molnupira-
vir for COVID-19 (10). After the Biomedical Advanced
Research and Development Authority declined to fund
these trials (11), Merck acquired the rights to molnupira-
vir and launched 2 phase 2 randomized trials in October
Annals.org
Annals of Internal Medicine © 2023 American College of Physicians 1
Annals of Internal Medicine
MEDICINE AND PUBLIC ISSUES


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Editorial
Journal of Medical Genetics, 1985, 22, 417
Sir Cyril Clarke and Journal of Medical Genetics: an appreciation
From its first issue in September 1964 until August this year - a span of 21 years - the Journal of
Medical Genetics has had only two editors, Arnold Sorsby and Sir Cyril Clarke. Fifteen of these
years have been under Sir Cyril's editorship, a time during which the Journal has grown steadily
both in reputation and in size. A glance through the contents pages of these years shows how
many landmarks in the field had their first publication in the Journal, as well as how many young
contributors to the early issues now hold distinguished positions throughout the world.
From its inception, the role of Journal ofMedical Genetics was seen as a unifying one, bringing
together under a single cover work on all aspects of Medical Genetics which, even at that time,
was scattered and often inaccessible. That the Journal should have maintained and strengthened
this role, giving its readers both a high scientific standard and a high degree of clinical interest, is
a formidable achievement, and one of which Sir Cyril can justly be proud.
This issue marks Sir Cyril's retirement from the editorship by a collection of papers which
differs somewhat from the usual form of 'festschrift'. The contributions come from those who
worked with him as colleagues in Liverpool over varying periods, and whose present work and
interests cover - as can be seen from the papers - a wide variety of fields. Contributors were
asked for a paper that reflected their own current work, rather than attempting to select a single
theme;
as an acknowledgement to a person whose own work and interests have ranged
exceptionally widely in both Medical and basic Genetics, this is perhaps appropriate.
While most people will associate Sir Cyril's name with his work on the prevention of Rhesus
haemolytic disease, his studies on inherited oesophageal cancer, on genetic linkage in man, on


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ABO blood group associations, as well as his research on the genetic control of mimicry in the
genus Papilio, are but a few examples of his original talent.
Some of Sir Cyril's colleagues, notably Phillip Sheppard, are sadly no longer living to
contribute, while Stanley Walker's death occurred shortly after completing his paper for this
issue. Not all the contributors are currently working in Medical Genetics departments, a fact that
perhaps reflects Sir Cyril's own view that Medical Genetics should be a subject that permeates
and influences the whole of medicine, rather than develop in isolation. Finally, the contribution
from Victor McKusick emphasises the close and fruitful link over many years that resulted in a
succession of young workers (myself included) travelling between Liverpool and Baltimore to
gain further experience in a still embryonic specialty.
The Journal of Medical Genetics and its readers wish Sir Cyril and his wife Feo a long, happy,
and active retirement.
PETER S HARPER
417


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Journal of Medical Genetics (1975).
12, 162.
Genetical components of physiological tremor
P. J. TYRER* and JUDITH KASRIELt
Summary.
Physiological tremor was measured in 14 pairs of monozygotic
twins, 14 pairs of dizygotic twins, and 14 pairs of control subjects matched for age
and sex.
Postural finger tremor was measured in each pair using a sub-miniature
accelerometer and subjecting the amplified signals to power spectral analysis.
Sig-
nificant genetical influences were found for the proportion oftremor at frequencies
near the peak frequency of9-10 Hz.
No genetical effects were found at other fre-
quencies or for the absolute amount of tremor.
The results suggest that physio-
logical tremor between 8 and 13 Hz is genetically influenced and that tremor at
these frequencies is determined by different mechanisms than those at other fre-
quencies.
Although tremor has been studied scientifically
for over a century (Ferrand, 1868), we are still not
certain how normal physiological tremor is caused
and how it is maintained.
The evidence that we
have is conflicting but it appears that such tremor is
the resultant of several different mechanisms. They
include
'hunting'
in the servo-loop between
a
stretch stimulus and reflex corrective muscular
action (Halliday and Redfearn,
1956; Lippold,
Redfearn, and Vuco, 1957; Lippold, 1970a), ballisto-
cardiographic
effects transmitted passively from
cardiorespiratory
forces
(Brumlik,
1962;
Van
Buskirk and Fink, 1962; Yap and Boshes, 1967), and
mechanical influences, particularly at higher fre-
quencies of tremor (Stiles and Randall, 1967).
One
consistent feature of physiological tremor is the pre-
sence of a main peak at or near 9 Hz which persists
under a wide range of experimental conditions
(Sutton and Sykes, 1967).
The relationship be-
tween tremor near the peak frequency and that at
other frequencies has also been disputed; some
workers have suggested that fast and slow physio-
logical tremors are harmonics of the peak frequency
of tremor (Merton, Morton, and Rashbass, 1967).


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In view of the uncertainty about the origins of
the different frequencies of physiological tremor
it is surprising that there have been no genetical
studies of the phenomenon.
Essential or heredo-
familial tremor, which appears to be qualitatively
similar to physiological tremor but exaggerated
in degree (Marshall, 1962), has long been known
to be influenced strongly by genetical factors (Dana,
1887; Critchley, 1949; Larsson and Sjogren, 1960)
but even in this condition the precise nature of the
genetical components is unclear.
Genetical investigations have potential value in
elucidating many of the unanswered questions
posed by these studies, not the least of which is the
relationship
between
tremor
and
the
electro-
encephalogram (EEG).
Lippold (1970b; 1973) has
provided
strong evidence, which although
dis-
puted has yet to be explained, that the alpha rhythm
is an artifact produced by tremor ofthe extra-ocular
muscles.
As the alpha rhythm is genetically in-
fluenced (Lennox, Gibbs, and Gibbs, 1945; Young,
Lader, and Fenton,
1972) physiological tremor
would have to be similarly influenced if it was the
cause of the alpha rhythm.
Method
Fourteen pairs ofmonozygotic (MZ) twins, 14 pairs of
dizygotic (DZ) twins, and 14 pairs ofcontrols, aged 17-44
(mean 24.1 years) were investigated.
All the twins were
contacted through the Medical Research Council Twin
Register at the Institute ofPsychiatry, apart from the two
authors and their co-twins.
All measurements oftremor
were made by P.J.T. who was unaware of the zygosity
of the twins until the end of the study.
J.K. selected
the twins so that there were 14 pairs of supposed or pre-
viously confirmed monozygotic and dizygotic twins with
162
Received 22 May 1974.
* Department of Psychiatry, University of Southampton, South
Academic Block, Southampton General Hospital, Tremona Road,
Southampton S09 4XY.
t Institute of Psychiatry, De Crespigny Park, London SE5 8AF.


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Brit. J. Ophthal. (1976) 60, 428
Central retinal artery occlusion (reversible) in
sickle trait with glaucoma
RONALD L. RADIUS AND DANIEL FINKELSTEIN
From the Wilmer Ophthalmological Institute, The Johns Hopkins University
and Hospital, Baltimore, Maryland, USA
Retinal vascular occlusions of several types occur
in the various sickle haemoglobin disorders (SS,
SA, SC, S-Thal) (Edington and
Sarkies,
I952;
Henry
and
Chapman,
1954;
Goldberg,
197Ia;
Kennedy and Cope,
I958;
Isbey,
Clifford, and
Tanaka,
I958;
Lieb,
Geeraets,
and
Guerry,
I959). Occlusions
are primarily of small vessels
in the peripheral retina preceding the development
of neovascularization (Welch and Goldberg, I966;
Goldberg,
197Ia, b). Occlusions of small vessels
in the macula have also been reported in SS and
SC
haemoglobinopathy
(Knapp,
I972;
Acacio
and
Goldberg,
I973;
Ryan,
1974).
They
are
probably due
to intravascular emboli of sickled
red
blood
cells
(Welch
and
Goldberg,
I966;
Goldberg,
I97ia, b).
Factors known
to enhance
sickling
of
red
cells
thereby
influencing
the
risk
of
vascular
thrombosis
include
hypoxia,
dehydration,
lowered
pH,
and
hyperviscosity
(Sherman, I940; Griggs and Harris, 1956; Perillie
and Epstein, I962; Charache and Conley, I964).
Not surprisingly, therefore, under certain condi-
tions occlusion of large vessels such as the central
retinal artery occur (Kabakow, Van Wiemokly, and
Lyons,
I955;
Conrad and Penner,
I967;
Stein
and
Gay,
I970;
Michelson
and
Pfaffenbach,
I972). We report a case of central retinal artery
occlusion in SA disease immediately after diuretic
and
hyperosmotic
therapy
for
acute
traumatic
glaucoma. We believe that this treatment must be
used cautiously in the sickle patient whose ocular
circulation
is
already
compromised
by
acute
glaucoma.
Case report
An i8-year-old black woman presented to the Wilmer
emergency room with a traumatic hyphaema 24 hours
after a blunt ocular injury. Her medical and family
history were unremarkable. Uncorrected visual acuity
was light perception
right
eye, 20/20
left
eye. The


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eye. The
Address for reprints: D. Finkelstein, MD, The Wilmer Ophthal-
mological
Institute,
Johns
Hopkins
University
and
Hospital,
Baltimore, Maryland 21205, USA
anterior chamber contained diffuse haemorrhage with
a layered hyphaema
of about
io per
cent.
Ocular
motility was normal. Intraocular pressure by applanation
was 55 mmHg. The fundus could not be visualized
through the anterior chamber blood. Examination of
the left eye was normal in all respects.
Treatment consisted of atropine cycloplegia, topical
dexamethasone,
and
I 20 ml
glycerol
75
per
cent.
Emesis followed shortly after osmotic therapy. Intra-
ocular pressure remained raised at 55 mmHg. Subse-
quent treatment consisted of intravenous acetazolamide
500 mg and mannitol I00 g (1-5 g/kg) at an infusion
rate of 5 g/minute. One hour later ocular tension was
55 mmHg. Vision in the right eye was no light percep-
tion when tested with the American optical indirect
ophthalmoscope at maximum voltage.
The
clinical impression was that the patient had
suffered a central retinal artery occlusion. An anterior
chamber paracentesis of o-o8 ml was performed. After
this vision returned to brisk light perception.
Intra-
ocular pressure was subsequently controlled with oral
acetazolamide. With clearing of the ocular haemorrhage
fundus examination showed disc hyperaemia. Fluores-
cein angiography was essentially unremarkable. Elec-
troretinography could not be performed. Subsequent
serum
electrophoresis
showed
sickle-cell
trait
(SA)
haemoglobinopathy with 42 per cent sickle-cell haemo-
globin. Visual acuity returned to the 20/30 level.
Discussion
Our patient presented with an intraocular pressure
of 55 mmHg and brisk light perception vision.
One hour after diuretic and osmotic therapy the
pressure remained
at 55 mmHg with no
light
perception.
This
time
sequence
suggests
that
increased intravascular tonicity and altered haemo-
dynamics after these medications may have pre-
cipitated an occlusion in an already compromised
vascular system. Intravenous infusion of mannitol


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is
known
to
increase
serum
osmolarity
from
Io-40 mosm/l depending on rate of infusion and
dosage
(Galin,
Davidson, and Pasmanik,
I963;
Weiss and Wise, I962; Kolker and Hetherington,
1970).
Increased serum
tonicity
occurs
rapidly
throughout the infusion and returns to equilibrium


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Brit. 7. Ophthal. (I975) 59, 560
Ocular motor dysfunction in total and
hemicerebellectomized monkeys
RONALD M. BURDE, MALCOLM H. STROUD, G. ROPER-HALL,
FREMONT P. WIRTH, AND JAMES L. O'LEARY*
From the Departments of Ophthalmology, Otolaryngology, Neurology and Neurosurgery, Washington
University School of Medicine, St Louis, Missouri, USA
Recent reports (Kornhuber, 1971; Robinson, I974;
Westheimer and Blair,
1973) have implied con-
flicting roles for the cerebellum in the control of eye
movements. Recently, as part of a large multispecies
study to determine the effects of cerebellectomy on
power
and
co-ordinating functions (Wirth
and
O'Leary, 1974), we had the opportunity to study
prospectively the effects of this surgery on the ocular
motor system in primates. Altogether 2I monkeys
(Macaque mullata) were observed both during the
acute stage (the first postoperative 8 weeks) and
during the compensated stage (as late as 6 months
after surgery). Our observations
on the
ocular
motor function of these primates are reported in
this paper and an attempt is made to draw some
pathophysiological correlations with human ocular
motor dysfunction.
Material and methods
The details of animal preparation have been reported
separately (Wirth and O'Leary, 1974). The extent of
cerebellar removal was confirmed in each animal with a
thorough neuroanatomical study at the conclusion of
each experiment. These findings are also reported in
detail elsewhere (Wirth and O'Leary, I974) and will be
referred to here only when relevant to specific ocular
movement disorders.
The animals were studied in a primate chair with or
without head restraint. Motion pictures (i6 mm) were
taken of all animals during the tests. Electro-oculography
was performed in three animals and frame projection
comparisons were made to estimate speed of movement
in all animals. The following ocular motor functions,
for the most part, were routinely studied:
I. Movements during spontaneous gaze (that
is, the
presence or absence of nystagmus, strabismus, skew


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deviation, conjugate gaze weakness, or deviation)
Dr James L. O'Leary died in May '975
Supported in part by grants NS-o8o33, NS-o6563, NS-O4513 from
the National Institutes for Neurological Disease and Stroke, Bethesda,
Maryland, and by grant EY-oooi6 from the National Eye Institute,
Bethesda, Maryland
Address
for reprints: Ronald M. Burde, MD, Department of
Ophthalmology, Washington University School of Medicine, 66o
South Euclid Avenue, St Louis, Missouri 63110, USA
z. Horizontal and vertical saccades
3. Following movements
4. Convergence function
5. Horizontal and vertical optokinetic nystagmus
6. Doll's head manoeuvre
7. Cold water calorics (30°C).
Warm water calorics (44°C) were performed when
indicated. A special canaliculus extension earpiece of
Teflon tubing (I mm internal diameter) was inserted
into the external auditory meatus of each animal and
threaded into the bony canal to ensure adequate stimulus
delivery. Then I 5 cms water was flushed into the external
ear in approximately
IS seconds.
Animals were divided arbitrarily into two groups:
Acute-xamined within the first 8 weeks of surgery.
Compensated-examined in the ninth week or later.
Results
Adequate data were available in io animals which
underwent total cerebellectomy and in two animals
that underwent hemicerebellectomy. The results
are summarized in Tables I and II.
In animals that underwent total cerebellectomies
and which were studied during the acute period the
following eye movement abnormalities were noted:
I. Spontaneous gaze
Spontaneous nystagmus was noted constantly in
two
of seven
animals. This was an upbeating
nystagmus
(fast phase up). An
additional four
animals had intermittent nystagmus, usually hori-
zontal, but occasionally with an oblique component.
Gaze-paretic nystagmus (that is, nystagmus in
conjugate lateral gaze with a small amplitude and
high frequency) was present in four of six monkeys.
This was a horizontal movement in lateral gaze
(fast phase right in right gaze and vice versa), and a


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vertical movement in vertical gaze, usually up-
beating when looking up and downbeating when
looking
down.
Generally
the
nystagmus
was
bilateral and symmetrical. In one animal (No. 13),
that underwent first a left cerebellectomy followed
by a right cerebellectomy, the gaze-paretic nystag-


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Journal ofMedical Genetics, 1978, 15, 189-201
Hereditary pancreatitis in England and Wales'
J. R. SIBERT
From the Department ofChild Health, University Hospital of Wales, Cardiff
SUMMARY
Information from 72 patients from 7 families in England and Wales confirms that
hereditary pancreatitis is inherited as an autosomal dominant condition with limited penetrance. The
degree of penetrance is approximately 80%. These patients have had recurrent attacks of abdominal
pain starting from childhood or young adult life. The mean age of onset in the 7 families studied was
13.6 years. There were two peaks, with maximum numbers at 5 years and 17 years. The second peak
was thought to represent genetically susceptible individuals having pain brought on by alcohol rather
than representing evidence of genetic heterogeneity. Five of the 7 families had members with both
childhood and adult ages of onset. Only 4 patients out of 72 had life-threatening disease and in the
majority of cases the attacks of pain were of nuisance value only. Hereditary pancreatitis was
implicated in only 1 patient's death and this was not definite. Patients appear to get better after a
period of symptoms usually as they approach middle age, or after a severe attack. In older patients
alcohol, emotional upsets, and fatty food appear to precipitate attacks. Pancreatic insufficiency
(5.5%), diabetes mellitus (12.5%), pseudocysts (5.5%), and haemorrhagic pleural effusion
are
uncommon complications. Portal vein thrombosis occurred definitely in 2 patients and was suspected
in 3 others. Carcinoma of the pancreas was not found in any of 72 patients studied in detail; however,
2 members from a family not visited personally had chronic pancreatitis and malabsorption going on
to carcinoma. They may have suffered from a different disease.
Genetic linkage information was too slight for many definite conclusions. However, there was no
suggestion of linkage with any of the markers tested.
Hereditary pancreatitis was first described by Comfort


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and Steinberg (1952), from the Mayo Clinic. They
described a family with 4 definite and 2 suspected
cases of chronic relapsing pancreatitis starting in child-
hood or young adult life. The pattern of inheritance
appeared to be an autosomal dominant one with
incomplete
penetrance.
Four
other
families
were
described in the ensuing years from the Mayo Clinic
(Gross and Comfort, 1957; Gross, 1958; Gross et al.,
1962). The findings in those families confirmed the
dominant mode of inheritance of the condition and the
early age of onset of the attacks of pain. Gross et al.
(1962) reported in addition that 4 of these 5 families
had aminoaciduria. Most of their work on this was
confined to one family (the K kindred) who appeared
to have a lysine-cystine aminoaciduria.
'This work forms part of an MD Thesis accepted by the University of
Cambridge.
Received for publication 7 July 1977
The first family described outside the Mayo Clinic
was reported by Cornet et al. in 1962 from Nantes in
France.
Information on
another
family from the
United States was published soon after (Gerber, 1963)
and
since then there have been over 35
families
reported in the world literature. These families have
come from all over the world; however, apart from the
Indian family of Choudhry et al. (1971) all have come
from patients of European ancetry. There have been
two relatively recent reviews of the condition from the
United States by McElroy and Christiansen (1972)
and Kattwinkel et al. (1973).
The only reports of hereditary pancreatitis from the
United Kingdom have been by Nash (1971) and a
large family from Newcastle (Sibert, 1975). A study
was made on these and other families in England and
Wales to investigate the natural history of the con-
dition, the evidence for genetic heterogeneity, the
pattern of inheritance, and the urine amino acids. A
189


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Bringing Gene Therapies for HIV Disease to Resource-Limited
Parts of the World
Joseph M. McCune,* Emily H. Turner, Adam Jiang, and Brian P. Doehle
HIV Frontiers, Global Health Innovative Technology Solutions, Bill & Melinda Gates Foundation, Seattle, Washington, USA.
INTRODUCTION
DESPITE THE FACT that 59% of people living with HIV
(PLHIV) currently achieve viral suppression on anti-
retroviral therapy (ART), recent gains in controlling the
global HIV/AIDS epidemic may be threatened: key HIV
incidence rates are declining only modestly, the sustain-
ability of programs to expand ART remains unclear, and
the ‘‘youth bulge’’ in sub-Saharan Africa contributes to a
growing at-risk population.1 Although much effort has
been devoted to prevention interventions, these face major
technical and/or implementation challenges.
A complementary approach is a safe, effective, and
durable intervention that completely eliminates HIV in-
fection (‘‘eradication’’) or that suppresses viremia in the
absence of ART (‘‘remission’’) (both of these states are
referred to as an ‘‘HIV cure’’ herein). Although a daunting
goal, the scientiﬁc basis is clear: long-term remission if not
eradication has been observed in the ‘‘Berlin patient’’2 as
well as the ‘‘London patient’’3 following transplantation
of bone marrow progenitor cells lacking the viral cor-
eceptor, CCR5; and durable remission occurs in tens of
thousands of PLHIV (so-called ‘‘Elite Controllers’’), some
of whom (‘‘Exceptional Elite Controllers’’) may have
eliminated their infections through natural immunity.4,5
Ongoing work over the past decade suggests that HIV
cure might be induced by some interventions, alone or in
combination, including provision of broadly neutralizing
antibodies, generation of effective antiviral CD8+ T cell
responses, and knockout of the viral coreceptor, CCR5.6
Little is known, however, about the nature and vulner-
abilities of the rebound-competent viral reservoir that
persists despite ART and about the immunologic control


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of virus in the absence of ART; today’s ‘‘best bets,’’ in
other words, must still be viewed as long shots.
Initiated by the Bill & Melinda Gates Foundation in
2019, the HIV Frontiers Program aims to move work on
HIV cure toward interventions that will ultimately be
available to all, most especially those in resource-limited
parts of the world where the prevalence of disease is high
(Fig. 1). It starts with the premise that the journey will
be long (15–25 years) and that it will ultimately yield a
‘‘single-shot cure,’’ that is, a product that is delivered
percutaneously (in vivo) in a single encounter, safely, and
effectively modifying selected cells in the body so that
viral replication and spread are suppressed and reinfection
blocked.
This aspirational goal is likely to be realized through a
series of progressive interventions that move from com-
bination therapies provided over a longer duration of time
to those in which cells are modiﬁed outside of the body (ex
vivo) before reinfusion.7 The work will build on curr-
ent knowledge to advance through a series of technical and
practical hurdles while also gathering new knowledge to
best design a curative intervention for HIV and to deter-
mine whether and how it might be used.
To get to the point of having a ‘‘single-shot cure’’ for
HIV in hand, two interlocking areas of focus are being
pursued:
Current best bets
To minimize the expected time to impact, investments
are being made in all of the necessary elements of a
‘‘single-shot’’ HIV cure in parallel. As a critical enabler,
the Program is leveraging the considerable interest and
resources in biotech/pharma companies that are now
developing ex vivo genetic and cell-based interventions;
uniquely among ongoing efforts, it intends to shift the
emphasis of such interventions to delivery in vivo, an
*Correspondence: Dr. Joseph M. McCune, HIV Frontiers, Global Health Innovative Technology Solutions, Bill & Melinda Gates Foundation, 500 5th Avenue North, Seattle,


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WA 98109-4636, USA. E-mail: mike.mccune@gatesfoundation.org
ª Joseph M. McCune et al., 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
HUMAN GENE THERAPY, VOLUME 32, NUMBERS 1 and 2
DOI: 10.1089/hum.2020.252 j 21
2021 by Mary Ann Liebert, Inc.


key: title
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Changes in circulating forms of anti-Muullerian hormone and androgens in women with and without PCOS: a systematic longitudinal study throughout pregnancy


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Changes in circulating forms of
anti-Muu¨llerian hormone and
androgens in women with and without
PCOS: a systematic longitudinal study
throughout pregnancy
M. Peigne´
1,2,3, V. Simon1,3, P. Pigny
4, N. E. H. Mimouni
1,
C. Martin5, D. Dewailly
1,3, S. Catteau-Jonard1,3,†, and
P. Giacobini
1,†*
1Laboratory of Development and Plasticity of the Neuroendocrine Brain, University of Lille, Inserm, CHU Lille, Lille Neuroscience and
Cognition, UMR-S 1172, Lille, France 2Department of Reproductive Medicine and Fertility Preservation, AP-HP—Universite´ Sorbonne
Paris-Nord, Jean Verdier Hospital, Bondy, France 3Department of Medical Gynecology, CHU Lille, Jeanne de Flandre Hospital, Lille,
France
4Department of Biochemistry and Hormonology, CHU Lille, Centre de Biologie Pathologie, Lille, France
5Department of
Biostatistics, CHU Lille, Lille, France
*Correspondence address. Laboratory of Development and Plasticity of the Neuroendocrine Brain, University of Lille, Inserm, CHU Lille,
Lille Neuroscience and Cognition, UMR-S 1172, Building Biserte, 1 Place de Verdun, 59045 Lille Cedex, France. E-mail: paolo.giacobini@inserm.fr
https://orcid.org/0000-0002-3075-1441
Submitted on December 12, 2022; resubmitted on February 23, 2023; editorial decision on March 1, 2023
STUDY QUESTION: What are the changes in serum concentration of total and cleaved anti-Muu¨llerian hormone (AMH) molecular
forms and of androgens before and throughout pregnancy in women with and without polycystic ovary syndrome (PCOS) in a longitudinal
follow-up investigation?
SUMMARY ANSWER: Serum levels of total and cleaved AMH are higher from preconception to the third trimester of pregnancy in
women with PCOS as compared to controls, whereas testosterone and androstenedione levels are higher in women with PCOS than in
control women before pregnancy and during the second and third trimester of pregnancy.
WHAT IS KNOWN ALREADY: Cross-sectional or partial longitudinal studies have shown higher AMH and androgen levels in pregnant


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women with PCOS as compared with non-PCOS women. To date, no complete longitudinal dynamic monitoring of the circulating forms
of AMH and androgens from pre-conception to the third trimester of pregnancy have compared women with and without PCOS.
STUDY DESIGN, SIZE, DURATION: This systematic prospective quarterly longitudinal monocentric study was a comparative
follow-up of 30 women with PCOS and 29 controls before and during pregnancy from April 2019 to July 2022.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Women aged 18–43 years with a pre-conception measurement of AMH
were included during the ﬁrst trimester of a singleton pregnancy. The PCOS group was deﬁned according to the Rotterdam diagnostic
criteria. The control group patients included in the study had normal ovarian reserves. Circulating total and cleaved AMH, and serum
estradiol, LH, and androgen levels were measured during the ﬁrst, second, and third trimester of pregnancy in all study participants.
MAIN RESULTS AND THE ROLE OF CHANCE: Before pregnancy, patients with PCOS had higher levels of AMH than controls. The
total and cleaved AMH forms were signiﬁcantly higher in women with PCOS than controls from pre-conception to the third trimester
of pregnancy (all P < 0.001). Androgens (total testosterone and androstenedione) were higher in women with PCOS than controls from
mid-pregnancy onwards.
LIMITATIONS, REASONS FOR CAUTION: Our control population was a population of infertile women with no ovarian problems
but most of them had undergone ART treatments to achieve pregnancy.
†These authors contributed equally to this work.
V
C The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which


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permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com
Human Reproduction, Vol.38, No.5, pp. 938–950, 2023
Advance Access Publication on March 15, 2023
https://doi.org/10.1093/humrep/dead050
ORIGINAL ARTICLE Reproductive endocrinology


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Changes in circulating forms of anti-Muullerian hormone and androgens in women with and without PCOS: a systematic longitudinal study throughout


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Offspring physiology following the use
of IVM, IVF and ICSI: a systematic
review and meta-analysis of animal
studies
Kiri H. Beilby
1,*, Ezra Kneebone1, Tessa J. Roseboom2,
Indah M. van Marrewijk3,4, Jeremy G. Thompson3,5,
Robert J. Norman3, Rebecca L. Robker
3, Ben Willem J. Mol
1,6,
and Rui Wang
1
1Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia 2Department of Obstetrics and Gynaecology,
Academic Medical Centre, Amsterdam, The Netherlands
3The Robinson Research Institute, School of Biomedicine, University of
Adelaide, Adelaide, Australia 4Department of Obstetrics and Gynaecology, Erasmus MC University Medical Centre, Rotterdam, The
Netherlands 5ARC Centre of Excellence for Nanoscale BioPhotonics, University of Adelaide, Adelaide, Australia 6Aberdeen Centre for
Women’s Health Research, University of Aberdeen, Aberdeen, UK
*Correspondence address. Department of Obstetrics and Gynaecology, Monash University, Level 5, Monash Medical Centre, 246 Clayton
Road, Clayton, VIC 3168, Australia. E-mail: kiri.beilby@monash.edu
https://orcid.org/0000-0002-1378-5586
Submitted on March 29, 2022; resubmitted on November 24, 2022; editorial decision on December 5, 2022
TABLE OF CONTENTS
................................................................................................................................
• Introduction
• Methods
Search strategy
Eligibility criteria
Experimental ART protocols
Outcomes
Study selection, data extraction and risk of bias assessment
Data synthesis
• Results
Search results
Study characteristics
Risk of bias
Birthweight
Length of gestation
Blood pressure
Metabolic outcomes
Behaviour outcomes
Lifespan
• Discussion
Summary of key findings
Interpretation
V
C The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which


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permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com
Human Reproduction Update, Vol.29, No.3, pp. 272–290, 2023
Advance Access Publication on January 5, 2023
https://doi.org/10.1093/humupd/dmac043


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Cyclic processes in the uterine tubes, endometrium, myometrium, and cervix: pathways and perturbations


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Cyclic processes in the uterine tubes, endometrium,
myometrium, and cervix: pathways and perturbations
Sarah J. Holdsworth-Carson
1,2,*, Ellen Menkhorst
2, Jacqueline A. Maybin
3, Anna King4, Jane E. Girling
2,5
1Julia Argyrou Endometriosis Centre, Epworth HealthCare, Melbourne, Australia
2Department of Obstetrics and Gynaecology, University of Melbourne and Gynaecology Research Centre, Royal Women’s Hospital, Melbourne, Australia
3Institute for Regeneration and Repair, MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh, UK
4Department of Obstetrics and Gynaecology, NHS Lothian, Edinburgh, UK
5Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
*Correspondence address. Julia Argyrou Endometriosis Centre, Epworth HealthCare, Ground Floor, 175-185 Hoddle St, Richmond 3121 VIC, Australia.
E-mail: sarah.holdsworth-carson@epworth.org.au
https://orcid.org/0000-0002-3533-0486
Abstract
This review leads the 2023 Call for Papers in MHR: ‘Cyclical function of the female reproductive tract’ and will outline the complex and fas-
cinating changes that take place in the reproductive tract during the menstrual cycle. We will also explore associated reproductive tract
abnormalities that impact or are impacted by the menstrual cycle. Between menarche and menopause, women and people who menstru-
ate living in high-income countries can expect to experience 450 menstrual cycles. The primary function of the menstrual cycle is to
prepare the reproductive system for pregnancy in the event of fertilization. In the absence of pregnancy, ovarian hormone levels fall, trig-
gering the end of the menstrual cycle and onset of menstruation. We have chosen to exclude the ovaries and focus on the other structures
that make up the reproductive tract: uterine tubes, endometrium, myometrium, and cervix, which also functionally change in response
to ﬂuctuations in ovarian hormone production across the menstrual cycle. This inaugural paper for the 2023 MHR special collection will


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discuss our current understanding of the normal physiological processes involved in uterine cyclicity (limited speciﬁcally to the uterine
tubes, endometrium, myometrium, and cervix) in humans, and other mammals where relevant. We will emphasize where knowledge
gaps exist and highlight the impact that reproductive tract and uterine cycle perturbations have on health and fertility.
Keywords: menstrual cycle / endometrium / myometrium / cervix / uterine tube / fertility / fallopian tube / menstruation
Introduction
The menstrual cycle encompasses the series of changes in hor-
mone production that result in alteration of the structure and
function of the female reproductive tract, including the uterine
tubes (oviduct or fallopian tube), uterus, and uterine cervix.
Women and others who menstruate in high-income countries
can expect to experience 450 menstrual cycles in their lifetime
(reviewed by Eaton et al. (1994) and Critchley et al. (2020a)). The
first occurrence of menstruation is termed menarche and the av-
erage age of menarche in high-income countries is 12.5 years
(Morris et al., 2011; Colich et al., 2020; Martinez, 2020). At the other
end of the reproductive lifespan, menopause is defined as the
permanent cessation of menstruation resulting from the loss of
ovarian follicular activity. Natural menopause is recognized to
have occurred after 12 consecutive months of amenorrhoea, for
which there is no other obvious pathological or physiological
cause. The average age of menopause in high-income countries
is 50.5 years, but this is affected by demographic, reproductive,
genetic, and environmental factors (Gold, 2011).
The primary function of the menstrual cycle is to prepare the re-
productive system for pregnancy should fertilization occur. The first
day of menstrual bleeding is designated as Day 1 of the menstrual
cycle and the typical length of a cycle is 24–38days (Munro et al.,
2018). During the first part of the menstrual cycle, the follicular or


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menstrual/proliferative phase, oestradiol (E2) is the dominant ovar-
ian hormone (Fig. 1). Following ovulation, the corpus luteum
secretes high levels of progesterone (P4) during the luteal/secretory
phase to decidualize and prepare the endometrium for possible im-
plantation (Fig. 1). The corpus luteum also secretes relaxin in the
latter half of the secretory phase; its levels are otherwise low or
undetectable in humans during the other phases of the cycle
(Marshall et al., 2017). In the absence of pregnancy, the ovarian hor-
mone levels fall, with the fall in P4 triggering the end of the men-
strual cycle and onset of menstruation (Fig. 1). The changes in
uterine tubal, endometrial, myometrial, and cervical structure and
function in response to fluctuations in ovarian hormone production
are the subject of this review. Here, we cover the complex, highly
coordinated processes of vascular function, inflammation, and tis-
sue remodelling that occur throughout the menstrual cycle in the
reproductive tract. Current understanding of the physiology of the
menstrual cycle will be reviewed before examination of the patholo-
gies caused, linked, or exacerbated by cyclical pathways. We hope
this review will not only illustrate the current knowledge of the
functional and mechanistic pathways related to the reproductive
tract and menstrual cycle but also highlight the historical lack of re-
search focus in this area, particularly with respect to the uterine
tube, myometrium, and cervix. Our review is the first paper in the
Received: December 19, 2022. Revised: March 01, 2023. Editorial decision: March 31, 2023.
V
C The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.


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SKF96365 modulates activity of CatSper channels in
human sperm
Elis Torrezan-Nitao1, Sean G. Brown
2, Linda Lefievre3, Jennifer Morris1, Joao Correia
1,4, Claire V. Harper
5, and
Stephen Publicover
1,*
1School of Biosciences, University of Birmingham, Birmingham, UK
2School of Applied Sciences, Division of Health Sciences, Abertay University, Dundee, UK
3Institute of Clinical Sciences, School of Biomedical Sciences, University of Birmingham, Birmingham, UK
4Institute of Metabolism and Systems Research, School of Biomedical Sciences, University of Birmingham, Birmingham, UK
5Department of Biology, Faculty of Arts and Sciences, Edge Hill University, Ormskirk, Lancashire, UK
*Correspondence address. School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK. E-mail: s.j.publicover@bham.ac.uk
https://orcid.org/0000-0002-2911-5288
Abstract
Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2þ
concentration ([Ca2þ]i) transient followed by repetitive [Ca2þ]i activity (oscillations), which are believed to be functionally important.
We investigated the potential signiﬁcance of store-operated Ca2þ-entry in these oscillations using the inhibitor SKF96365 (30 mM;
SKF). Following pre-treatment of human sperm with 3 mM P4, exposure to SKF doubled the proportion of oscillating cells (P ¼ 0.00004).
In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2þ]i transient in >80% of cells which was followed by oscilla-
tions in 50% of cells. The CatSper blocker RU1968 (11 mM) inhibited the SKF-induced [Ca2þ]i increase and reversibly arrested [Ca2þ]i
oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then
decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased


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(by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking
bovine serum albumin (BSA), both P4 and SKF induced a [Ca2þ]i transient in >95% of cells but the ability of SKF to induce oscillations
was greatly reduced (P ¼ 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but
that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce
oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.
Keywords: human sperm / SKF96365 / CatSper / progesterone / calcium / calcium oscillations / patch clamp / cation channel of
sperm
Introduction
Intracellular Ca2þ concentration ([Ca2þ]i) signalling is crucial to
sperm function, being a key regulator of motility, acrosome reac-
tion, and capacitation (Darszon et al., 2005; Publicover et al., 2008;
Prajapati et al., 2022). A number of stimuli, including zona pellu-
cida (ZP), are believed to regulate human sperm function by this
pathway, the best characterized being the steroid progesterone
(P4), which is secreted by the cells of the cumulus and is present
throughout the female tract (Correia et al., 2007). P4 acts on hu-
man sperm by activation of the sperm-specific Ca2þ-permeable
channel CatSper (Lishko et al., 2011; Strunker et al., 2011), which,
in human sperm and those of many other species, is the principal
mechanism of Ca2þ mobilization (Lishko et al., 2012; Lishko and
Mannowetz, 2018).
In addition to CatSper, Ca2þ stores and capacitative (store-
operated) Ca2þ entry (CCE) are implicated in sperm [Ca2þ]i signal-
ling (Costello et al., 2009; Correia et al., 2015). CCE may be
triggered by CatSper-mediated elevation of [Ca2þ]i, serving to am-
plify, propagate and/or prolong the [Ca2þ]i signal (Alasmari et al.,
2013; Correia et al., 2015). No specific pharmacological agents are


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available for blockade of CCE, but 10–30 mM SKF96365 (SKF) is a
known and well-characterized antagonist of CCE both in cell lines
and in a range of acutely cultured cell types (Labelle et al., 2007;
Ng et al., 2008, 2010; Li et al., 2010; Sabourin et al., 2016). SKF has
also been shown to inhibit calcium-release activated Ca2þ (CRAC)
current (a form of CCE; Parekh and Putney, 2005) in cells where
CRAC currents occur naturally and also in cells transfected with
Orai, a pore forming channel which is involved in CRAC and other
CCE channels (Prakriya and Lewis, 2002; Yeromin et al., 2006;
Varnai et al., 2009).
SKF has been used as a tool to investigate the participation of
CCE in sperm functions. Treatment with SKF inhibited acrosome
reaction in sea urchin sperm (Hirohashi and Vacquier, 2003) and
suppressed the increase in [Ca2þ]i induced by mitochondrial
inhibitors (Ardon et al., 2009). In mouse sperm, SKF inhibited
Ca2þ influx and acrosome reaction induced by maitotoxin
(Trevino et al., 2006) and suppressed motility (Ru et al., 2015). In
human sperm, SKF has been reported to suppress motility
(Castellano et al., 2003) to inhibit acrosomal swelling (believed to
be a key stage in acrosome reaction) induced by P4 and by thap-
sigargin, which mobilizes stored Ca2þ; Sosa et al., 2016), and to
Received: December 07, 2022. Revised: March 17, 2023. Editorial decision: April 24, 2023.
V
C The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Human Reproduction, 2023, 29(6), gaad015
https://doi.org/10.1093/molehr/gaad015
Advance Access Publication Date: April 27, 2023
Original Research


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Biennial Review of Pain
Molecular taxonomy of nociceptors
and pruriceptors
Jussi Kupari, Patrik Ernfors*
1. Introduction
The ability to feel the warmth of the sun or the coolness of a
breeze on a summer’s day; a pleasant caress of skin; to explore
the texture, size, and shape of an object; and to feel pain as a
mechanism to protect us from what is dangerous around us has
fascinated mankind for centuries. Perhaps this fascination is
rooted in the fact that the somatic senses of temperature, pain,
touch, itch, and proprioception endow us with the ability to
continuously stay in contact with the world within and around us.
Attempting to explain how we react to heat, the 17th century
philosopher Ren ´e Descartes depicted a thread connecting the
skin with the brain and particles of fire pulling on the thread.20 In
the 1880s, distinct sensory spots on the skin were recognized
and shown to respond to specific stimuli, such as touch, heat, or
cold. This finding was taken as evidence for different sensations
relying on the existence of distinct nerves tuned to specific types
of stimuli.83,94 In the beginning of the 20th century, the discovery
of different nerve fiber types with distinct conduction velocities,
activation thresholds, and refractory periods made it possible to
link specific fiber types to unique qualities of sensation, such as
proprioception and different kinds of touch. Unlike innocuous
touch and pressure, which are communicated through nerve
fibers responding only to mechanical stimuli, noxious stimuli were
found to activate a class of polymodal nociceptive nerve fibers
responding to several kinds of unpleasant stimuli, including high-
threshold mechanical force and intense heat.7,12 This work led to
the general idea that the capacity to discriminate perceptual
qualities arises from unique functions of a variety of sensory
neurons involved in somatic sensation. This is possible because
different neuron types are tuned to respond and transduce
certain, but not all, kinds of stimuli, such as warm and cool


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temperatures,
noxious
heat,
noxious
cold,
light
punctate
sensation, painful pressure, sharp objects, hair pull, chemical
irritants, inflammatory substances, chemical and mechanical
itch, and various kinds of touch and vibration.29,38
Apart from providing sensory awareness about our body and
its surroundings, the somatosensory system is also essential for
other functions we perform effortlessly and without much
thought. For example, a continuous flow of information in a
sensory–motor feedback loop involving several dozen muscles is
necessary to coordinate movements when taking a sip from a
glass of water or for the seemingly simple task of walking.
Somatosensory neurons are also organized in discreet pathways
with autonomic sympathetic and parasympathetic motor neu-
rons driving reflexes involved in physiological homeostasis, such
as thermoregulation through reflex control of sweat glands and
selective vasoconstrictor systems in muscle and skin active when
standing up and during hypothermia, respectively.16,49,74 Fur-
thermore, pleasant and painful sensations involve not only
discriminative perception and coordination of efferent reflex
functions but also emotional and motivational components
important for behavior. For example, social touch such as
hugging and caressing induce sensations of pleasantness to
facilitate emotional bonding, affiliative behavior, and well-be-
ing,82,88 while noxious stimuli not only convey sensory discrim-
inative and protective reflexes but also include an affective-
motivational dimension of unpleasantness contributing to long-
term behavior avoiding harm.112,125
The existence of molecularly unique neuron types coding for
each quality and dimension of somatosensation is unlikely.
Instead, summation of activity and inactivity from different types of
nerve fibers is likely to contribute to the sensory qualities and
dimensions involved. Several recent studies support this conjec-
ture, eg. the discovery of warmth sensation relying on the


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simultaneous activation of warmth-sensing and inhibition of cold-
sensing nerves97; the requirement of different sensory neurons
for protective reflexes and affective coping behavior elicited by the
same stimuli45; the suppression of noxious cold (210 to 10˚C) by
Calca1 neurons18,81; the modulation of touch-evoked itch by
Merkel cells and Slc17a8-lineage (low-threshold mechanorecep-
tor [LTMR]) sensory neurons32,33,108; and alleviation of Npy2r1
neuron-dependent acute pain behavior by the simultaneous
activity of the A-LTMR touch neurons.3 With a thorough
molecular categorization of sensory neuron types, experiments
can be designed with even greater precision, opening possibilities
for gaining new, deeper insights into the cellular basis for
nociception and pruriception.
2. Categorizing sensory neurons
Sensory neurons have historically been classified based on
physiological properties, morphology, target innervation, spinal
termination, developmental hierarchy, and functional and neuro-
chemical properties (reviewed by Emery and Ernfors, 202029).
Sponsorships or competing interests that may be relevant to content are disclosed
at the end of this article.
Division of Molecular Neurobiology, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm, Sweden
*Corresponding author. Address: C2 Medicinsk biokemi och biofysik, C2 Molekyl ¨ar
neurobiologi Ernfors, Solnav ¨agen 9, 6C, 17177 Stockholm, Sweden. Tel.: 146 8-
524 876 59. E-mail address: patrik.ernfors@ki.se (P. Ernfors).
PAIN 164 (2023) 1245–1257
Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf
of the International Association for the Study of Pain. This is an open access article
distributed under the Creative Commons Attribution License 4.0 (CCBY), which
permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
http://dx.doi.org/10.1097/j.pain.0000000000002831
June 2023·Volume 164·Number 6
www.painjournalonline.com
1245


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Brit. J. Ophthal. (I 975) 59, 439
An epidemic of picornavirus and adenovirus conjunctivitis
U. C. CHATURVEDI, ASHA MATHUR, U. K. SINGH, A. K. KAPOOR,
R. M. L. MEHROTRA, AND R. C. SAXENA
From the Upgraded Department of Pathology and Bacteriology, K.G. Medical College, Lucknow, India
A pandemic of acute haemorrhagic conjunctivitis
occurred all over south-east Asia and Japan during
1970 and
1971
(Kono, Sasagawa,
Ishii, Sugiura,
Ochi,
Matsumiya,
Uchida, Kameyama,
Kaneko,
and Sakurai, 1972). A similar epidemic occurred in
I969
in Ghana
(Chatterjee,
Quarcoopome,
and
Apenteng, I97oa, b) and Nigeria (Parrott,
I971).
In the Indian subcontinent the epidemic of con-
junctivitis was first reported in Bombay during April
1971, and later spread almost all over India. Cases
of
conjunctivitis
started
increasing
at
Lucknow
during the last week of May I97i, reaching a peak
in June, and declining by July. Picorna-like viruses
have been isolated from cases in Singapore (Lim
and Yin-Murphy, 1971; Yin-Murphy, 1972) and in
Japan (Kono and others, I972). The epidemic at
Lucknow was peculiar in that adenoviruses as well
as picornaviruses appear to have been present in
these cases.
Material and methods
The study was carried out by the ophthalmologist (RCS)
on 253 typical cases of conjunctivitis which presented at
Gandhi Memorial and associated hospitals for treatment.
Out of 253 patients 84-5 per cent were aged between
I I
and 40 years. The youngest was 3 years old and the oldest
65 years. The illness started unilaterally, and most of the
population at Lucknow was affected. There were multiple
cases in a family and the infection could be transmitted
even by casual conversation. The incubation period was less
than 24 hours in most cases. The chief complaints were a
gritty sensation in the eyes, watering, and photophobia.
A few patients had upper respiratory symptoms. Ocular
examination revealed redness, oedema of the conjunctiva
and lids, subconjunctival haemorrhage in a number of
cases, and enlarged preauricular lymph nodes. The cornea


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remained unaffected. The detailed clinical findings have
been reported by Saxena, Bhatia, and Chaturvedi (1972).
LABORATORY STUDIES
The laboratory investigations included total and differ-
ential leucocyte counts and conjunctival swab cultures
on blood agar and Robertson's cooked meat medium.
Address for reprints: Dr U. C. Chaturvedi, Upgraded Department of
Pathology and Bacteriology,
K.G.
Medical
College,
Lucknow-
226003, India
Bacteria were identified by their morphology, cultural
characters,
and
biochemical
reactions.
Smears
were
prepared from conjunctival scrapings of 6i patients and
stained with Giemsa stain to study the pattern of cellular
infiltration and to determine whether inclusion bodies
suggestive of viral infection would be found.
VIROLOGICAL INVESTIGATIONS
Conjunctival swabs and scrapings
were
collected
for
virological study within 48 hours of the onset of illness.
Paired sera were collected from I5 patients for study of
antibody titres.
VIRUS ISOLATION
This was done on primary rhesus monkey kidney tissue
culture. Each sample was inoculated into a group of four
tissue culture tubes. These were incubated stationary at
370C and were examined daily for 15 days. Tubes showing
cytopathic effects were subcultured.
ADENOVIRUS SEROLOGY
Haemagglutination test
The cytopathic agents were tested for haemagglutinin
using albino rat and rhesus monkey erythrocytes. It was
found that only rat erythrocytes were agglutinated by the
isolates; therefore for further experiments only erythro-
cytes from albino rats were used, taking the precautions
described by Rosen (I960). Hyperimmune antisera were
raised against two of the isolates in rabbits by the tech-
nique of Rosen (I960). After treating the hyperimmune
sera with 25 per cent kaolin and absorption with rat
erythrocytes, a haemagglutination inhibition (HI)
test
was performed on all the isolates. The HI
tests were
repeated in the presence of heterologous antisera (Rosen,
1960). With eight of the selected
isolates chessboard


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titrations were done using the hyperimmune sera described
above. Paired sera collected during the acute phase and in
convalescence were tested for HI antibodies against one
of the isolates.
ELECTRON MICROSCOPY
One ofthe agents was studied under the electron microscope
in the monkey kidney tissue culture fluid and after passage
in HeLa cells at Birmingham.
IDENTIFICATION
This agent was finally identified at the Virus Reference
Laboratory, London, by a neutralization test.


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Transciptional Profiling of Human Liver Identifies Sex-Biased Genes Associated with Polygenic Dyslipidemia and Coronary Artery Disease


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Transcriptional Profiling of Human Liver Identifies Sex-
Biased Genes Associated with Polygenic Dyslipidemia
and Coronary Artery Disease
Yijing Zhang1, Kathrin Klein2, Aarathi Sugathan1, Najlla Nassery1, Alan Dombkowski3, Ulrich M. Zanger2,
David J. Waxman1*
1 Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts, United States of America, 2 Dr. Margarete Fischer-Bosch
Institute of Clinical Pharmacology, Stuttgart, Germany, 3 Division of Clinical Pharmacology and Toxicology, Department of Pediatrics, Wayne State University, Detroit,
Michigan, United States of America
Abstract
Sex-differences in human liver gene expression were characterized on a genome-wide scale using a large liver sample
collection, allowing for detection of small expression differences with high statistical power. 1,249 sex-biased genes were
identified, 70% showing higher expression in females. Chromosomal bias was apparent, with female-biased genes enriched
on chrX and male-biased genes enriched on chrY and chr19, where 11 male-biased zinc-finger KRAB-repressor domain
genes are distributed in six clusters. Top biological functions and diseases significantly enriched in sex-biased genes include
transcription, chromatin organization and modification, sexual reproduction, lipid metabolism and cardiovascular disease.
Notably, sex-biased genes are enriched at loci associated with polygenic dyslipidemia and coronary artery disease in
genome-wide association studies. Moreover, of the 8 sex-biased genes at these loci, 4 have been directly linked to
monogenic disorders of lipid metabolism and show an expression profile in females (elevated expression of ABCA1, APOA5
and LDLR; reduced expression of LIPC) that is consistent with the lower female risk of coronary artery disease. Female-biased
expression was also observed for CYP7A1, which is activated by drugs used to treat hypercholesterolemia. Several sex-


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biased drug-metabolizing enzyme genes were identified, including members of the CYP, UGT, GPX and ALDH families. Half of
879 mouse orthologs, including many genes of lipid metabolism and homeostasis, show growth hormone-regulated sex-
biased expression in mouse liver, suggesting growth hormone might play a similar regulatory role in human liver. Finally,
the evolutionary rate of protein coding regions for human-mouse orthologs, revealed by dN/dS ratio, is significantly higher
for genes showing the same sex-bias in both species than for non-sex-biased genes. These findings establish that human
hepatic sex differences are widespread and affect diverse cell metabolic processes, and may help explain sex differences in
lipid profiles associated with sex differential risk of coronary artery disease.
Citation: Zhang Y, Klein K, Sugathan A, Nassery N, Dombkowski A, et al. (2011) Transcriptional Profiling of Human Liver Identifies Sex-Biased Genes Associated
with Polygenic Dyslipidemia and Coronary Artery Disease. PLoS ONE 6(8): e23506. doi:10.1371/journal.pone.0023506
Editor: Xin Wei Wang, National Cancer Institute, United States of America
Received December 31, 2010; Accepted July 19, 2011; Published August 12, 2011
Copyright:  2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported in part by National Institutes of Health grant DK33765 (to D.J.W.), the German Federal Ministry of Education and Research
grant ‘‘Virtual Liver’’ 0315755 and the Robert Bosch Foundation, Stuttgart, Germany (to U.M.Z.). These funding agencies had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: djw@bu.edu
Introduction


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Mammalian sex determination is initiated by the SRY gene,
which activates a developmental pathway leading to testis
formation and establishes life-long sex differences in the patterns
of gonadal hormone secretion [1]. Gonadal hormones, in turn,
exert permanent differentiating effects (‘organizational’ actions) as
well as short-term stimulatory effects that lead to sex differences in
gene expression in multiple tissues [2]. Sex differences are also
induced by non-gonadal signals and factors, including direct sex-
biased effects of individual X and Y-chromosome genes [3].
Epigenetic modifications also play an important role in the
development and maintenance of sexual dimorphism [4] by
processes such as genetic imprinting [5,6] and X-chromosome
inactivation [7,8,9]. Sex differences characterize histone acetyla-
tion and histone methylation [10] and the expression of certain
histone demethylases [11,12]. Sexual differentiation is thus
achieved through a complex interplay of multiple mechanisms
[13].
Global gene expression studies in mouse and rat liver have
identified .1,000 sex-dependent transcripts, which collectively
have a major impact on hepatic physiology, inflammatory
responses, diseased states, and the metabolism of steroids, drugs
and environmental chemicals [14,15,16]. However, very little is
known about the sex-dependence of gene expression in human
liver. Small but pharmacologically significant sex differences in the
expression of certain human hepatic drug-metabolizing CYP
enzymes have been reported, most notably for CYP3A4 [17,18],
however, only limited efforts have been made to identify sex
differences in human liver on a larger scale [19]. Such studies have
the potential to elucidate clinically important sex differences in
human hepatic physiology and pathophysiology, including sex
differences in circulating lipid profiles, which are more favorable in
women [20,21] and are associated with their lower risk of
PLoS ONE | www.plosone.org
1
August 2011 | Volume 6 | Issue 8 | e23506


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886 
VOLUME 20 | NUMBER 6 | JUNE 2017   nature neurOSCIenCe
Humans differ from other primates in a number of aspects, most 
strikingly with respect to their cognitive abilities. The human brain 
is a highly heterogeneous organ comprising many anatomically  
and functionally distinct structures. These include the neocortex,  
a 2–5-mm thick structure covering brain hemispheres. Given that it is 
the most recently evolved part of the brain, present only in mammals1, 
the neocortex might plausibly harbor those changes responsible for 
uniquely human cognition. Specifically, cognitive functions unique to 
humans or particularly pronounced in humans have been mapped to 
specific neocortical areas, including specific areas of prefrontal cortex 
(PFC), using functional imaging experiments or cases of localized 
brain damage2,3.
In search of mechanisms underlying human cognitive abilities,  
a number of studies have investigated differences in neocortex  
composition between humans and nonhuman primates at different 
levels of molecular organization, including gene expression, iden-
tifying a number of human-specific gene expression features4–6. 
These studies, performed on bulk tissue samples representing one  
or more specific neocortical regions, were insufficient to yield a  
comprehensive picture of the molecular mechanisms underlying 
human cognition.
The neocortex itself has a complex laminar architecture, tradition-
ally divided into six layers based on visual examination of its histo-
logical organization1. Neocortical layers differ from each other in 
terms of both cell type composition and the direction of neuronal 
connections7. Several studies have assessed gene expression in specific 
cortical layers. More than 5,000 genes were shown to have differential 
expression across layers in the mouse neocortex based on cortical 
layer samples dissected using laser capture microdissection (LCM) 
and measured using microarrays8,9 or high-throughput sequenc-


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ing8. Similarly, 4,923 genes showed significant expression differences 
among cortical layers 2–5 when dissected using LCM from ten corti-
cal regions of the macaque brain and measured using microarrays, 
with most of the differences shared among regions10.
In addition to transcriptome-wide studies, expression differences 
among cortical layers in the mouse11 and human12 brains have pre-
viously been assessed using in situ hybridizations, revealing several 
hundred mouse and 76 human genes showing visibly differential 
expression among layers. Only one study so far, based on abundance 
quantification of 39 proteins in four different brain regions (cingu-
late, primary motor, somatosensory and primary visual cortices) of 
eight humans and three chimpanzees, examined recent evolutionary 
changes in gene expression across cortical layers13. That study did not 
quantitatively estimate the protein abundance differences between 
species in each layer, however. Thus, at present, we still lack system-
atic analysis of layer-related expression in the human brain, as well 
as identification of layer-related expression changes unique to the 
human brain.
To address this, we adopted an experimental procedure allowing 
comprehensive profiling of transcripts present in different strata  
of the cortical samples and applied this to assess gene expression  
in a specific area of human, chimpanzee and macaque PFC across  
all histologically defined cortical layers and the underlying  
white matter.
1CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, SIBS, CAS, Shanghai, China. 2Big Data Decision Institute, 
Jinan University, Guangzhou, China. 3Skolkovo Institute of Science and Technology, Skolkovo, Russia. 4University of Chinese Academy of Sciences, Beijing, China. 
5Department of Neuroscience, National Research Center, Kurchatov Institute, Moscow, Russia. 6School of Life Science and Technology, ShanghaiTech University,


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Shanghai, China. 7Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. 8Immanuel Kant Baltic Federal University, Kaliningrad, Russia. 9Comparative 
Biology group, PICB, SIBS, CAS, Shanghai, China. 10These authors contributed equally to this work. Correspondence should be addressed to P.K. (khaitovich@eva.mpg.de).
Received 27 June 2016; accepted 17 March 2017; published online 17 April 2017; doi:10.1038/nn.4548
Comprehensive transcriptome analysis of neocortical 
layers in humans, chimpanzees and macaques
Zhisong He1,10  , Dingding Han1,2,10, Olga Efimova3, Patricia Guijarro1, Qianhui Yu1,4, Anna Oleksiak1,  
Shasha Jiang1, Konstantin Anokhin5, Boris Velichkovsky6, Stefan Grünewald1 & Philipp Khaitovich3,6–9
While human cognitive abilities are clearly unique, underlying changes in brain organization and function remain unresolved. 
Here we characterized the transcriptome of the cortical layers and adjacent white matter in the prefrontal cortexes of humans, 
chimpanzees and rhesus macaques using unsupervised sectioning followed by RNA sequencing. More than 20% of detected 
genes were expressed predominantly in one layer, yielding 2,320 human layer markers. While the bulk of the layer markers were 
conserved among species, 376 switched their expression to another layer in humans. By contrast, only 133 of such changes 
were detected in the chimpanzee brain, suggesting acceleration of cortical reorganization on the human evolutionary lineage. 
Immunohistochemistry experiments further showed that human-specific expression changes were not limited to neurons but 
affected a broad spectrum of cortical cell types. Thus, despite apparent histological conservation, human neocortical organization 
has undergone substantial changes affecting more than 5% of its transcriptome.
r e S Ou rC e
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.


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RESEARCH PAPER
Assembly of novel microbial genomes from gut metagenomes of rhesus 
macaque (Macaca mulatta)
Shengzhi Yanga†, Zhenxin Fana,b†, Jiawei Lia, Xinqi Wanga, Yue Lana, Bisong Yuea,b, Miao Hec, Anyun Zhanga,d, 
and Jing Lia
aKey Laboratory of Bioresources and Eco-Environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu, Sichuan, 
China; bSichuan Key Laboratory of Conservation Biology on Endangered Wildlife, College of Life Sciences, Sichuan University, Chengdu, 
Sichuan, China; cInstitute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, Sichuan, China; dAnimal Disease Prevention 
and Food Safety Key Laboratory of Sichuan Province, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of 
Life Sciences, Sichuan University, Chengdu, Sichuan, China
ABSTRACT
Rhesus macaque (RM, Macaca mulatta), as an important model animal, commonly suffers from 
chronic diarrheal disease, challenging the breeding of RMs. Gut microbiomes play key roles in 
maintaining intestinal health of RMs. However, it is still unclear about more features of gut 
microbiome as responsible for intestinal health of RMs. In this study, we performed de novo 
assembly of metagenome-assembled genomes (MAGs) based on fecal metagenomes from chronic 
diarrheal RMs and asymptomatic individuals. In total of 731 non-redundant MAGs with at least 80% 
completeness were reconstructed in this study. More than 97% MAGs were novel genomes 
compared with more than 250,000 reference genomes. MAGs of Campylobacter and 
Helicobacteraceae from RM guts mainly carried flagella-associated virulence genes and chemo-
taxis-associated virulence genes, which might mediate motility and adhesion of bacteria. 
Comparing to MAGs of Campylobacter from humans, distributions and functions of these MAGs 
of Campylobacter from RMs exhibited significant differences. Most members of Bacteroidota, 
Spirochaetota, Helicobacteraceae, Lactobacillaceae and Anaerovibrio significantly decreased in


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guts of chronic diarrhea RMs. More than 92% MAGs in this study were not contained in 2,985 
MAGs previously reported from other 22 non-human primates (NHPs), expanding the microbial 
diversity in guts of NHPs. The distributions and functions of gut microbiome were prominently 
influenced by host phylogeny of NHPs. Our results could help to more clearly understand about the 
diversity and function of RMs gut microbiome.
ARTICLE HISTORY 
Received 17 August 2022  
Revised 28 November 2022  
Accepted 3 March 2023 
KEYWORDS 
Macaca mulatta; 
Metagenomic binning; MAG; 
non-human primate; gut 
microbiome; Campylobacter
Introduction
Due to many populations and the similarity of 
genetic evolution to human, rhesus macaque 
(RM, Macaca mulatta) generally serves as an 
important model animal to reveal physiology of 
human, research clinical disease and develop new 
drugs.1–3 Therefore, RMs with huge needs are 
widely raised in captivity as model animal. 
However, many captive RMs usually suffer from 
chronic diarrhea with high morbidity and mortal-
ity to pose serious challenges in breeding and man-
agement of RMs.4 Chronic diarrhea of RMs is 
prominently manifested by long-term and recur-
rent diarrhea.5 Previous studies indicated that 
chronic diarrhea disease of RMs was accompanied 
by significant changes of gut microbial composi-
tion and metabolic function.6,7 The significant 
decrease 
of 
carbohydrate-active 
enzyme 
(CAZyme) genes and significant increase of anti-
biotic resistance genes (ARGs) were found in guts 
of chronic diarrhea RMs comparing with asympto-
matic RMs.7 However, these changes of microbial 
abundance and diversity might not mean that 
emergence and continuation of diarrhea disease 
are caused by the increased/decreased microbiome. 
In addition, taxonomic annotation of gut micro-
biome based on metagenomic short reads might 
lose many important microbial taxa due to 
CONTACT Jing Li 
ljtjf@126.com


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Key Laboratory of Bioresources and Eco-Environment (Ministry of Education, College of Life Sciences, Sichuan 
University, Chengdu, Sichuan, China; Anyun, Zhang 
zhanganyun@scu.edu.cn 
Key Laboratory of Bioresources and Eco-Environment (Ministry of 
Education), College of Life Sciences, Sichuan University, Sichuan, China
†Contributed equally to this work
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2188848
GUT MICROBES                                              
2023, VOL. 15, NO. 1, 2188848 
https://doi.org/10.1080/19490976.2023.2188848
© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.  
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, 
distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted 
Manuscript in a repository by the author(s) or with their consent.


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Mocrobial metabolites as modulators of the infant gut microbiome and host-microbiome interations in early life


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REVIEW
Microbial metabolites as modulators of the infant gut microbiome and 
host-microbial interactions in early life
Henrik M. Roager
a, Catherine Stanton
b, and Lindsay J. Hall
c,d,e
aDepartment of Nutrition, Exercise and Sports, University of Copenhagen, Frederiksberg, Denmark; bAPC Microbiome Ireland, Teagasc 
Moorepark Food Research Centre, Fermoy, Co. Cork, Ireland; cGut Microbes & Health, Quadram Institute Biosciences, Norwich, UK; dIntestinal 
Microbiome, School of Life Sciences, ZIEL – Institute for Food & Health, Technical University of Munich, Freising, Germany; eNorwich Medical 
School, University of East Anglia, Norwich, UK
ABSTRACT
The development of infant gut microbiome is a pivotal process affecting the ecology and function 
of the microbiome, as well as host health. While the establishment of the infant microbiome has 
been of interest for decades, the focus on gut microbial metabolism and the resulting small 
molecules (metabolites) has been rather limited. However, technological and computational 
advances are now enabling researchers to profile the plethora of metabolites in the infant gut, 
allowing for improved understanding of how gut microbial-derived metabolites drive microbiome 
community structuring and host-microbial interactions. Here, we review the current knowledge on 
development of the infant gut microbiota and metabolism within the first year of life, and discuss 
how these microbial metabolites are key for enhancing our basic understanding of interactions 
during the early life developmental window.
ARTICLE HISTORY 
Received 3 October 2022  
Revised 3 February 2023  
Accepted 6 March 2023 
KEYWORDS 
Metabolites; metabolomics; 
infants; microbiota; health; 
host; diet
Introduction
A relatively simple microbial community at birth 
develops into a diverse and complex microbial eco-
system across the early life window1, which plays 
a vital role in postnatal maturation and development 
of the endocrine, nervous and immune systems2,3.


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The gut microbiome thus has a profound influence 
on both early and long-term health and 
development4,5. In addition to influencing develop-
ment and maturation of the host immune system, 
the gut microbiota has a number of important func-
tions in the host, including breakdown of ingested 
complex carbohydrates, protection against patho-
genic bacteria and metabolic functions including 
production of vitamins, short chain fatty acids 
(SCFAs) and bile acid biotransformation6. The 
importance of neonatal bacterial colonization for 
the growth of a healthy individual is clear and exhi-
bits the power that the microbiome has on its host 
from an early stage in development.
The gut microbiota composition is initially known 
to be in a state of flux and consequently stabilizes, so 
that by age 2.5–4 years, it begins to resemble the 
composition and characteristics of an adult gut7,8. In 
this early phase, the gut microbiota is more vulnerable 
to modulation than in the adult state9, suggesting that 
the first 1000 days of life is a critical window of 
opportunity for optimal bacterial colonization. 
Disruption to microbial development during this per-
iod is believed to have long-lasting consequences, 
impacting on normal immune and neurocognitive 
development and has been associated with increased 
risk of developing diseases such as asthma and meta-
bolic syndrome later in life10,11.
The gut microbiota may also be modulated at 
this critical phase in a beneficial manner. Strategies 
to optimize early microbial acquisition during this 
key developmental window of opportunity may 
help to prevent and treat disease. Interventions to 
modulate the early microbiome include the use of 
probiotic and prebiotic interventions or combina-
tions of both, as either synbiotics12 or postbiotics13. 
The most promising results for probiotics have 
been seen in the prevention or treatment of 
CONTACT Henrik M. Roager 
hero@nexs.ku.dk


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Department of Nutrition, Exercise and Sports, University of Copenhagen, Rolighedsvej 30, Frederiksberg 
DK-1958, Denmark
GUT MICROBES                                              
2023, VOL. 15, NO. 1, 2192151 
https://doi.org/10.1080/19490976.2023.2192151
© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.  
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, 
distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted 
Manuscript in a repository by the author(s) or with their consent.


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Genetic structure of the population with rheumatoid arthritis in north east England: a genetic approach to define different subtypes


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Annals of the Rheumatic Diseases,
1986; 45, 881-891
Genetic structure of the population with rheumatoid
arthritis in north east England: a genetic approach to
define different subtypes
S S PAPIHA,' J S LANCHBURY,' AND B PAL2
From the 'Department of Human Genetics, Medical School, University of Newcastle upon Tyne; and the
2Department of Rheumatology, Dryburn Hospital, Durham
SUMMARY
Clinically and immunologically rheumatoid arthritis (RA) is possibly a heterogeneous
disorder. Despite numerous efforts clearer definition of this heterogeneity has been of limited
success. Measurements of rheumatoid factor (RF) and antinuclear antibodies (ANA) by
conventional methods define subpopulations of patients with RA and in a few recent studies an
association of human leucocyte antigens (HLA) undoubtedly indicates the immunogenetic
differences in the susceptibility of RA patients with different status of autoantibodies. The
studies on a few isolated non-HLA genetic markers in RA are controversial. To understand the
role of genetic factors
in susceptibility 24 single
gene characters other than HLA were
investigated in 225 patients with RA classified by humoral status (presence or absence of RF and
ANA) into three groups and in 104 healthy control individuals from the north east of England.
Locus by locus comparison suggested associations of MN, Lewis, and Bf system with RF positive
patients.
Although
the
associations
with MN and
Lewis
blood
groups
require
further
investigations,
the involvement of the Bf locus
is
in agreement with the immunological
component of the disease suggested by HLA associations and it could be due to the phenomenon
of linkage disequilibrium. Measures of genetic distance applied to the subpopulations of patients
with RA, divided according to the presence
or absence of humoral factors, suggest that
RF+ANA+, RF-ANA-, and RF+ANA- subgroups are distinct genetic diseases, each affecting a
different subsection of the population which is genetically distinct. Such genetic heterogeneity


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may suggest a different pathogenetic mechanism for each of these subpopulations of rheumatoid
arthritis.
Key words: blood groups, serum proteins, red cell enzymes, phenotypic variation, genetic
heterogeneity.
Present evidence that genetic factors may play an
important part in the development of rheumatoid
arthritis (RA) comes from twin studies, pedigree
analysis, and associations with it of marker variants
in genetic polymorphic systems. 1-7 The initial inves-
tigations on monozygotic cotwins showed an inci-
dence of the disease 33 times that expected, and
family studies showed a relative risk ranging be-
tween 1 and 15*2 for the first degree relatives. These
Accepted for publication 21 April 1986.
Correspondencc
to Dr S
S
Papiha,
Dcpartment
of Human
Genetics, 19 Claremont Placc, Newcastlc upon Tync NE2 4AA
observations suggested strong genetic susceptibility
to the disease.
In
the
last two decades
a few
retrospective
analyses of the literature cast doubt on the validity
of the existing data on familial aggregation of RA,
and
suggested
a weak
genetic
element
in
the
aetiology of the disease.8 9 Meanwhile, research on
its association with human leucocyte antigens once
again drew attention to the possibility of genetic
predisposition coded at or near the HLA-D locus
situated on chromosome 6 in man, or a defective
immune response gene which may be in linkage
disequilibrium with the HLA-D locus.4 1i-12
881


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This journal is © The Royal Society of Chemistry 2014
Mol. BioSyst., 2014, 10, 653--662 | 653
Cite this: Mol. BioSyst., 2014,
10, 653
Unravelling the proteome of adult rhesus monkey
ovaries†
Hui He,‡a Hui Teng,‡a Tao Zhou,‡a Yueshuai Guo,‡a Gaigai Wang,a Min Lin,a
Yujie Sun,a Wei Si,b Zuomin Zhou,a Xuejiang Guo*a and Ran Huo*a
Ovarian physiology and pathology are important areas of scientific research. Eﬀorts have been made to
identify the ovary-related transcriptomes in diﬀerent species. However, the proteomic studies are limited. The
rhesus monkey is very similar to humans, and it is widely used in the study of reproductive biology and
medicine. In this study, using an optimized proteomics platform, we successfully identified 5723 rhesus
ovarian proteins, of which 4325 proteins were consistently identified in all three replicates and with at least 2
unique peptides. The 4325 proteins were chosen for further analysis. Through gene ontology and pathway
analyses,
we
obtained
a
preliminary
understanding
of
the
function
of
these
proteins.
A
random
immunohistochemistry analysis was used to determine the expression of proteins in various cell types. By
comparing the genes identified in this study with genes that were reported to have relatively high levels of
expression in human oocytes, we obtained genes that were predicted to play roles in maintenance of normal
ovarian physiology. Searching the identified genes from this study against the MGI database gave us a list of
proteins those exist in the rhesus monkey ovary and are important for female mouse reproduction as well.
The overlap of genes in this study and the genes whose abnormal expression or dysfunction were reported
to be associated with human polycystic ovary syndrome (PCOS) and premature ovarian failure (POF)
prompted us to use the rhesus monkey to study these two common causes of female infertility. This study
may provide a basis for future studies of human reproductive disorders using the rhesus monkey as a model.
1. Introduction


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Oocytes are stored and grow in the mammalian ovary. Ovulation
takes place under the control of the hypothalamus–pituitary–
gonadal axis and involves the participation of many important
molecules. The mammalian ovary plays a key role in oogenesis
and the secretion of gonadal hormones, such as estrogen,
progesterone, and small amounts of androgens, all of which
are important for the maintenance of normal female sexual
characteristics and normal pregnancies. Follicle, containing
oocyte and granulosa cells, is the basic functional unit of the ovary.
Folliculogenesis consists of a series of events including the activa-
tion of the primordial follicle, pre-antral follicle development,
dominant follicle selection, subordinate follicle apoptosis, antral
follicle development and ovulation, mechanisms of which involve
many molecules and pathways.1,2 The World Health Organization
stated that infertility could become the third most prevalent disease
after cancer and cardiovascular disease. It was estimated that
approximately 15% of reproductive age couples who live in
industrialized countries are infertile.3 Infertility caused by
female anatomy accounts for more than half of all cases of
infertility. There are many reasons for female infertility, within
which ovarian factors including polycystic ovary syndrome
(PCOS),4 premature ovarian failure (POF),5 ovary cancer etc.
account for a large proportion.6 Researchers have identified
some genes that are involved in female infertility, as evidenced
by KO mice and other model systems.7,8 Although there have
been several reports, a considerable amount of information
about the ovary remains unknown. To gain more information,
a State Key Laboratory of Reproductive Medicine, Department of Histology and
Embryology, Nanjing Medical University, Nanjing 210029, Jiangsu, China.
E-mail: huoran@njmu.edu.cn, guo_xuejiang@njmu.edu.cn; Fax: +86-25-86862908;
Tel: +86-25-86862038
b Kunming Primate Research Center and Kunming Institute of Zoology, Chinese


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Academy of Sciences, Kunming, Yunnan, China
† Electronic supplementary information (ESI) available: Fig. S1 shows the details
of a selected group of over represented pathways in the ovary. Table S1 shows the
information from the identified proteins and the proteins that overlap with the
human ovarian proteome. Table S2 shows the identified peptide information.
Table S3 shows the GO and pathway analysis of the identified proteins. Table S4
shows the primary antibodies used in immunohistochemistry studies. Table S5
shows the information of the high abundance proteins. Table S6 shows the
overlapping genes between genes identified in this study and the reported oocyte
overexpressed genes and the basic functional analysis and classification of these
overlapping genes. Table S7 shows the female reproductive phenotype annota-
tions of rhesus ovarian homologous mouse genes. Table S8 shows proteins that
not only were identified in this proteome but also were reported to be diﬀerently
expressed in PCOS ovary and normal ovary. This material is available free of
charge via the internet at http://pubs.acs.org. See DOI: 10.1039/c3mb70312f
‡ These authors contributed equally to the work.
Received 27th July 2013,
Accepted 22nd November 2013
DOI: 10.1039/c3mb70312f
www.rsc.org/molecularbiosystems
Molecular
BioSystems
PAPER
Published on 25 November 2013. Downloaded by West Lake University on 3/10/2023 5:53:54 AM. 
View Article Online
View Journal  | View Issue


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Current Evidence and Rationale to Guide Perioperative Management, Including Transfusion Decisions, In Patients With Sickle Cell Disease


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Copyright © 2023 International Anesthesia Research Society. Unauthorized reproduction of this article is prohibited.
June 2023 • Volume 136 • Number 6 
www.anesthesia-analgesia.org 1107
E   NarratiVe reView article
Patient Blood Management
Current Evidence and Rationale to Guide Perioperative 
Management, Including Transfusion Decisions, in 
Patients With Sickle Cell Disease
Roberta C. G. Azbell, MD,* Sophie M. Lanzkron, MD, MHS,† and Payal C. Desai, MD‡
Sickle cell disease (SCD) is a collection of inherited hemoglobin disorders that results in chronic 
hemolytic anemia, vaso-occlusion, pain, and end organ damage. Surgery in the SCD population 
requires careful planning, as perioperative stressors can lead to increased sickling and risk of 
inducing or further exacerbating vaso-occlusive episodes (VOEs). Additionally, the underlying 
hypercoagulability and immunocompromised state due to SCD places patients at increased 
risk of both venous thromboembolism and infection. Judicious fluid administration, temperature 
regulation, thorough preoperative and postoperative analgesic planning, and preoperative trans-
fusion are all crucial components of decreasing risks of surgery in patients with SCD. (Anesth 
Analg 2023;136:1107–14)
GLOSSARY
ACS = acute chest syndrome; ASH = American Society of Hematology; CI = confidence interval; 
CT = computed tomography; DHTR = delayed hemolytic transfusion reactions; DVT = deep vein 
thrombosis; eGFR = estimated glomerular filtration rate; EQ-5D = EuroQol-5D; HbC = hemoglobin C;  
HbS = sickle hemoglobin; HELLP = hemolysis, elevated liver enzyme and low platelets; IV = intrave-
nous; LR = lactated Ringer; NS = normal saline; RCT = randomized control trial; RR = relative risk; 
SCD = sickle cell disease; VOE = vaso-occlusive episode; VTE = venous thromboembolic disease
S
ickle cell disease (SCD) is a collection of inher-
ited hemoglobin disorders that affects up to 
100,000 people in the United States.1The abnor-
mality resides in the hemoglobin molecule with the


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substitution of valine for glutamic acid in the beta glo-
bin chain. The resultant abnormal hemoglobin (called 
sickle hemoglobin or HbS) is less soluble than the wild 
type and forms a polymer upon deoxygenation and 
that results in shape change from the normal red cell 
disk to an elongated sickle form. SCD occurs when 
the HbS mutation is homozygous or is coinherited 
with another beta globin chain gene mutation such 
as beta thalassemia or hemoglobin C. Patients with 
SCD have a chronic hemolytic anemia and intermit-
tent episodes of vaso-occlusion of small blood vessels 
leading to acute pain episodes, as well as acute and 
chronic injury to lungs, heart, kidneys, bone, and the 
central nervous system.2
When preparing a patient with SCD for surgery, there 
are many factors to consider. Intraoperatively, surgery 
may lead to hypotension, acidosis, hypoxia, frequent 
exposure to low temperatures, vasoconstriction, venous 
stasis, and increased stress, all of which can promote 
increased sickling and potentially induce vaso-occlusive 
episodes (VOEs).3,4 Additionally, patients with SCD have 
higher risks of perioperative infection due to functional 
asplenia,5 and increased risk of perioperative throm-
bosis as a result the underlying hypercoagulable state. 
Preoperative planning is required to help minimize the 
risks of perioperative morbidity and mortality.
While standard tools are used to consider peri-
operative risks for all patients,6,7 none of these tools 
include SCD and likely underestimate the periopera-
tive risks for this specific patient population. And cur-
rently, there are no specific tools for perioperative risk 
estimation in SCD.
GENERAL CONSIDERATION
Fluid Management
Fluid management in the SCD population can 
be complicated by the presence of underlying 
Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, 
Inc. on behalf of the International Anesthesia Research Society. This is an 
open-access article distributed under the terms of the Creative Commons


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string_value:
Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), 
where it is permissible to download and share the work provided it is prop-
erly cited. The work cannot be changed in any way or used commercially 
without permission from the journal.
DOI: 10.1213/ANE.0000000000006463
From the *Department of Internal Medicine, The Ohio State University 
Wexner Medical Center, Columbus, Ohio; †Department of Internal 
Medicine, Division of Hematology, Johns Hopkins School of Medicine; and 
‡Department of Internal Medicine, Division of Hematology, The Ohio State 
Wexner Medical Center, Columbus, Ohio.
Accepted for publication February 8, 2023.
Funding: None.
Conflicts of Interest: See Disclosures at the end of the article.
Reprints will not be available from the authors.
Address correspondence to Roberta C. G. Azbell, MD, Department of Internal 
Medicine, The Ohio State University Wexner Medical Center, 1800 Cannon 
Dr, Lincoln Tower, Suite 1110L, Columbus, OH 43210. Address e-mail to 
roberta.azbell@osumc.edu.
CME


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Social threat, fronto-cingulate-limbic morphometry, and symptom course in depressed adolescents: a longitudinal invesetigation


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Psychological Medicine
cambridge.org/psm
Original Article
Cite this article: Ojha A, Teresi GI, Slavich GM,
Gotlib IH, Ho TC (2023). Social threat, fronto-
cingulate-limbic morphometry, and symptom
course in depressed adolescents: a
longitudinal investigation. Psychological
Medicine 53, 5203–5217. https://doi.org/
10.1017/S0033291722002239
Received: 12 November 2021
Revised: 5 May 2022
Accepted: 28 June 2022
First published online: 19 September 2022
Key words:
Adolescence; amygdala; anterior cingulate
cortex; depression; nucleus accumbens;
social stress
Author for correspondence:
Tiffany C. Ho, Ph.D.,
E-mail: tiffany.ho@psych.ucla.edu
© The Author(s), 2022. Published by
Cambridge University Press. This is an Open
Access article, distributed under the terms of
the Creative Commons Attribution licence
(http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted re-use, distribution
and reproduction, provided the original article
is properly cited.
Social threat, fronto-cingulate-limbic
morphometry, and symptom course in
depressed adolescents: a
longitudinal investigation
Amar Ojha1,2
, Giana I. Teresi3, George M. Slavich4, Ian H. Gotlib5 and
Tiffany C. Ho6
1Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA; 2Center for Neural Basis of Cognition,
University of Pittsburgh, Pittsburgh, PA, USA; 3Department of Psychology, University of Pittsburgh, Pittsburgh, PA,
USA; 4Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA, USA;
5Department of Psychology, Stanford University, Stanford, CA, USA and 6Department of Psychiatry and Behavioral
Sciences, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA
Abstract
Background. Psychosocial stressors characterized by social threat, such as interpersonal loss
and social rejection, are associated with depression in adolescents. Few studies, however, have
examined whether social threat affects fronto-cingulate-limbic systems implicated in adoles-
cent depression.


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Methods. We assessed lifetime stressor severity across several domains using the Stress and
Adversity Inventory (STRAIN) in 57 depressed adolescents (16.15 ± 1.32 years, 34 females),
and examined whether the severity of social threat and non-social threat stressors was asso-
ciated with gray matter volumes (GMVs) in the anterior cingulate cortex (ACC), amygdala,
hippocampus, and nucleus accumbens (NAcc). We also examined how lifetime social threat
severity and GMVs in these regions related to depressive symptoms at baseline and over
9 months.
Results. General stressor severity was related to greater depression severity at baseline and
over 9 months. Moreover, greater severity of social threat (but not non-social threat) stressors
was associated with smaller bilateral amygdala and NAcc GMVs, and smaller bilateral surface
areas of caudal and rostral ACC (all pFDR ⩽ 0.048). However, neither social threat nor non-
social threat stressor severity was related to hippocampal GMVs (all pFDR ⩾ 0.318). All
fronto-cingulate-limbic structures that were associated with the severity of social threat
were negatively associated with greater depression severity over 9 months (all pFDR ⩽ 0.014).
Post-hoc analyses suggested that gray matter morphometry of bilateral amygdala, NAcc,
and rostral and caudal ACC mediated the association between social threat and depression
severity in adolescents over 9 months (all pFDR < 0.048).
Conclusions. Social threat specifically affects fronto-cingulate-limbic pathways that contribute
to the maintenance of depression in adolescents.
Introduction
Depression is a leading cause of disability worldwide (World Health Organization, 2017), with
nearly one in five people experiencing depression during their lifetime (Friedrich, 2017). The
lifetime prevalence of depression is especially alarming in adolescents, with a cumulative inci-
dence of 13.6% in males and 36.1% in females by age 18 (Breslau et al., 2017). Moreover, indi-


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viduals who develop depression during adolescence, as compared to those who develop
depression later in life, tend to experience more severe and recurrent episodes of the disorder
(Naicker, Galambos, Zeng, Senthilselvan, & Colman, 2013; Zisook et al., 2007). Viewed in this
context, adolescence represents an important developmental period marked by heightened
vulnerability for the onset and adverse consequences of depression. Therefore, it is critical
to identify psychosocial and neurobiological mechanisms that contribute to depression in
adolescents.
Social stressors and depression: the role of social threat
Although stressful life events often precede and increase risk for depressive episodes across the
lifespan (Hammen, 2005, 2006), this effect is especially pronounced in adolescents
(Lewinsohn, Allen, Seeley, & Gotlib, 1999; Monroe & Harkness, 2005). This finding is not sur-
prising given that adolescence is a period during which environmental demands have outsized


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Drori et al., Sci. Adv. 8, eabm1971 (2022)     15 July 2022
SCIE N C E A D V A NCES | RESEA R CH  A RT ICL E
1 of 14
N E UR OSCIENCE
Mapping microstructural gradients of the human 
striatum in normal aging and Parkinson’s disease
Elior Drori*, Shai Berman, Aviv A. Mezer
Mapping structural spatial change (i.e., gradients) in the striatum is essential for understanding the function of 
the basal ganglia in both health and disease. We developed a method to identify and quantify gradients of micro-
structure in the single human brain in vivo. We found spatial gradients in the putamen and caudate nucleus of the 
striatum that were robust across individuals, clinical conditions, and datasets. By exploiting multiparametric 
quantitative MRI, we found distinct, spatially dependent, aging-related alterations in water content and iron con-
centration. Furthermore, we found cortico-striatal microstructural covariation, showing relations between striatal 
structural gradients and cortical hierarchy. In Parkinson’s disease (PD) patients, we found abnormal gradients in 
the putamen, revealing changes in the posterior putamen that explain patients’ dopaminergic loss and motor 
dysfunction. Our work provides a noninvasive approach for studying the spatially varying, structure-function re-
lationship in the striatum in vivo, in normal aging and PD.
INTRODUCTION
The striatum, consisting of the caudate nucleus and the putamen, is 
a major input structure of the basal ganglia, playing a crucial role in 
motor and cognitive aspects of goal-directed behavior (1). Changes 
in striatal tissue and interhemispheric asymmetries are associated 
with motor and cognitive dysfunctions that take place in normal 
aging (2) and in neurodegenerative disorders such as Parkinson’s 
disease (PD), dystonia, and Huntington’s disease (1, 3). The function 
of the striatum is tightly related to its cellular and biochemical prop-
erties: Histochemical studies highlight the distinct neurochemical


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compartments of the striatal tissue (i.e., striosomes and matrix), 
which differ in dopaminergic and cholinergic expression and change 
in distribution and chemical properties along the anatomical axes 
of the striatum (4–6). These compartments are thought to constitute 
an organizational feature of striatal connectivity (6–8). The connec-
tivity of the striatum has been linked to the hierarchal organization 
of the cortex, with gradients of sensorimotor, associative, and lim-
bic domains along the anterior-posterior (i.e., rostral-caudal) and 
ventromedial-dorsolateral axes (9, 10). Accordingly, electrophysiol-
ogy and functional magnetic resonance imaging (fMRI) studies have 
provided evidence for functional and cognitive gradients in the stri-
atum (11–14).
Changes in the striatum’s biological spatial variation (i.e., gradi-
ents; we use these terms interchangeably) are notably apparent in PD, 
mainly along the anterior-posterior axis, which emphasizes acute 
degeneration in posterior parts of the putamen (15–19). This degen-
eration is characterized by the depletion of dopaminergic innerva-
tion to the striatum, due to the patients’ loss of dopaminergic neurons 
in the substantia nigra pars compacta (1). Positron emission tomog-
raphy (PET) and single-photon emission computed tomography 
(SPECT) studies in PD patients demonstrate the decrease of dopa-
mine intake in the striatum (20–23). Striatal deterioration, in turn, 
affects motor function, leading to a range of movement impairments 
(1, 24). Hence, gradients in the cellular and neurochemical content 
level (i.e., the microenvironment) of the striatum are a key feature 
in understanding the striatum’s dysfunction in PD.
Microstructural gradients of the striatum are studied mainly using 
invasive animal research and human postmortem analyses. In living 
humans, MRI is the primary noninvasive tool for structural research 
of the brain. Developments in quantitative MRI (qMRI) provide para-
metric mappings of biophysical tissue properties, such as the longi-


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tudinal relaxation rate R1, which is sensitive to a variety of biological 
sources in the microenvironment of the tissue, including myelin, 
water content, and iron concentration. Therefore, qMRI has been 
proposed to provide “in vivo histology” of the human brain (25). We 
hypothesized that incorporating a spatial approach in qMRI methods 
to map the heterogeneous microstructural properties of subcortical 
regions, and specifically the striatum, would have important effects 
for our understanding of aging and PD in vivo.
Here, we present an in vivo method to quantify microstructural 
gradients in the caudate and putamen in normal aging and 
PD. Our innovative approach allows the measurement of spatial vari-
ation in microstructure along the main axes of the striatum. Using 
this approach, we characterize striatal gradients that are related to 
multiple biological sources, in healthy younger and older adults. Next, 
to detect PD-related microstructural changes, we generalize our re-
sults to widely available clinical datasets. We investigate how changes 
in striatal gradients are related to PD motor symptomatology, as well 
as to striatal dopamine loss in individual patients. Last, our analysis 
expands to the relationship between the striatum and the cerebral 
cortex, identifying a gradient of cortico-striatal microstructure net-
works, that shows dependency between striatal position and the cor-
tical circuit hierarchical organization (9, 13).
RESULTS
Microstructural gradients in the striatum
Previous studies have identified spatial gradients in the microenvi-
ronment along the axes of the striatum ex vivo (4–6). To assess spa-
tial variability in humans in vivo, we developed an automatic tool 
to express the main axes of the striatum on the single-subject level 
(Fig. 1A). Our tool uses a region-of-interest (ROI) mask and per-
forms singular value decomposition (SVD) on the voxel coordi-
nates to find the main orthogonal axes of a given region. Voxels are


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then divided into seven equally spaced segments along each of the 
axes (see section S1), and the median value of a qMRI parameter 
The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of 
Jerusalem, Jerusalem, Israel.
*Corresponding author. Email: elior.drori@mail.huji.ac.il
Copyright © 2022 
The Authors, some 
rights reserved; 
exclusive licensee 
American Association 
for the Advancement 
of Science. No claim to 
original U.S. Government 
Works. Distributed 
under a Creative 
Commons Attribution 
NonCommercial 
License 4.0 (CC BY-NC).


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Open camera or QR reader and
scan code to access this article
and other resources online.
Telemedicine for First-Trimester Medical Abortion in Canada:
Results of a 2019 Survey
Regina M. Renner, MD, MPH,1,2,* Madeleine Ennis, PhD,1,2,*
Ama Kyeremeh, MPH,2 Wendy V. Norman, MD, MHSc,2–4
Sheila Dunn, MD, MSc,2,5 Helen Pymar, MD, MPH,2,6
and Edith Guilbert, MD, MSc2,7
Departments of 1Obstetrics and Gynaecology and 3Family
Practice, University of British Columbia, Vancouver, Canada.
2Contraception and Abortion Research Team, Women’s Health
Research Institute, British Columbia Women’s Hospital
and Health Centre, Vancouver, Canada.
4Faculty of Public Health and Policy, London School of Hygiene
and Tropical Medicine, London, United Kingdom.
5Department of Family and Community Medicine, University
of Toronto, Ontario, Canada.
6Department of Obstetrics, Gynecology and Reproductive
Sciences, University of Manitoba, Manitoba, Canada.
7Department of Obstetrics, Gynecology and Reproduction,
Universite´ Laval, Que´bec City, Canada.
*Co-first authors.
Abstract
Introduction: Telemedicine has the potential to improve
abortion access disparities in Canada. We aimed to explore
the provision of telemedicine for first-trimester medical abor-
tion and related barriers in 2019.
Methods: We conducted a national, cross-sectional, anon-
ymized, web-based survey of clinicians who provided abortion
care in 2019 in Canada. We distributed our survey through
professional health organizations to maximize identification
of possible eligible respondents and used a modified Dillman
technique to foster responses. Questions elicited provider
demographics, clinical characteristics, including telemedicine
first-trimester medical abortion and perceived related barri-
ers. Descriptive statistics were analyzed using R software.
Results: Among 465 respondents, 388 reported providing
first-trimester medical abortion across Canada; 44.0% rep-
orted experience using telemedicine for some components of
care: 49.3% of primary care clinicians and 28.7% of spe-


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cialists. Telemedicine was used for initial consultation
(86.0%), prescription (82.2%), or follow-up (92.2%). The
median percentage of telemedicine providers’ patients who
underwent a dating ultrasound was 90.0. The majority usu-
ally followed up with patients through quantitative human
chorionic gonadotropin (hCG) (84.2%). Seventy-eight percent
perceived barriers to telemedicine; the most common being
inability to confirm gestational age with ultrasound (43.0%),
and lack of provincial telemedicine abortion fee code to pay
practitioners (30.2%), timely access to serum hCG testing
(24.6%), and nearby emergency services (23.3%).
Discussion: In 2019, fewer than half of respondents reported
providing some aspects of first-trimester medical abortion
through telemedicine and the majority perceived barriers. Our
results can inform knowledge translation activities to reduce
barriers and increase telemedicine abortion care in Canada.
Keywords: telemedicine, abortion induced, surveys, ques-
tionnaires, Canada, health care, mifepristone
ª Regina M. Renner et al. 2022; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative
Commons Attribution Noncommercial License [CC-BY-NC] (http://creativecommons.org/licenses/by-nc/4.0/) which permits any non-
commercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are cited.
686 TELEMEDICINE and e-HEALTH
MARY ANN LIEBERT, INC.  VOL. 29
NO. 5  MAY 2023
DOI: 10.1089/tmj.2022.0245


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RESEARCH ARTICLE SUMMARY
◥
PROTEOMICS
Tissue-based map of the
human proteome
Mathias Uhlén,* Linn Fagerberg, Björn M. Hallström, Cecilia Lindskog, Per Oksvold,
Adil Mardinoglu, Åsa Sivertsson, Caroline Kampf, Evelina Sjöstedt, Anna Asplund,
IngMarie Olsson, Karolina Edlund, Emma Lundberg, Sanjay Navani,
Cristina Al-Khalili Szigyarto, Jacob Odeberg, Dijana Djureinovic,
Jenny Ottosson Takanen, Sophia Hober, Tove Alm, Per-Henrik Edqvist, Holger Berling,
Hanna Tegel, Jan Mulder, Johan Rockberg, Peter Nilsson, Jochen M. Schwenk,
Marica Hamsten, Kalle von Feilitzen, Mattias Forsberg, Lukas Persson,
Fredric Johansson, Martin Zwahlen, Gunnar von Heijne, Jens Nielsen, Fredrik Pontén
INTRODUCTION: Resolving the molecular
details of proteome variation in the different
tissues and organs of the human body would
greatly increase our knowledge of human bi-
ology and disease. Here, we present a map of
the human tissue proteome based on quantita-
tive transcriptomics on a tissue and organ level
combined with protein profiling using micro-
array-based immunohistochemistry to achieve
spatial localization of proteins down to the
single-cell level. We provide a global analysis
of the secreted and membrane
proteins, as well as an analysis
of the expression profiles for all
proteins targeted by pharmaceu-
tical drugs and proteins impli-
cated in cancer.
RATIONALE: We have used an
integrative omics approach to
study the spatial human pro-
teome. Samples representing all
major tissues and organs (n =
44) in the human body have been
analyzed based on 24,028 anti-
bodies corresponding to 16,975
protein-encoding genes, comple-
mented with RNA-sequencing
data for 32 of the tissues. The
antibodies have been used to
produce more than 13 million
tissue-based immunohistochemis-
try images, each annotated by
pathologists for all sampled tis-
sues. To facilitate integration
with other biological resources,
all data are available for down-
load and cross-referencing.
RESULTS: We report a genome-
wide analysis of the tissue


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specificity of RNA and protein
expression covering more than
90% of the putative protein-
coding genes, complemented with analyses
of various subproteomes, such as predicted
secreted proteins (n = 3171) and membrane-
bound proteins (n = 5570). The analysis shows
that almost half of the genes are expressed in
all analyzed tissues, which suggests that the
gene products are needed in all cells to main-
tain “housekeeping” functions such as cell
growth, energy generation, and basic metab-
olism. Furthermore, there is enrichment in
metabolism among these genes, as 60% of all
metabolic enzymes are expressed in all ana-
lyzed tissues. The largest number of tissue-
enriched genes is found in the testis, followed
by the brain and the liver.
Analysis of the 618 pro-
teins targeted by clinically
approved drugs unexpect-
edly showed that 30% are
expressed in all analyzed
tissues. An analysis of me-
tabolic activity based on genome-scale meta-
bolic models (GEMS) revealed liver as the most
metabolically active tissue, followed by adipose
tissue and skeletal muscle.
CONCLUSIONS: A freely available interactive
resource is presented as part of the Human
Protein Atlas portal (www.proteinatlas.org), of-
fering the possibility to explore the tissue-
elevated proteomes in tissues and organs and
to analyze tissue profiles for specific protein
classes. Comprehensive lists of proteins ex-
pressed at elevated levels in the different tis-
sues have been compiled to provide a spatial
context with localization of the proteins in the
subcompartments of each tissue and organ
down to the single-cell level.▪
RESEARCH
394
23 JANUARY 2015 • VOL 347 ISSUE 6220
sciencemag.org SCIENCE
ON OUR WEB SITE
◥
Read the full article
at http://dx.doi.
org/10.1126/
science.1260419
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The list of author affiliations is available in the full article online.
*Corresponding author. E-mail: mathias.uhlen@scilifelab.se
Cite this article as M. Uhlén et al., Science 347, 1260419
(2015). DOI: 10.1126/science.1260419


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The human tissue–enriched proteins. All tissue-enriched proteins are shown for 13 representative tissues or
groups of tissues, stratified according to their predicted subcellular localization. Enriched proteins are mainly
intracellular in testis, mainly membrane bound in brain and kidney, and mainly secreted in pancreas and liver.
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efficacy of bupropion and cognitive behavioral therapy in the treatment of methamphetamine use disorder


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REVIEW ARTICLE
Efﬁcacy of bupropion and cognitive behavioral therapy
in the treatment of methamphetamine use disorder:
a systematic review and meta-analysis
Luis Fernando Maya Apuy,0000-0000-0000-0000 Marie Antouannet Bernabe´ Barreto, Luis Alejandro Hurtado Merino
Facultad de Medicina, Universidad de Piura, Lima, Peru.
Objectives: We assessed the efﬁcacy of cognitive behavioral therapy and bupropion compared to
cognitive behavioral therapy alone for methamphetamine use disorder.
Methods: The selection criteria for this systematic review study with meta-analysis were randomized
clinical trials on the efﬁcacy of cognitive behavioral therapy and bupropion in the treatment for
methamphetamine use disorder (assessed by urine metabolites). The search was conducted in
PubMed, PubMed Central, LILACS, SciELO, Cochrane Library, SCOPUS, Google Scholar, Ovid
Medline, Clinicaltrials.gov, and the International Clinical Trials Registry Platform. The primary outcome
was relapse. Risk of bias was assessed with the RoB 2 tool. The results of each clinical trial were input
into an Excel spreadsheet. We performed a meta-analysis using relative risk and a 95%CI.
Results: Of the 597 initial articles (498 after removing duplicate records), ﬁve were included in the
meta-analysis, with an aggregate sample of 539 patients. An overall relative risk of 0.91 (95%CI 0.78-
1.05) was estimated for relapse.
Conclusion: Our study limitations included publication bias and heterogeneous populations. We
found no evidence that cognitive behavioral therapy and bupropion reduced the risk of relapse
compared to cognitive behavioral therapy and placebo.
Keywords: Substance-related
disorders;
antidepressive
agents,
second-generation;
cognitive
behavioral therapy; methamphetamine
Introduction
Methamphetamine is a highly stimulating derivative of
amphetamine with greater bioavailability in the central
nervous system (CNS).1 It includes the amino group of
(S)-amphetamine and bears a methyl substituent. Metham-


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phetamine is stronger than amphetamine because a
greater amount reaches the brain at the same dose.2 Its
mechanism of action consists in increasing monoamine
levels in the CNS, mainly dopamine, due to monoamine
oxidase enzyme inhibition and an additive effect on the
tyrosine hydroxylase enzyme. After use, a feeling of
euphoria, energy, and alertness is experienced.3
A 2020 survey by the U.S. National Institute on Drug
Abuse found a methamphetamine use disorder preva-
lence of 0.6%.4 According the United Nations Ofﬁce on
Drugs and Crime,5 the prevalence of amphetamine use
disorder was 2.31, 6.10, 5.65, and 2.31% in Australia
(2020), Haiti (2018), the United States (2020), and Austria
(2020), respectively.
Chronic methamphetamine use is associated with
medical
problems,
such
as
neuronal
damage
and
cognitive disorder, cardiovascular involvement (especially
strokes), dental disease, and infection (HIV, hepatitis B
virus, and hepatitis C virus). Methamphetamine use is
also linked to increased crime.6
The pathology of use disorder is associated with posi-
tive reinforcement and stimulation of the reward system,
in which dopamine plays an important role during initial
drug use. As use becomes more habitual, stimulation of
the dorsal striatal regions occurs.7 The dopaminergic
system exhibits modulatory effects on many brain regions
related to cognitive functioning, including front striatal and
limbic structures. This is associated with complaints of
cognitive dysfunction, including memory problems and
self-reported deﬁcits in everyday functioning.8 Moreover,
methamphetamine consumption can create a psycholo-
gical obsession due to drug-induced elation.9
The ﬁrst-line treatment for methamphetamine use
disorder is psychotherapy; there is no effective drug for
treatment.1 However, psychotherapy is not always avail-
able, which leads to a search for other treatment options.
In addition, the efﬁcacy of psychotherapy decreases after
discharge.10
Correspondence: Marie Antouannet Bernabe´ Barreto, San Felipe


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Avenue 1079/1804, Jesus Maria, Lima, 15072, Peru.
E-mail: marie.bernabe@alum.udep.edu.pe
Submitted Nov 28 2022, accepted Feb 13 2023.
How to cite this article: Apuy LFM, Barreto MAB, Merino LAH.
Efﬁcacy of bupropion and cognitive behavioral therapy in the
treatment of methamphetamine use disorder: a systematic review
and meta-analysis. Braz J Psychiatry. 2023;45:280-285. http://doi.
org/10.47626/1516-4446-2022-2979
Braz J Psychiatry. 2023 May-Jun;45(3):280-285
doi:10.47626/1516-4446-2022-2979
Brazilian Psychiatric Association
00000000-0002-7316-1185


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Nature | www.nature.com | 1
Article
Nasopharyngeal lymphatic plexus is a hub 
for cerebrospinal fluid drainage
Jin-Hui Yoon1,7, Hokyung Jin1,2,7, Hae Jin Kim3,7, Seon Pyo Hong1, Myung Jin Yang1,2, 
Ji Hoon Ahn1, Young-Chan Kim1,2, Jincheol Seo4, Yongjeon Lee4, Donald M. McDonald5,6, 
Michael J. Davis3,8 ✉ & Gou Young Koh1,2,8 ✉
Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long  
been known to drain through the lymphatics to cervical lymph nodes1–17, but the 
connections and regulation have been challenging to identify. Here, using fluorescent 
CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal 
lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This 
plexus had unusual valves and short lymphangions but no smooth-muscle coverage, 
whereas downstream deep cervical lymphatics had typical semilunar valves, long 
lymphangions and smooth muscle coverage that transported CSF to the deep cervical 
lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells 
regulated CSF drainage through the transport properties of deep cervical lymphatics. 
During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical 
lymphatics were not similarly altered, and CSF outflow could still be increased by 
adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in 
lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed 
increased type I interferon signalling and other inflammatory cytokines. The 
importance of evidence for the nasopharyngeal lymphatic plexus functioning as a 
CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant 
pharmacological activation of deep cervical lymphatic transport towards lymph 
nodes can still increase CSF outflow, offering an approach for augmenting CSF 
clearance in age-related neurological conditions in which greater efflux would be 
beneficial.


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CSF is essential for mechanical protection, nourishment and clearing 
of neurotransmitters, metabolites and protein aggregates such as 
amyloid-β and tau from the central nervous system17,19. CSF is continu-
ously secreted by the choroid plexus into the cerebral ventricles and 
subarachnoid space, circulates within and around the brain and spinal 
cord, and turns over 3 to 5 times per day19. The regulation of CSF produc-
tion, circulation and drainage is receiving increasing attention due to 
evidence that reduced CSF secretion and/or clearance during ageing 
could contribute to the development of Alzheimer’s disease and other 
neurodegenerative conditions20–23.
Among the known routes for CSF drainage from the subarach-
noid space are lymphatics in the cribriform plate, where olfactory 
nerves pass through the ethmoid bone, and in the perineurium of 
cranial nerves8,17,24. Lymphatics in the dura mater also function as a 
CSF drainage route9–17,25. However, despite solid documentation of the 
contribution of lymphatics to CSF clearance, the connections between 
the subarachnoid space and extracranial lymphatics involved in CSF  
clearance have been challenging to elucidate.
A landmark study1 provided evidence for CSF outflow to the naso-
pharynx by finding Richardson’s blue dye in the lymphatics of the nasal 
and palatal mucosa after injection of the tracer into the subarachnoid 
space1. Other studies4–7 revealed that dye or Microfil silicone rubber 
injected into the subarachnoid space subsequently appears in the nasal 
lymphatics and in the perineurium of cranial nerves. A previous study12 
reported that CSF can drain through lymphatics of the pharynx. A mag-
netic resonance imaging (MRI) study26 provided further evidence of 
CSF outflow through the cribriform plate to the nasopharynx. A recent 
study27 provided additional observations on the connections between 
the subarachnoid space and lymphatic vessels around olfactory nerves 
that cross the cribriform plate en route to the nasal mucosa. Another


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study16 observed connections between intracranial lymphatics and 
extracranial lymphatics using light-sheet fluorescence microscopy 
after injection of ovalbumin–Alexa Fluor 555 (OVA–Alexa Fluor 555) con-
jugate into CSF or by MRI after systemic injection of gadolinium-based 
contrast agent. However, tracing OVA–Alexa Fluor 555 in fixed tissues 
was confounded by phagocytosis of the tracer by macrophages. Our 
https://doi.org/10.1038/s41586-023-06899-4
Received: 28 March 2023
Accepted: 24 November 2023
Published online: xx xx xxxx
Open access
 Check for updates
1Center for Vascular Research, Institute for Basic Science, Daejeon, Republic of Korea. 2Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and 
Technology (KAIST), Daejeon, Republic of Korea. 3Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO, USA. 4National Primates Research Center, Korea 
Research Institute of Bioscience and Biotechnology, Cheongju, Republic of Korea. 5Department of Anatomy, Cardiovascular Research Institute, University of California, San Francisco, San 
Francisco, CA, USA. 6Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. 7These authors contributed equally: Jin-Hui Yoon, 
Hokyung Jin, Hae Jin Kim. 8These authors jointly supervised this work: Michael J. Davis, Gou Young Koh. ✉e-mail: davismj@health.missouri.edu; gykoh@kaist.ac.kr


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Unbiased transciprtion factor CRISPR screen identifies ANF800 as master repressor of enteroendocrine differentiation


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DIFFERENTIATION
Unbiased transcription factor CRISPR screen
identifies ZNF800 as master repressor of
enteroendocrine differentiation
Lin Lin1,2,3*, Jeff DeMartino2,3, Daisong Wang1,2, Gijs J. F. van Son2,3, Reinier van der Linden1,2,
Harry Begthel1,2, Jeroen Korving1,2, Amanda Andersson-Rolf1,2, Stieneke van den Brink1,2,
Carmen Lopez-Iglesias4, Willine J. van de Wetering4, Aleksandra Balwierz3, Thanasis Margaritis3,
Marc van de Wetering2,3, Peter J. Peters4, Jarno Drost2,3, Johan H. van Es1,2, Hans Clevers1,2,3*†
Enteroendocrine cells (EECs) are hormone-producing cells residing in the epithelium of stomach,
small intestine (SI), and colon. EECs regulate aspects of metabolic activity, including insulin levels,
satiety, gastrointestinal secretion, and motility. The generation of different EEC lineages is not
completely understood. In this work, we report a CRISPR knockout screen of the entire repertoire
of transcription factors (TFs) in adult human SI organoids to identify dominant TFs controlling
EEC differentiation. We discovered ZNF800 as a master repressor for endocrine lineage commitment,
which particularly restricts enterochromaffin cell differentiation by directly controlling an endocrine
TF network centered on PAX4. Thus, organoid models allow unbiased functional CRISPR screens
for genes that program cell fate.
E
nteroendocrine cells (EECs) are special-
ized epithelial cells of the intestinal tract
that, like all other epithelial cell lineages,
derive from regionally specified Lgr5+ in-
testinal stem cells (ISCs) (1–5). Balanced
differentiation of EEC lineages from ISCs is
governed by a network of transcription fac-
tors (TFs) (6–12). RNA sequencing and organ-
oid technology have illuminated the temporal
hierarchy of gene expression profiles during
EEC development (13, 14). By using a time-
resolved reporter allele of Neurog3 (master
regulator of endocrine development) (15, 16),
a real-time and lineage-specific map of mouse
EEC differentiation in vivo was constructed at


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single cell–level resolution (12). Overexpres-
sion of NEUROG3 can generate endocrine
cells for functional characterization in human
pancreatic duct cells (17). Leveraging human
gut organoids, this strategy enabled profiling
of EEC subtypes along the proximal-distal
gastrointestinal axis (18). TFs known to specify
EEC subtypes in mice were further examined in
human organoids, revealing a predominantly
conserved regulatory mechanism downstream
of NEUROG3 activation. However, discrepancies
between mouse and human data were also ob-
served. The regulatory mechanisms upstream
of NEUROG3 and the endogenous repressive
factors that control NEUROG3 expression at
the adult stage remain largely unknown. In this
work, we established an organoid-based plat-
form in combination with high-throughput ge-
netic screening for the unbiased discovery of TFs
that govern human EEC lineage commitment.
Results
TFome-wide CRISPR knockout screen in
human SI organoids
Optimized human SI organoids exhibit the
spontaneous generation of all major cell line-
ages, including EEC subtypes, without TF over-
expression (19). For the current study, we
aimed to establish a CRISPR screening platform
spanning the entire repertoire of transcription
factors (TFome) in human intestinal organoids
to allow for unbiased and systematic discov-
ery of TFs that govern cell lineage commitment
from adult LGR5+ intestinal stem cells. We
used a CRISPR knockout library comprising
7210 single-guide RNAs (sgRNAs) targeting
1800 human TFs, alongside 100 negative con-
trols (20–22). In the human ileum organoid
line used, EECs were marked by CHGA-IRES-
iRFP670, goblet cells by MUC2-mNeonGreen,
and Paneth cells by DEFA5-IRES-DsRed (fig.
S1A). We performed lentiviral-mediated library
integration in expanding SI organoids (Fig. 1A).
Library coverage was assessed by sequencing
the integrated sgRNAs, comparing the organ-
oids after transduction with the plasmid pool.
Fate separation of CHGA+ EECs and MUC2+
cells occurs early in secretory progenitors,


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whereas DEFA5+ Paneth cells emerge later from
MUC2+ progenitors (19), illustrating sequential
fate decisions of ISCs (23, 24). We aimed to
track the early CHGA+/MUC2+ binary fate
decision. After two-step organoid differentia-
tion (19), we analyzed bulk populations as well
as sorted EECs (CHGA+), goblet cells (MUC2+)
and the CHGA–/MUC2–/DEFA5– population
(fig. S1B). 0 to ~0.7% sgRNAs yielded zero
reads in bulk populations, indicating robust
coverage (fig. S2A). Normalized read-count
distribution and Pearson correlations demon-
strated high concordance between biological
replicates (fig. S2, B and C). sgRNAs that target
essential ISC genes [KLF5 (25) and TCF7L2/
TCF4 (26–29)] were depleted, along with
“generic” cell fitness genes (POLR2L, MYC,
and RAD51) at both expansion and differen-
tiation culturing stages (b score < –1; FDR <
0.05 versus plasmid library) (fig. S2D and table
S1). Furthermore, sgRNAs targeting IRF2, an
interferon pathway TF, were highly enriched
upon differentiation, consistent with existing
literature (30, 31).
sgRNAs were then assessed in CHGA+ EECs
and MUC2+ goblet cells versus the triple
reporter–negative cell population (Fig. 1B
and table S2). As expected, NEUROG3, SOX4,
and INSM1 sgRNAs were depleted in EECs
(8, 15, 16, 32). Additionally, NFIC, TEF, and
ZHX2 appeared essential for EEC commitment.
For each of these, clonal knockout organoids
revealed a mild yet significant reduction of
CHGA+ EECs (Fig. 1, B and C, and fig. S3, A to
C). Conversely, GFI1 sgRNAs were enriched in
EECs (Fig. 1, B and C). Mouse Gfi1 is crucial
for goblet cell differentiation by suppression
of Neurog3-driven EEC cell fate (23, 33).
Indeed, GFI1 knockout in human organoids
abrogated goblet cell formation while increas-
ing EEC numbers (fig. S3, A and C, and fig. S14,
C and D).
ZNF800 represses EEC differentiation
The strongest repressor of EEC differentiation
in the screen, ZNF800, is a C2H2 zinc-finger TF
of unknown function (Fig. 1, B and C) (34).
Human ZNF800 is broadly expressed, includ-


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ing in the SI and colon epithelium (fig. S4, A
and B). ZNF800−/− organoids contained in-
creased EECs and strongly reduced goblet and
Paneth cell numbers (Fig. 1, D and E, and fig.
S4, C and G). Transmission electron micros-
copy confirmed the absence of goblet and
Paneth cells with the characteristic apical
secretory vesicles and the increase of EECs
with the basolateral secretory granules (Fig. 1F).
Similarly, ZNF800 knockout in colon organoid
lines from two different donors also resulted
in a significant increase in EECs (fig. S4, D to G).
We next performed single-cell RNA sequenc-
ing (scRNA-seq) to study the phenotype of
ZNF800−/− organoids. For equal representa-
tion, CHGA+ cells were sorted from wild type
(WT) and ZNF800−/− organoids and pooled
with CHGA– cells from the same lines in a 1:4
ratio (Fig. 2A and fig. S5A). Major intestinal
cell types were identified by graph-based clus-
tering analysis (Fig. 2B and fig. S5B). We
observed the expected reduction of goblet cells
RESEARCH
1Hubrecht Institute, Royal Netherlands Academy of Arts and
Sciences (KNAW) and UMC Utrecht, Utrecht, Netherlands.
2Oncode Institute, Utrecht, Netherlands. 3Princess Maxima
Center for Pediatric Oncology, Utrecht, Netherlands. 4The
Maastricht Multimodal Molecular Imaging Institute,
Maastricht University, Maastricht, Netherlands.
*Corresponding author. Email: l.lin@hubrecht.eu (L.L.);
h.clevers@hubrecht.eu (H.C.)
†Current address: Pharma Research and Early Development of
F. Hoffmann–La Roche Ltd., Basel, Switzerland.
Lin et al., Science 382, 451–458 (2023)
27 October 2023
1 of 8
Downloaded from https://www.science.org at Tsinghua University on October 26, 2023


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Nature | www.nature.com | 1
Article
TNRC18 engages H3K9me3 to mediate 
silencing of endogenous retrotransposons
Shuai Zhao1,2,3,4,12, Jiuwei Lu5,12, Bo Pan1,2, Huitao Fan4,11, Stephanie D. Byrum6, Chenxi Xu1,2,3,4, 
Arum Kim1,2,4, Yiran Guo1,2,7, Krishna L. Kanchi4, Weida Gong4, Tongyu Sun8, Aaron J. Storey6, 
Nathaniel T. Burkholder9, Samuel G. Mackintosh6, Peyton C. Kuhlers10, Ricky D. Edmondson6, 
Brian D. Strahl4,9, Yarui Diao2,8, Alan J. Tackett6, Jesse R. Raab4,10, Ling Cai2,3,10, Jikui Song5,13 ✉ & 
Gang Greg Wang1,2,3,4,9,13 ✉
Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene 
repression and heterochromatin formation, cell-fate determination and organismal 
development1. H3K9me3 also provides an essential mechanism for silencing 
transposable elements1–4. However, previous studies have shown that canonical 
H3K9me3 readers (for example, HP1 (refs. 5–9) and MPP8 (refs. 10–12)) have limited 
roles in silencing endogenous retroviruses (ERVs), one of the main transposable 
element classes in the mammalian genome13. Here we report that trinucleotide- 
repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes 
H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14). 
Biochemical, biophysical and structural studies identified the carboxy-terminal 
bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3- 
specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for 
the direct recruitment of co-repressors such as HDAC–Sin3–NCoR complexes, thus 
enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis 
that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal 
death in mice and, in multiple mammalian cell models, led to derepressed expression 
of ERVs, which affected the landscape of cis-regulatory elements and, therefore, 
gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and


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regulatory pathway that operates to epigenetically silence evolutionarily young ERVs 
and exert substantial effects on host genome integrity, transcriptomic regulation, 
immunity and development.
Almost half of the human genome is made up of transposable elements 
(TEs), many of which are retrotransposons such as long terminal repeats 
(LTRs) and non-LTRs15–17. Non-LTRs include long interspersed elements 
(LINEs) and short interspersed elements (SINEs), whereas LTRs consist 
of ERVs. To maintain host genome integrity, TEs are tightly regulated 
by various silencing mechanisms enforced by histone modification, 
DNA methylation, histone chaperone and/or noncoding RNAs15,18. 
Notably, H3K9me3, a histone mark characteristic of constitutive het-
erochromatin, is enriched at TEs1,15. H3K9me3 is deposited by its writer 
enzymes such as SETDB1 (also known as ESET)1,19–21 and recognized by 
the H3K9me3-reading chromodomain harboured within heterochro-
matin protein 1 (HP1) family proteins5–9. SETDB1 loss causes global TE 
activation13; however, such a large TE-activation phenotype cannot be 
recapitulated by HP1 loss13, which indicates the existence of unknown 
H3K9me3-associated mechanisms for mediating TE repression. Chro-
modomain within M-phase phosphoprotein 8 (MPP8), a component 
of the human silencing hub (HUSH) complex2, was identified as an 
H3K9me3-specific reader10–12. However, depletion of the HUSH complex 
mainly caused LINE1 derepression, which demonstrates that MPP8–
HUSH has a major role in silencing non-LTR (LINE1) TEs2–4. It remains 
elusive what H3K9me3 reader mediates silencing of LTRs (ERVs), a 
TE class that occupies approximately 8% of the human genome and 
potentially carries gene regulatory functions15,16,18.
CasID identifies TNRC18 as an ERV binder
We sought to identify mammalian ERV regulators using CasID22 
(Extended Data Fig. 1a), in which a fusion protein of endonuclease- 
https://doi.org/10.1038/s41586-023-06688-z
Received: 25 July 2022
Accepted: 27 September 2023


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Published online: xx xx xxxx
 Check for updates
1Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA. 2Duke Cancer Institute, Duke University School of Medicine, Durham, NC, USA. 
3Department of Pathology, Duke University School of Medicine, Durham, NC, USA. 4Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, 
Chapel Hill, NC, USA. 5Department of Biochemistry, University of California, Riverside, CA, USA. 6Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 
Little Rock, AR, USA. 7Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 8Department of Cell Biology, Duke University School of 
Medicine, Durham, NC, USA. 9Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. 10Department of Genetics, 
University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA. 11Present address: The First Affiliated Hospital of Harbin Medical University, Harbin, P. R. China. 12These 
authors contributed equally: Shuai Zhao, Jiuwei Lu. 13These authors jointly supervised this work: Jikui Song and Gang Greg Wang. ✉e-mail: jikui.song@ucr.edu; greg.wang@duke.edu


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ABCG2 is an itaconate exporter that limits antibacterial innate immunity by alleviating TFEB-dependent lysosomal biogenesis


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Article
ABCG2 is an itaconate exporter that limits
antibacterial innate immunity by alleviating TFEB-
dependent lysosomal biogenesis
Graphical abstract
Highlights
d A custom-designed CRISPR screen identiﬁes ABCG2 as an
itaconate exporter
d ABCG2 directly exports itaconate depending on its ATPase
activity
d ABCG2-mediated itaconate export alleviates TFEB-
dependent lysosomal biogenesis
d ABCG2-mediated itaconate export limits antibacterial innate
immunity
Authors
Chao Chen, Zhenxing Zhang,
Caiyun Liu, Pengkai Sun, Ping Liu,
Xinjian Li
Correspondence
lixinjian@ibp.ac.cn
In brief
It has been reported that itaconate is a
lysosomal inducer that promotes
antibacterial innate immunity. Here, Chen
et al. reveal that ATP-binding cassette
transporter G2 (ABCG2) is an itaconate
exporter. ABCG2-mediated itaconate
export limits antibacterial innate
immunity by alleviating itaconate-
induced TFEB activation and lysosomal
biogenesis.
Chen et al., 2024, Cell Metabolism 36, 1–13
March 5, 2024 ª 2023 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2023.12.015
ll


