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https://openalex.org/W2947448204
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https://digital.csic.es/bitstream/10261/240118/1/Cucumber_Montes_PV_Art2019.pdf
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English
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Cucumber mosaic virus infection as a potential selective pressure on Arabidopsis thaliana populations
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PLOS pathogens
| 2,019
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cc-by
| 12,980
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Editor: Hui-Shan Guo, Institute of Microbiology,
CHINA Editor: Hui-Shan Guo, Institute of Microbiology,
CHINA Received: January 17, 2019
Accepted: May 1, 2019
Published: May 28, 2019
Copyright: © 2019 Montes 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. Copyright: © 2019 Montes 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. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: This work was funded by grants
BFU2015-64018-R (Plan Estatal de I+D+i, MINECO,
Spain) to FGA, and BIO2016-75754-P (Agencia
Estatal de Investigacio´n, Spain and FEDER, UE) to
CAB. NM was in receipt of a FPI contract (BES-
2009-026698) from MEC, Spain. The funders had
no role in study design, data collection and Nuria Montes1¤, Carlos Alonso-BlancoID2, Fernando Garcı´a-ArenalID1* Nuria Montes1¤, Carlos Alonso-BlancoID2, Fernando Garcı´a-ArenalID1* 1 Centro de Biotecnologı´a y Geno´mica de Plantas (UPM-INIA), and E.T.S.I. Agrono´mica, Alimentaria y de
Biosistemas, Campus de Montegancedo, Universidad Polite´cnica de Madrid, Pozuelo de Alarco´n (Madrid),
Spain, 2 Departamento de Gene´tica Molecular de Plantas, Centro Nacional de Biotecnologı´a, Consejo
Superior de Investigaciones Cientı´ficas (CNB-CSIC), Campus Universidad Auto´noma, Cantoblanco, Madrid,
Spain a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ¤ Current address: Fisiologı´a Vegetal, Departamento Ciencias Farmace´uticas y de la Salud, Facultad de
Farmacia, Universidad San Pablo-CEU, Boadilla del Monte (Madrid), Spain and Servicio de Reumatologı´a,
Hospital Universitario de la Princesa, Instituto de Investigacio´n Sanitaria (IIS-IP), Madrid, Spain. * fernando.garciaarenal@upm.es OPEN ACCESS It has been proposed that in wild ecosystems viruses are often plant mutualists, whereas
agroecosystems favour pathogenicity. We seek evidence for virus pathogenicity in wild eco-
systems through the analysis of plant-virus coevolution, which requires a negative effect of
infection on the host fitness. We focus on the interaction between Arabidopsis thaliana and
Cucumber mosaic virus (CMV), which is significant in nature. We studied the genetic diver-
sity of A. thaliana for two defence traits, resistance and tolerance, to CMV. A set of 185 indi-
viduals collected in 76 A. thaliana Iberian wild populations were inoculated with different
CMV strains. Resistance was estimated from the level of virus multiplication in infected
plants, and tolerance from the effect of infection on host progeny production. Resistance
and tolerance to CMV showed substantial genetic variation within and between host popula-
tions, and depended on the virus x host genotype interaction, two conditions for coevolution. Resistance and tolerance were co-occurring independent traits that have evolved indepen-
dently from related life-history traits involved in adaptation to climate. The comparison of the
genetic structure for resistance and tolerance with that for neutral traits (QST/FST analyses)
indicated that both defence traits are likely under uniform selection. These results strongly
suggest that CMV infection selects for defence on A. thaliana populations, and support
plant-virus coevolution. Thus, we propose that CMV infection reduces host fitness under the
field conditions of the wild A. thaliana populations studied. Citation: Montes N, Alonso-Blanco C, Garcı´a-
Arenal F (2019) Cucumber mosaic virus infection
as a potential selective pressure on Arabidopsis
thaliana populations. PLoS Pathog 15(5):
e1007810. https://doi.org/10.1371/journal. ppat.1007810 RESEARCH ARTICLE
Cucumber mosaic virus infection as a potential
selective pressure on Arabidopsis thaliana
populations RESEARCH ARTICLE Introduction It is commonly accepted that hosts and pathogens coevolve [1]. This concept rests on the
assumption that pathogens are virulent parasites, defining virulence as the negative impact of
infection on the host fitness. As a consequence, hosts will evolve defences to limit pathogen
infection, or to compensate for its costs [2]. In plants, the two major defences against patho-
gens are resistance, defined as the ability of the host to limit infection and/or parasite multipli-
cation and tolerance, which limits the fitness effect of a given parasite burden, i.e., specifically
decreases virulence [3,4]. As host defences may reduce the parasite’s fitness, hosts and parasites
may coevolve, coevolution being the process of reciprocally adaptive genetic change in two or
more species [1]. Evidence for host pathogen coevolution is not abundant. For plants it derives
mostly from studies of agroecosystems in which the pathogen evolves in response to the
deployment of resistance in the host population [5]. These studies have provided the bases for
theory on host-pathogen coevolution, including the gene-for-gene model of host-pathogen
interaction [6,7]. However, coevolution requires certain conditions to be met [1]: i) genetic
variation in the relevant host (e.g., resistance, tolerance) and pathogen (e.g., infectivity, viru-
lence) traits; ii) reciprocal effects of the relevant traits of the interaction on the fitness of host
and pathogen; iii) dependence of the outcome of the host-pathogen interaction on the combi-
nation of host and pathogen genotypes involved. These conditions must be analysed in wild
systems, in which the host may evolve in response to environmental pressures, including path-
ogen infection, at odds with agricultural systems. Evidence for plant-pathogen coevolution
from wild pathosystems is limited to a few instances, all involving fungal, oomycete or bacterial
pathogens [8,9]. To our knowledge, plant-virus coevolution has not been demonstrated in any wild system. In fact, it has been proposed that viruses often would be mutualistic symbionts, rather than
pathogens, in wild plant ecosystems [10–12], and it has been shown that virus infection may
be detrimental or positive for the host depending on the environment [13,14]. Hence the inter-
est in seeking evidence about whether viruses are plant pathogens in wild ecosystems and
viruses and plants co-evolve, or if virus virulence is the result of the specific conditions of
agroecosystems. analysis, decision to publish, or preparation of the
manuscript. analysis, decision to publish, or preparation of the
manuscript. ecosystems and, consequently, plants have evolved defences against virus infection. To
test this hypothesis, we studied the genetic diversity of Arabidopsis thaliana for two
defence traits, resistance and tolerance, to Cucumber mosaic virus (CMV) at a regional
scale in the Iberian Peninsula. Resistance and tolerance to CMV showed substantial
genetic variation within and between host populations, and depended on the virus x host
genotype interaction, two conditions for coevolution. Resistance and tolerance were inde-
pendent traits that co-occurred at the population and regional scales, and that have
evolved independently from other adaptive life-history traits. Analyses also indicated that
resistance and tolerance are likely under selection, most likely due to virus infection. These results support a hypothesis of plant-virus coevolution and contribute to demon-
strate that plant viruses may be virulent parasites of plants in wild ecosystems. Competing interests: The authors have declared
that no competing interests exist. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Author summary Plant-virus coevolution has not been demonstrated in any wild system, and it has been
proposed that viruses often would be mutualistic symbionts, rather than pathogens, in
wild plant ecosystems. We analyse if viruses are virulent pathogens of plants in wild 1 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations analysis, decision to publish, or preparation of the
manuscript. Virus infection as a selective pressure on Arabidopsis wild populations (CMV). A. thaliana is an annual semelparous species with two distinct developmental periods:
the vegetative growth period, producing a rosette of leaves, and the reproductive period in
which the inflorescence grows, new flowers are produced continuously, and older flowers
develop into siliques [24]. It is a cosmopolitan species, with a broad native distribution in Eur-
asia and Africa [25–27]. The Iberian Peninsula has been shown to contain the largest A. thali-
ana diversity in Eurasia due to its colonization from different refugia after the last glaciations
[26–29]. In Iberia, A. thaliana occurs in a variety of habitats, and substantial genetic variation
has been described, within and among populations, for relevant adaptive traits including phe-
nological traits like flowering time and seed dormancy [30–33]. Although wild populations of
A. thaliana have been shown to contain ample genetic and phenotypic diversity for responses
to herbivores and pathogens [34–38], the diversity for traits related to plant-virus interactions
has not been systematically analysed. CMV is an RNA virus with the broadest host range including about 1,200 species in more
than 100 plant families. CMV is horizontally transmitted by many species of aphids in a non-
persistent manner, and through the seed with efficiencies that depend on the genotypes of
CMV and the plant species [39]. In A. thaliana, seed transmission rates vary between 2 and 8%
[40, 41]. CMV isolates are highly diverse and have been classified into subgroups IA, IB and II,
based on the nucleotide sequence similarity of their genomic RNAs [39,42]. Analyses of the incidence of five viruses in six wild A. thaliana populations from central
Spain during 10 years showed that CMV was most prevalent, up to 80% according to popula-
tion and year [43,44], indicating that the A. thaliana–CMV interaction is significant in nature. As in other hosts monitored in the Iberian Peninsula, Subgroup IA isolates are most prevalent
[44–46]. Our group has analysed the role of resistance and tolerance in this interaction. The
infection of 21 wild genotypes of A. thaliana representing the variation of the species in Eur-
asia with three CMV strains, showed that quantitative resistance to CMV depended on the
interaction between host genotype x virus strain, and was a host trait with moderate to high
heritability [47]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Introduction Reports of negative effects of virus infection in wild plants in their natural
habitats are not abundant [e.g., [15–22] and indicate that effects may largely depend on site or
host population [23], but the genetic variation of defence and virulence has not been analysed
in these systems. To analyse plant-virus coevolution in wild ecosystems we have chosen the system Arabidop-
sis thaliana L. Heynh. (Brassicaceae)-Cucumber mosaic virus (Cucumovirus, Bromoviridae), PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 2 / 24 Virus infection as a selective pressure on Arabidopsis wild populations PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virulence, estimated as the effect of infection on viable seed production, did
not correlate with virus load, due to host genotype x virus strain-specific tolerance and, again,
tolerance was a host trait with moderate to high heritability [47]. Interestingly, tolerance was
positively correlated with the length of post-embryonic development (life span) of the host
genotypes [47], and was due, at least in part, to host life-history trait modification upon infec-
tion: long life span genotypes delayed flowering upon infection and re-allocated resources
from vegetative growth to reproduction, thus decreasing the effects of infection on progeny
production, i.e., attaining tolerance [48]. It remains to be shown that defence polymorphisms
result from the selection applied by CMV infection, and not by any other environmental factor
known to modulate plant developmental architecture and phenology, such as life span, flower-
ing time and plant size, which are known to have a role in adaptation of A. thaliana to the abi-
otic environment [49–51]. In this work we study the genetic diversity of A. thaliana for resistance and tolerance to
CMV at a regional scale in the Iberian Peninsula. To this end, we exploit a collection of 76 nat-
ural populations covering the wide ecological, environmental and genetic diversities of A. thaliana in this region [32,49]. We address the following questions: i) Which is the amount of
genetic diversity for resistance and tolerance to different CMV genotypes? ii) Are the geo-
graphic and environmental climatic patterns of resistance and tolerance similar or different
from those of related adaptive life history traits of A. thaliana? iii) Are resistance and tolerance
traits under natural selection? Addressing these questions is crucial to determine if CMV infec-
tion has a negative impact on its host fitness under natural field conditions and, consequently,
if there is coevolution between A. thaliana and CMV. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 3 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Table 1. Values (mean and range of variation), heritability and autocorrelation, of life history and defence traits to CMV in the Iberian population of A. thaliana. Autocorrelation
Traita
nb
Mean±SEc
Min-Maxd
h2
b
e
Moran's I f
Distanceg
RW
76
0.50±0.04
0.04–1.80
0.96
0.43–0.85
219.31
IW
76
1.83±0.06
0.66–3.48
0.71
0.41–0.89
204.69
SW
76
0.85±0.02
0.31–1.23
0.61
0.23–0.33
153.47
GP
76
117.72±0.80
103.40–140.71
0.92
0.43–0.59
191.75
LP
76
184.26±0.76
170.80–198.00
0.72
ns
ns
Resistance to Cdc-CMVh
76
19.28±1.37
0.56–56.28
0.81
ns
ns
Resistance to Lro-CMVh
76
7.53±0.71
0.26–27.83
0.78
ns
ns
Tolerance to Cdc-CMVi
76
0.35±0.02
0.03–0.77
0.70
ns
ns
Tolerance to Lro-CMVi
76
0.51±0.02
0.06–0.79
0.54
ns
ns ation), heritability and autocorrelation, of life history and defence traits to CMV in the Iberian population of A. thaliana a: Traits are: Rosette weight (RW), inflorescence without seed weight (IW) and viable seed weight (SW), expressed in g, and growth period (GP) and life span (LP),
expressed in days. b: number of individuals. c: Mean value and standard error of at least 5 replicated plants. c: Mean value and standard error of at least 5 replicated plants. e: Broad sense heritability expressed as percentage of genetic variation. e: Broad sense heritability expressed as percentage of genetic variation. f: Values of significant values of Moran's index (P<0.001). g: Geographic distance in km, showing significant values of Moran’s index. h: Resistance is expressed as virus accumulation (μg of virus RNA g fresh leaf weight-1). i: Tolerance is expressed as effect of infection on viable seed production (SWi/SWm). ns: non-significant. https://doi.org/10.1371/journal.ppat.1007810.t001 the variance (24.6% for A. thaliana genotype, 38.9% for virus isolate and 24.5% for their inter-
action). On average, Cdc-CMV accumulated to higher levels than Lro-CMV (19.28±1.37 and
7.53±0.71 μg virus RNA g fwt-1, respectively) (Fig 2 and Table 1). Virus accumulation in the
host plant showed high heritability for both CMV isolates, with an average of 0.8 (Table 1),
heritability being defined as the genetic component of the variance of the trait. Tolerance was estimated from the effect of virus infection on progeny production. Since
CMV infection does not affect seed viability or the weight of single seeds in a large number of
A. Genetic variation for resistance and tolerance to CMV in A. thaliana To estimate the genetic diversity for resistance and tolerance to CMV, 76 A. thaliana wild
genotypes collected in different natural populations from the Iberian Peninsula (Fig 1 and S1
Table) were assayed. We consider here a population as the set of A. thaliana plants growing in
a specific geographical site. Plants were inoculated after eight-week vernalisation, using two
CMV isolates from Iberian A. thaliana populations, Cdc-CMV and Lro-CMV. All 76 geno-
types were systemically infected by both CMV isolates, no immunity or hypersensitive resis-
tance reaction being detected. Resistance was estimated from the levels of virus multiplication, quantified as virus RNA
accumulation. RNA accumulation varied between 0.26 and 56.28 μg of virus RNA g fwt-1
(Table 1, Fig 2 and S2 and S3 Tables), variation significantly depending on the A. thaliana
genotype (F75,816 = 2.98, P<10−4), the virus isolate (F1,816 = 113.30, P<10−4) and the interac-
tion virus isolate x host genotype (F75,816 = 75.82, P<10−4), which together explained 88% of Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population locations. Circles with asterisk indicate the 12
populations used for within/between population analyses, their names appearing next to them. https://doi.org/10.1371/journal.ppat.1007810.g001 Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population loca
populations used for within/between population analyses, their names appearing next to them. Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population locations. Circles with asterisk indicate the 12
populations used for within/between population analyses, their names appearing next to them. Fig 1. Geographic distribution of Arabidopsis thaliana populations analysed in this study. Circles indicate population locations. Circles with asterisk indicate the 12
populations used for within/between population analyses, their names appearing next to them. https://doi.org/10.1371/journal.ppat.1007810.g001 https://doi.org/10.1371/journal.ppat.1007810.g001 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 4 / 24 thaliana genotypes [14,47,52], tolerance was estimated as the ratio of seed weight in infected
to mock-inoculated plants (SWi/SWm), which varied between 0.03 and 0.79 (Table 1, Fig 2, S2
and S3 Tables). As for resistance, tolerance significantly depended on A. thaliana genotype
(F76,816 = 3.10, P<10−4), virus isolate (F1,816 = 71.40, P<10−4) and their interaction (F75,816 =
5.18, P<10−4), which together explained 73% of the variance (26.9% for A. thaliana genotype,
25.0% for virus isolate and 21.2% for their interaction). Tolerance to Cdc-CMV (0.35±0.02) was
lower than tolerance to Lro-CMV (0.51±0.02) (Fig 2 and Table 1). Tolerance in the host showed
medium to high heritability, between 0.70 and 0.54 for Cdc-CMV and Lro-CMV, respectively. No significant relationship was detected between virus RNA accumulation and SWi/SWm
across genotypes nor within genotypes (r-0.11, P0.358). Together, results show that natural
populations of A. thaliana contain substantial but independent genetic variation for resistance
and tolerance to CMV. scence without seed weight (IW) and viable seed weight (SW), expressed in g, and growth period (GP) and life span (LP), https://doi.org/10.1371/journal.ppat.1007810.t001 a: Traits are: Rosette weight (RW), inflorescence without seed weight (IW) and viable seed weight (SW), expressed in g, and growth period (GP) and life span (LP),
expressed in days PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Genetic diversity and geographic or climatic patterns of life history traits in
A. thaliana Tolerance to CMV in A. thaliana is related to the host life history traits, as tolerance is due in
part to a reallocation of resources from growth to reproduction, which depends on the allome-
try of the vegetative to reproduction organs, (SW+IW)/RW [48]. In the 76 wild genotypes,
resource reallocation upon infection by both CMV isolates also depended on plant allometry,
being more efficient in genotypes with lower (SW+IW)/RW (F1,76>14.58, P<10−4), and the
effect of infection by both CMV isolates on RW correlated positively with LP and RW of
mock-inoculated plants (r0.29, P0.022). Neither resistance nor tolerance correlated with
viable seed production of mock-inoculated controls (r0.02, P0.540). We then analysed several life history traits related with growth and phenology in eight-week
vernalised mock-inoculated plants of the 76 wild genotypes. Rosette weight (RW), inflorescence
without seeds weight (IW) and seed weight (SW), growth period (GP) and life-span (LP) signifi-
cantly differed between genotypes (F75,3999.23, P<10−4) (S2 and S3 Tables). Heritability of
these traits was high, between 0.61 and 0.96 (Table 1). Overall, A. thaliana populations display a
large genetic variation for the analysed life history traits, as in previous works [33,49,50]. g g
y
y
p
Rosette weight (RW), inflorescence without seeds weight (IW) and seed weight (SW) signif-
icantly differed between genotypes (F75,3999.23, P<10−4) (S2 Table). To determine if life his-
tory traits related to tolerance showed similar or different geographic patterns than CMV
defences, we analysed their autocorrelation. All growth and phenological traits showed signifi-
cant spatial autocorrelation up to 153 km (Table 1). Furthermore, we analysed the relationship
between these life history traits and climate using Dutilleul’s t-test, univariate SAR models and
Mantel tests (S4 Table). Overall, RW, IW, SW and GP, but not LP, were positively correlated
with altitude and negatively correlated with most climatic variables, including annual mean,
minimal and maximal temperature, and precipitation seasonality (Fig 3 and S4 Table). These
analyses showed A. thaliana genotypes from higher altitude, lower temperatures and higher
precipitation seasonality flowered later, developed larger rosettes and inflorescences and pro-
duced more seeds. Moreover, Mantel tests showed that genetic distance was positively corre-
lated with IW, SW, GP (r>0.15, P<0.008) and marginally with RW (r = 0.08, P = 0.078) but
not with LP (r<-0.02, P>0.700). In contrast to CMV defence traits, life history traits related with resource allocation vary
according to the climatic environment where populations evolved. Virus infection as a selective pressure on Arabidopsis wild populations Finally, we analysed if there is a relationship between the genetic diversity for CMV defence
traits and the overall genetic diversity of the A. thaliana wild genotypes estimated from neutral
markers (250 SNPs). Mantel tests between pair-wise genetic distances estimated from neutral
markers and pair-wise differences for virus RNA accumulation or SWi/SWm did not detect
any significant correlation in relation to Cdc-CMV or Lro-CMV (r-0.04, P0.403). Genetic diversity and geographic or climatic patterns of life history traits in
A. thaliana Therefore, resistance and
tolerance to CMV show different evolutionary histories than life history traits, likely reflecting
distinct abiotic and biotic environmental selective forces acting on each group of traits. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Geographic and climatic patterns of A. thaliana resistance and tolerance to
CMV To determine if there is a geographic pattern for the genetic diversity for CMV resistance or
tolerance, we first analysed the spatial autocorrelation of both variables. Neither virus 5 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations accumulation nor SWi/SWm showed significant spatial autocorrelation at any geographic scale
Fig 2. Variation for resistance and tolerance to CMV in A. thaliana. Frequency distributions are for accumulation (μg RNA g fresh leaf
weight-1) of (A) Cdc-CMV and (B) Lro-CMV RNA, and of the effect of infection by (C) Cdc-CMV and (D) to Lro-CMV on seed production. https://doi.org/10.1371/journal.ppat.1007810.g002 Fig 2. Variation for resistance and tolerance to CMV in A. thaliana. Frequency distributions are for accumulation (μg RNA g fresh leaf
weight-1) of (A) Cdc-CMV and (B) Lro-CMV RNA, and of the effect of infection by (C) Cdc-CMV and (D) to Lro-CMV on seed production. Fig 2. Variation for resistance and tolerance to CMV in A. thaliana. Frequency distributions are for accumulation (μg
weight-1) of (A) Cdc-CMV and (B) Lro-CMV RNA, and of the effect of infection by (C) Cdc-CMV and (D) to Lro-CMV accumulation nor SWi/SWm showed significant spatial autocorrelation at any geographic scale
(P>0.050) (Table 1). Second, to find if these defence traits might be associated with the climate, we analysed
their relationship with climatic variables from the original local populations. Neither the accu-
mulation of any of the two CMV isolates, nor the SWi/SWm ratio in plants infected by any of
them, significantly correlated with any of the analysed abiotic variables (see Methods and S4
Table) according to Dutilleul’s t-tests (r-0.30, P0.039), SARs (F7.185, P0.009), or Man-
tel tests (r-0.12, P0.019) (S4 Table). 6 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 A. thaliana population differentiation for resistance and tolerance to CMV To quantify the distribution of genetic diversity for CMV defence traits within and among A. thaliana populations we analysed ten randomly sampled individual plants (henceforth named
as “individuals”) from 10 or 12 Iberian populations, which were tested for their resistance and
tolerance to two CMV isolates. One isolate from an Iberian population of A. thaliana (Cdc-
CMV) and a reference isolate (Fny-CMV) were chosen because they had been used in previous
work [43,47,48]. Since CMV resistance and tolerance depend on the environment [14,48,53], PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 7 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Virus infection as a selective pressure on Arabidopsis wild populations Fig 3. Relationships between life-history traits and geographic or climatic factors. Correlations are shown (A, B, C) for rosette weight (RW), (D, E, F)
inflorescence weight (IW), (G, H, I) seed weight (SW) and (J, K, L) growth period (GP) with altitude, annual mean temperature and precipitation
seasonality. Values are means of at least five replicates per plant genotype. Fig 3. Relationships between life-history traits and geographic or climatic factors. Correlations are shown (A, B, C) for rosette weight (RW), (D, E, F)
inflorescence weight (IW), (G, H, I) seed weight (SW) and (J, K, L) growth period (GP) with altitude, annual mean temperature and precipitation
seasonality. Values are means of at least five replicates per plant genotype. Fig 3. Relationships between life-history traits and geographic or climatic factors. Correlations are shown (A, B, C) for rosette weight (RW), (D, E, F)
inflorescence weight (IW), (G, H, I) seed weight (SW) and (J, K, L) growth period (GP) with altitude, annual mean temperature and precipitation
seasonality. Values are means of at least five replicates per plant genotype. https://doi.org/10.1371/journal.ppat.1007810.g003 two experiments were performed with different vernalisation period lengths, as vernalisation
affects life history traits relevant to tolerance such as rosette size, rosette leaf number, flowering
time [33,49,54], and seed germination [55]. An eight-week vernalisation treatment simulated a
cold winter, whereas a four-week vernalisation simulated a mild winter, as often occur across
years in the original population locations. In both experiments, all individuals were systemi-
cally infected by both CMV isolates. The two experiments yielded similar results. For clarity
only results of the long vernalisation treatment experiment are presented in the text, but results
of the short-vernalisation are shown in S5 Table. Virus accumulation varied considerably among individuals within populations (Fig 4,
Table 2 and S6 Table). The average virus accumulation in each population ranged from 2.62 to
32.02 μg virus RNA g fwt-1. The heritability of virus accumulation varied between 0.23 and 0.90
depending on CMV isolate and host population (Table 2). Virus accumulation significantly
depended on the virus isolate (F1,884 = 67.88, P<104), the A. thaliana population (F9,884 = 2.40,
P = 0.059) (Fig 4 and Table 2), the A. thaliana individual nested to population (F88,884 = 2.54,
P<10-4), and on the interactions CMV isolate x A. thaliana population (F9,884 = 4.11, P<10-4)
and CMV isolate x A. thaliana individual nested to population (F88,884 = 7.08, P<10-4). CMV
isolate, A. thaliana individual nested to population and their interaction explained 49.7, 3.3 and
14.6% of the variance, respectively, while A. thaliana population and the interaction CMV iso-
late x population explained 7.0 and 5.5%. The average accumulation was higher for Cdc-CMV
than for Fny-CMV (18.61±2.76 and 7.37±1.74 μg virus RNA g fwt-1, respectively). Besides, aver-
age values of Cdc-CMV accumulation over individuals and populations correlated significantly,
or marginally, with the corresponding values of Fny-CMV accumulation (rs = 0.32, P = 0.001;
rs = 0.60, P = 0.067, respectively), indicating that, in general, individuals and populations that
were more resistant to Cdc-CMV were also more resistant to Fny-CMV. CMV tolerance also showed substantial variation among individuals within populations,
SWi/SWm values ranging between 0.02 and 0.93 for Cdc-CMV, and between 0.03 and 0.81, for
Fny-CMV-infected plants (Fig 4, Table 2 and S6 Table). Average SWi/SWm values in each pop-
ulation varied from 0.16 to 0.47 (Fig 4 and Table 2), indicating a lower range of variation
among than within populations. Heritability of tolerance varied between 0.10 and 0.80
depending on CMV isolate and host population (Table 2). SWi/SWm varied significantly
depending on virus isolate (F1,884 = 5.30, P = 0.046), A. thaliana population (F9,884 = 2.40,
P = 0.059) (Fig 4 and Table 2), A. thaliana individual nested within population (F88,884 = 2.445,
P = 0.023) and the interaction CMV isolate x A. thaliana individual (F88,884 = 2.65, P<10−4). A. thaliana individual and the interaction CMV isolate x A. thaliana individual explained a larger
proportion of SWi/SWm variance (32.2%, 16.1%, respectively) than CMV isolate or A. thaliana
population (1.6%, 6.4%, respectively). When averaged over all individuals, tolerance to Cdc-
CMV (0.36±0.06) and Fny-CMV (0.32±0.06) were similar. Average values of SWi/SWm in
Cdc-CMV-infected plants correlated across individuals and populations with those of Fny-
CMV-infected plants (rs = 0.73, P = 0.016; rs = 0.60, P<10−4, respectively). Thus, as for resis-
tance, the individuals and populations more tolerant to Cdc-CMV were also, in general, more
tolerant to Fny-CMV. However, values of virus accumulation and SWi/SWm did not correlate
over individuals for any CMV isolate (rs0.09, P0.129). Together, these results indicate that
A. thaliana defences against CMV infection depend on the host genetic variation determining
the specific defences. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations Virus infection as a selective pressure on Arabidopsis wild populations PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 8 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 9 / 24 Virus infection as a selective pressure on Arabidopsis wild populations PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019
10 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 10 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Fig 4. Variation for resistance and tolerance to CMV within and among wild A. thaliana populations. Frequency
distributions of resistance (virus accumulation, μg RNA g fresh leaf weight-1) and tolerance (SWi/SWm) to Cdc-CMV
(blue) and Fny-CMV (red). Three-letter code for each population is as in Fig 1. Fig 4. Variation for resistance and tolerance to CMV within and among wild A. thaliana populations. Frequency
distributions of resistance (virus accumulation, μg RNA g fresh leaf weight-1) and tolerance (SWi/SWm) to Cdc-CMV
(blue) and Fny-CMV (red). Three-letter code for each population is as in Fig 1. https://doi.org/10.1371/journal.ppat.1007810.g004 https://doi.org/10.1371/journal.ppat.1007810.g004 Comparison of genetic differentiation among A. thaliana populations
between quantitative traits and neutrals markers To find out if CMV defence traits of A. thaliana might be under selection, we compared the
genetic differentiation among populations for CMV resistance and tolerance, with that for
neutral genetic variation (Fig 5). Two-hundred and fifty genome-wide SNPs, distinguished 74
different genotypes among the 120 individuals analysed (S7 Table) and were used to calculate
FST values. As in the previous section, analyses are based on the results of the long vernalisation
treatment experiment, analyses based on the short-vernalisation treatments gave similar
results and are shown in S5 Table. The estimated average genetic differentiation among populations for neutral markers was
0.58 (95%CI = 0.54–0.62), which is presumed to reflect the demographic history of the popula-
tions. Genetic differentiation among populations for quantitative traits was estimated by QST
values, leading to a similar average value of 0.31 (95%CI = 0.22–0.52) for accumulation of both
CMV isolates. The average QST estimated for SWi/SWm was 0.18 (95%CI = 0.12–0.35) for Cdc-
CMV and 0.10 (95%CI = 0.06–0.21) for Fny-CMV-infected plants. Therefore, the genetic vari-
ation of A. thaliana for both defence traits is distributed mostly within populations. QST values
for resistance and tolerance to Cdc-CMV and Fny-CMV were significantly smaller than FST
values (Fig 5), thus indicating that A. thaliana populations are genetically less differentiated
for resistance and tolerance to two CMV isolates than for neutral markers. Furthermore, we analysed if FST and QST values followed a pattern of isolation-by-distance. Mantel tests detected a significant correlation between FST values and geographic distance
between pairs of populations (r = 0.50, P<10−4), a likely result of their demographic history. By contrast, no significant correlation was found between the pair-wise QST values for virus
accumulation or SWi/SWm, for any CMV isolate, and their geographic distances (r0.24,
P0.258). Therefore, factors other than demography contribute to the population differentia-
tion patterns observed for resistance and tolerance to CMV. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Discussion Host-pathogen coevolution determines the dynamics and genetics of infectious disease, and
may shape the genetic structure of host and pathogen populations [1]. Understanding this
topic, central in pathology and evolutionary biology, requires knowledge on the genetics of
defence and pathogenicity, and the dynamics of their change in populations [1]. The abun-
dance of theoretical analyses of host-pathogen coevolution (e.g., [1,56,57]) is not matched by a
similar amount of empirical and experimental studies. While there is abundant information
compatible with host-pathogen coevolution in plant systems (e.g. [58]), it mostly derives from
crops, in which the genetic composition of the host plant is manipulated by humans. Studies
from wild systems, in which the genetic composition of host and pathogen populations may be
determined by reciprocal selection, are much scarcer, particularly for plant-virus interactions
[5,59]. To address this question we challenged A. thaliana individuals collected from a high
number of local populations with different CMV isolates. We chose to inoculate plants
mechanically rather than by aphid transmission, which is the natural means of horizontal
transmission [39]. Mechanical inoculation ensures a high rate of infection and minimises inoc-
ulum dose effects on virus accumulation, as opposed to aphid transmission, which is highly PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 11 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations Table 2. Values (mean and range of variation) and heritability of defence traits to CMV in Iberian populations of A. thalianaa. Population
Resistance to Cdc-CMVb
Resistance to Fny-CMVb
Tolerance to Cdc-CMVc
Tolerance to Fny-CMVc
Agu
Mean±SEd
32.02±3.64
11.85±2.84
0.43±0.07
0.36±0.07
Min-Maxe
7.98–45.64
2.16–26.11
0.14–0.78
0.08–0.74
nf
10
10
10
10
h2
b
g
0.74
0.90
0.49
0.44
Bis
Mean±SEd
17.05±2.31
10.91±1.11
0.34±0.04
0.37±0.07
Min-Maxe
9.37–30.04
3.31–14.14
0.12–0.48
0.03–0.71
nf
10
10
10
10
h2
b
g
0.74
0.58
0.38
0.8
Gra
Mean±SEd
15.77±1.68
6.24±1.15
0.40±0.07
0.26±0.05
Min-Maxe
6.69–24.45
2.42–13.97
0.12–0.84
0.04–0.54
nf
10
10
10
10
h2
b
g
0.66
0.68
0.64
0.741
Leo
Mean±SEd
22.36±1.90
9.48±1.40
0.32±0.07
0.22±0.05
Min-Maxe
15.45–32.05
3.59–16.29
0.05–0.74
0.03–0.46
nf
10
10
10
10
h2
b
g
0.62
0.79
0.61
0.77
Mer
Mean±SEd
17.58±2.56
11.55±2.35
0.16±0.04
0.19±0.06
Min-Maxe
5.37–36.76
3.89–24.15
0.04–0.46
0.05–0.67
nf
10
10
10
10
h2
b
g
0.79
0.89
0.32
0.67
Moc
Mean±SEd
15.49±2.25
5.15±0.43
0.35±0.04
0.32±0.05
Min-Maxe
4.08–24.43
2.92–7.07
0.13–0.49
0.12–0.57
nf
10
10
10
10
h2
b
g
0.71
0.34
0.27
0.47
Pob
Mean±SEd
14.91±1.71
2.62±0.60
0.36±0.07
0.33±0.05
Min-Maxe
5.33–22.55
0.94–7.01
0.02–0.59
0.07–0.57
nf
9
9
9
9
h2
b
g
0.52
0.23
0.74
0.10
Pra
Mean±SEd
15.04±2.15
4.78±0.93
0.47±0.09
0.41±0.07
Min-Maxe
5.19–24.98
1.99–10.26
0.06–0.93
0.04–0.68
nf
9
9
9
9
h2
b
g
0.635
0.63
0.55
0.66
Qui
Mean±SEd
15.23±2.34
6.21±0.78
0.47±0.06
0.46±0.06
Min-Maxe
6.26–27.61
3.87–11.85
0.20–0.69
0.24–0.81
nf
10
10
10
10
h2
b
g
0.68
0.34
0.64
0.52
San
Mean±SEd
20.00±2.48
4.16±1.46
0.36±0.06
0.32±0.05
Min-Maxe
11.38–38.89
0.08–12.18
0.11–0.66
0.10–0.57
nf
10
10
10
10
h2
b
g
0.69
0.81
0.47
0.32
Average of populations
Mean±SE
18.53±1.65
7.25±0.30
0.37±1.09
0.31±0.02
Min-Max
14.91–32.02
2.62–11.85
0.16–0.47
0.22–0.46
h2
b
g
0.60
0.62
0.51
0.55
(Continued) Values (mean and range of variation) and heritability of defence traits to CMV in Iberian populations of A. thalianaa. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 12 / 24 Virus infection as a selective pressure on Arabidopsis wild populations Table 2. (Continued)
Population
Resistance to Cdc-CMVb
Resistance to Fny-CMVb
Tolerance to Cdc-CMVc
Tolerance to Fny-CMVc
Over populations
h2
b
g
0.77
0.85
0.57
0.55
a: Values are for plants inoculated after an eight-week vernalisation period. b: Resistance is expressed as virus accumulation (μg of virus RNA g fresh leaf weight-1). c: Tolerance is expressed as effect of infection on viable seed production (SWi/SWm). d: Mean ± SE: Mean value and standard error (SE) of at least 5 replicated plants. e: Minimum and maximum values of the trait. f: number of individuals
g: Broad sense heritability expressed as percentage of genetic variation. https://doi.org/10.1371/journal.ppat.1007810.t002 inefficient for CMV [39, 40]. Moreover, there is no information on the aphid species that
transmit CMV in A. thaliana populations in central Spain. Also, A. thaliana genotypes were
assayed under common controlled conditions, which are not necessarily the same as in the
field. The challenge with different CMV strains of 185 individuals collected in 76 A. thaliana
local populations from the Iberian Peninsula, showed large differences in quantitative resis-
tance, as estimated from the level of virus multiplication in infected plants. Similarly, large dif-
ferences were found for tolerance to CMV, estimated as the effect of infection on host progeny
production. A large part of the variation of resistance and tolerance was explained by the virus
isolate, in agreement with previous results showing that CMV isolates vary largely in multipli-
cation rate and virulence in A. thaliana [41,47,48,53]. Variation for resistance and tolerance Fig 5. Genetic differentiation among wild A. thaliana populations for CMV resistance and tolerance traits, or for
neutral markers. Fig shows values of quantitative genetic differentiation (QST) for resistance (squares) and tolerance
(triangles), to Cdc-CMV (blue) or Fny-CMV (red), and for neutral genetic differentiation (FST) (black circle), among ten
A. thaliana populations. 95%CI are indicated. https://doi.org/10.1371/journal.ppat.1007810.g005 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations occurred at all analysed spatial scales: among individuals from a local population, among local
populations, and within the whole Iberian Peninsula region. These conclusions held for assays
conducted in different environments, a result to be underscored, as resistance and tolerance of
A. thaliana to CMV can be modulated by the abiotic environment [14]. The observed variation
in resistance and tolerance had a significant genetic component, as they showed medium to
high heritability values (0.23–0.81 for resistance, and 0.10–0.70 for tolerance) depending on
the spatial scale of the analysis and the isolate of CMV. Thus, our results show genetic variation
for presumably defence traits in the host population, a condition for host-pathogen coevolu-
tion. Regardless of spatial scales, our data also show that resistance and tolerance significantly
depend on the interactions between virus genotype and host genotype, another condition for
host-pathogen coevolution. It has been reported that A. thaliana genotypes showing high tolerance to CMV have a long
life span, and that tolerance is, at least in part, the result of resource re-allocation from vegeta-
tive growth to reproduction, which is more efficient in long-lived genotypes [14,47,48,52]. It
also has been shown that these life history and phenological traits have evolved as (direct or
indirect) responses to climatic conditions [33,49,50,60]. Accordingly, the analysis of 76 A. thaliana genotypes from different Iberian populations showed that resource reallocation upon
infection depended on the allometric ratio (SW+IW)/RW, and the effect of CMV infection on
vegetative growth correlated positively with LP and RW of mock-inoculated plants. Life span,
vegetative growth and seed production in non-infected plants were correlated with climatic
variables. In contrast, the genetic variation for CMV multiplication in the infected host, and
for the effect of CMV infection on seed production, was unrelated to those climatic factors. Therefore, the CMV defence traits have evolved, at least partly, independently from those
other adaptive traits which have evolved in response to climate. Accordingly, the evolution of
defence traits is not the result of A. thaliana responses to climate. These differential evolution-
ary histories strongly suggest that these traits are true resistance and tolerance defence
responses that may have evolved in response to CMV infection (see below). This conclusion
also agrees with the fact that resistance and tolerance are virus-specific traits of A. thaliana,
and not unspecific responses to the stress of virus infection [52]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Fig 5. Genetic differentiation among wild A. thaliana populations for CMV resistance and tolerance traits, or for
neutral markers. Fig shows values of quantitative genetic differentiation (QST) for resistance (squares) and tolerance
(triangles), to Cdc-CMV (blue) or Fny-CMV (red), and for neutral genetic differentiation (FST) (black circle), among ten
A. thaliana populations. 95%CI are indicated. Fig 5. Genetic differentiation among wild A. thaliana populations for CMV resistance and tolerance traits, or for
neutral markers. Fig shows values of quantitative genetic differentiation (QST) for resistance (squares) and tolerance
(triangles), to Cdc-CMV (blue) or Fny-CMV (red), and for neutral genetic differentiation (FST) (black circle), among ten
A. thaliana populations. 95%CI are indicated. https://doi.org/10.1371/journal.ppat.1007810.g005 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 13 / 24 Virus infection as a selective pressure on Arabidopsis wild populations CMV defence traits is that CMV infection plays a role as selective factor. Although we cannot
discard that the analysed defence traits may have evolved in response to selection from other
pathogens or pests, several arguments make a strong case for selection pressure due to CMV
infection: i) the high prevalence of CMV (up to 80%) in wild A. thaliana populations in the Ibe-
rian peninsula [43,44]; ii) the fact that the analysed defence traits are virus-specific in A. thaliana
[52]; and iii) the lack of correlation between CMV multiplication and virulence [47], virus mul-
tiplication being highly dependent on virus genotype and environment [14,47]. Selection for
resistance to pathogens has been best documented in populations of Linum marginale in
response to the fungus Melampsora lini, of Plantago lanceolata in response to the fungus Podo-
sphaera plantaginis, or of A. thaliana in response to the oomycete Hyaloperonospora arabidopsi-
dis or the bacterium Pseudomonas syringae [37,68,69,72–74,78,82,83]. Our results extend these
observations to plant-virus interactions. If defence in A. thaliana against CMV is under selec-
tion, a corollary is that CMV is a virulent pathogen of this plant under natural conditions, a rele-
vant conclusion that contributes significantly to understanding plant-virus interactions. Th
b
d
f
d
f
d
l
h h g
y
g p
The observed pattern of genetic diversity for resistance and tolerance to CMV, higher
within than between populations, can be explained by the features of the pathosystem. CMV is
ubiquitous in Iberia [45,84] and has been found in all monitored wild populations of A. thali-
ana, prevalence differing among populations and years [43]. Also, our present and past results
[47,48] show that CMV isolates differ in virulence to A. thaliana, and that virulence is modu-
lated by environmental factors as diverse as temperature, light intensity or host plant density
[14,53].Variation in the genetic composition of CMV populations would also result in varia-
tion for CMV infection-associated selection, as the outcome of the interaction depends on the
A. thaliana and CMV genotypes involved. These factors would explain the maintenance of
genetic variation in defence traits within host populations and the limited differentiation
among populations. Furthermore, this explanation suggests that resistance and tolerance to
CMV involve fitness penalties for A. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Thus, our study shows sub-
stantial genetic variation for resistance and tolerance to CMV within and between populations
of A. thaliana. Genetic variation within or/and between populations for resistance to a variety
of pathogens has been reported for a limited number of wild plants, including Amphicarpaea
bracteata, Eucalyptus globulus, Podophyllum peltatum, Linum marginale, Silene latifolia, Pha-
seolus vulgaris, Plantago laceolata or A. thaliana [61–78]. Analyses of the variation for plant
tolerance to pathogens are much rarer [47,75,79], and none of them has analysed within popu-
lation variation. All these studies refer to resistance or tolerance to fungi, oomycetes or bacte-
ria, and the only report we are aware of on variation for resistance to a virus, is our previous
analysis in the A. thaliana-CMV system [43], which involved a much more limited sample of
host populations. The analyses in this study also showed that resistance and tolerance display different evolu-
tionary histories than neutral genetic variation, since QST values for resistance and tolerance
are lower than FST values. Accordingly, resistance and tolerance are traits likely under uniform
selection, i.e., a selection which favours a higher diversity of traits within than among popula-
tions. This conclusion is supported by results for two CMV isolates and in different environ-
mental conditions. Also, neutral genetic differentiation follows a pattern of isolation by
distance, which is not detected for the genetic differentiation for resistance or tolerance. By
contrast, similar analyses have previously suggested diversifying selection on the genetic varia-
tion for quantitative traits such as flowering time, leaf number, specific leaf area or leaf succu-
lence in A. thaliana [32,80,81]. The most parsimonious explanation for uniform selection on PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 14 / 24 Plant material and environmental data Two different sets of A. thaliana samples from the Iberian Peninsula were analysed. First, 76
accessions or wild genotypes collected from different populations were selected to cover the
genetic and environmental diversity of the species in that region [30,49] (Fig 1 and S1 Table). This collection spanned 800 x 700 km, populations being spaced in the average 384.9±3.7
(20.2–1,038.1 km). Altitudes ranged from 123 to 1,670 m above sea level. Each sample was
genetically different based on previous SNP genotyping and genome sequences [51,96]. Sec-
ond, ten individuals plants (individuals) randomly sampled from 12 of these populations were
selected for intra and inter-population analyses (Fig 1 and S1 Table). Samples from eight of
these populations have been previously genotyped for 250 genome-wide SNPs that were segre-
gating in these populations [30,32]. For the remaining four populations (Bis, Mer, Moc, Pob)
10 individuals/population were genotyped in this study for the same set of SNPs. The climatic information from the locations of A. thaliana populations was obtained from
the digital climatic atlas of the Iberian Peninsula at 1 km2 resolution [50,97]. Thirty-three vari-
ables were used, related to temperature, precipitation and solar radiation (S3 Table). In addi-
tion, 19 bioclimatic variables derived by combination of annual trends, seasonality and
extreme conditions were also included (www.worldclim.org). Altitude was also analysed as a
proxy for climate. Annual mean temperature of the populations ranged 6.1–17.4˚C (12.5±0.3)
and annual precipitation ranged 405.7–1695.8 mm (753.8±33.9) (S8 Table). All accessions or individuals used in this study were propagated by selfing during two gen-
erations by the single seed descent procedure, in a glasshouse supplemented with lamps to pro-
vide a long-day photoperiod. This allowed reducing residual heterozygosity that might contain
some wild individuals but also removing any potential maternal and grand-mother effects. Seeds were stratified (darkness, 4ºC) for 7 days before germination at 25/20ºC day/night, 16 h
light. Ten day-old seedlings were transferred to 4ºC, 8 h light, for vernalisation during 4 or 8
weeks, depending on the experiment. After vernalisation, plants were transplanted to 0.43 L
pots and returned to the greenhouse, where they were kept (25/20ºC day/night, 16 h light)
until the end of the experiment. Virus infection as a selective pressure on Arabidopsis wild populations with variation for climatic factors, in contrast to variation for other adaptive life-history traits
of A. thaliana. In addition, we found evidence that these two defence traits are under uniform
selection. The results of this study are compatible with CMV infection acting as a selection
pressure for defence on populations of A. thaliana and, hence, we propose that CMV infection
likely reduces the host fitness under the field conditions of the analysed wild A. thaliana popu-
lations, although field experiments would be required to prove this fact. The results presented
here also show that some of the conditions for coevolution are met in the system A. thaliana-
CMV, but more work on the virus side is necessary to prove if coevolution occurs. These
results raise two challenging questions: what are the mechanisms that maintain polymor-
phisms for resistance and tolerance within A. thaliana populations, and what is the negative
impact of CMV infection on the host in nature and how does such an impact vary according
to field conditions. thaliana, which would hinder fixation of resistance/toler-
ance alleles and would contribute to the maintenance of defence polymorphisms within popu-
lations [85]. We have not found evidence for such costs under the assayed conditions, as
neither resistance nor tolerance were negatively correlated with viable seed production of
mock-inoculated controls. However, fitness costs might not be detectable under our experi-
mental conditions, and/or might be unveiled under the less favourable environment of the
field. Another interesting result is that resistance and tolerance to CMV co-occur in wild A. thali-
ana populations. Theory predicts that resources being limited, hosts would not invest in both
resistance and tolerance, which would be mutually exclusive defences. The conditions that
should favour the evolution of resistance or tolerance have been much modelled, and a nega-
tive correlation between both traits across host genotypes is expected [4,86,87]. We found no
correlation between resistance and tolerance in any experiment, indicating that they evolve as
independent traits. It has been proposed that resistance and tolerance could coexist if costs
and benefits of each defence were different and non-additive [88–91]. Models also propose
that tolerance alleles should become fixed in host populations, which would not be polymor-
phic for this trait under most assumptions [91–93]. A report on the tolerance to CMV in
Mimulus guttatus conforms to these predictions, as tolerance had no costs, but showed little
genetic variation [94,95]. On the contrary, our results do not agree with model predictions, as
we found large genetic variation for tolerance and evidence for coexistence of resistance and
tolerance. In conclusion, this work shows that in A. thaliana there is genetic variation, within and
among populations, for defences to CMV that result in lower virus multiplication or in lower
impact of infection on the plant fitness. Genetic variation for defence to CMV is not associated PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 15 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Quantification of CMV multiplication Virus multiplication in plants was estimated from virus RNA accumulation as described in
Paga´n et al., (2014) [41]. Briefly, at fifteen days post-inoculation 0.01 g fresh weight (fwt) of
leaf tissue was harvested from four different systemically infected leaves. Nucleic acids were
extracted from the pooled leaf tissue using TRI-reagent (Sigma-Aldrich, St Louis, MO, USA). Virus RNA was then quantified by dot-blot hybridization with 32P-labelled RNA probes com-
plementary to nucleotides 1933–2215 of Fny-CMV RNA3 (GeneBank Acc. No. D10538). In
each blot, internal standards for Fny-CMV, Cdc-CMV or Lro-CMV RNA were included as a
two-fold dilution series (1–0.001 μg) of purified virion RNA in nucleic acid extracts from non-
inoculated plants. Mock-inoculated samples served as negative controls. Nucleic acid extracts
were blotted at different dilutions to ensure that hybridization signal was on the linear portion
of the RNA concentration-hybridization curve. As loading controls, parallel membranes were
hybridized with a cDNA probe of β-tubulin chain 2 (TUB2) mRNA of A. thaliana (1086–1568
nt, GeneBank Acc. No. NM_125664.4). Virus infection as a selective pressure on Arabidopsis wild populations named At-CMV in [43]. Isolates were multiplied in Nicotiana clevelandii, Fny-CMV from
transcripts of cDNA clones and Cdc-CMV and Lro-CMV from biological clones derived from
local lesions in Chenopodium quinoa. Virions were purified as in [99]. A. thaliana plants were
mechanically inoculated at the five-leaf stage (stage 1.05, [100]) with 15 μl of sap from infected
N. clevelandii leaves in 0.01 M phosphate buffer pH 7.0, 0.2% sodium diethyldithiocarbamate. Fifteen μl of buffer were applied to mock-inoculated controls. The (unkown) virus concentra-
tion in leaf sap ensured infection of 100% of inoculated plants. Each treatment (virus-inocu-
lated or buffer mock-inoculated) involved at least five replicated plants from each original
sample, that is at least five plants derived from the same genotype or individual. All plants in
each experiment were grown in a completely randomized design. Quantification of life-history traits and tolerance to CMV Rosette weight was used to estimate vegetative growth effort, inflorescence plus seed weight to
estimate total reproductive effort, and seed weight to estimate progeny production [101]. Pre-
vious work has shown that CMV infection does not affect seed viability, nor the weight of indi-
vidual seeds, in a broad range of A. thaliana genotypes [14,47,52]. Plants were harvested at
complete senescence and dry weight of rosettes (rosette weight, RW), inflorescence structures
without seeds (inflorescence weight, IW) and seeds (seed weight, SW) were measured sepa-
rately (g). Two phenological parameters of A. thaliana life cycle were quantified: Growth
period (GP) and life-span (LP) were measured as the time (days) between planting seedlings in
soil and opening of the first flower (GP), or complete senescence (LP). Tolerance was mea-
sured by the effect of virus infection on progeny production: SWi/SWm, where i and m denote
infected and mock inoculated plants, respectively [48]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus isolates and inoculations Three subgroup IA CMV isolates were used, Fny-CMV, Cdc-CMV and Lro-CMV, which dif-
fer in the sequence of their genomic RNAs in about 1% of positions. Fny-CMV is a well-char-
acterized reference isolate [98]. Cdc-CMV and Lro-CMV were isolated from field-infected A. thaliana plants of the Cdc and Cho populations, respectively, in 2008 and 2011, Cdc-CMV was PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 16 / 24 Virus infection as a selective pressure on Arabidopsis wild populations genotype (nested within populations) as random factors. The 95% confidence intervals (95%
CI) for QST values were estimated as P S2ðn 1Þ
w2
n 1 s2 S2ðn 1Þ
w2
n 1
h
i
¼ 0:95 [106]. Genetic differentia-
tion for neutral markers was estimated as FST [107] using the analysis of molecular variance
(AMOVA) as implemented in ARLEQUIN v3.5.1.2 [108]. AMOVA were performed using
multilocus genotypes for 250 segregating SNPs [30,32] and their significances were estimated
from 1,000 permutations. The relationships between Euclidean geographical distance and FST or QST values among
population pairs were determined by Mantel correlation test using PASSaGE v.2 [109] with
1,000 permutations. Genetic distances between individuals were calculated as the proportion
of allelic differences over the total number of alleles in the corresponding set of polymorphic
loci, using GGT v. 2.0 [110]. Statistical analyses Differences in RW, IW, SW, GP, LP, virus accumulation or tolerance to CMV, according to
host individual/genotype and virus isolate, were analysed by general linear models (GLM) con-
sidering host individual/genotype as a random factor, and virus isolate as a fixed factor. Differ-
ences in RW, IW, SW, GP, LP, virus accumulation or tolerance to CMV according to
population, host individual/genotype, and virus isolate, were analysed by GLM considering iso-
late as a fixed factor, and population and individual/genotype nested to population, as random
factors. Relationships between values of different traits were tested using Spearman’s correlation
test. GLMs and Spearman’s correlation tests were performed using SPSS 20 software package. Spatial autocorrelation patterns of environmental variables, life-history traits, virus accu-
mulation and tolerance to virus, were analysed using correlograms [111] generated with PAS-
SaGE v.2. For each variable, Moran’s I autocorrelation coefficients [112] were calculated and
their significance tested from 1,000 permutations. Correlation between pairs of environmental
variables, between pairs of different traits and between environmental variable and different
traits were tested with Dutilleul’s modified t-test using SAM v.4 [113,114]. Simultaneous auto-
regressive models (SAR) [115] were performed to test the relationship between environmental
variables and different traits using SAM v.4. Bonferroni correction was applied for multiple
comparisons. The relationships between environmental variables and life-history or defence traits were
analysed by partial Mantel tests controlling for the location of populations given by the geo-
graphic distance matrix using PASSaGE v.2. For that, matrices of euclidean distances were
derived for each environmental variable and phenotypic trait and significance was evaluated
from 1,000 permutations. Genetic analyses Broad sense heritability of each trait was estimated as h2
b = VG/(VG+VE), where VG is the
among-genotypes or among-populations variance component and VE is the residual variance. Variance components were determined using the REML method [102] of SPSS 20 package
(SPSS Inc., Chicago, USA). Genetic differentiation between populations for quantitative traits was measured by QST
values [103], estimated as VB/(VB+VW) [104,105], where VB is the between-population vari-
ance and VW is the within-population variance. VB and VW were estimated by the REML
method from a nested analysis of variance performed using population and individual or PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 17 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Acknowledgments Antolı´n Lo´pez Quiro´s and Miguel A´ngel Mora provided excellent technical support. Author Contributions Conceptualization: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Conceptualization: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Formal analysis: Nuria Montes. Funding acquisition: Fernando Garcı´a-Arenal. Funding acquisition: Fernando Garcı´a-Arenal. Investigation: Nuria Montes, Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Writing – original draft: Nuria Montes. Investigation: Nuria Montes, Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Writing – original draft: Nuria Montes. Writing – review & editing: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Writing – review & editing: Carlos Alonso-Blanco, Fernando Garcı´a-Arenal. Virus infection as a selective pressure on Arabidopsis wild populations S4 Table. Coefficients of the correlation/regression between values of mock-inoculated A. thaliana life-history or defence to CMV traits, and those of the environmental variables of
the corresponding population obtained by SARs, Correlation with Dutilleul´s correction,
and Mantel test. A threshold significance value (α = 0.0014) was set after applying Bonferro-
ni’s correction (1– [1– a] 1/n) for multiple comparisons. (XLSX) S5 Table. Mean, minimum and maximum values of resistance and tolerance to CMV of A. thaliana individuals inoculated after a four-week vernalisation period. Mean, minimum
and maximum values, heritability and QST of resistance and tolerance to CMV for twelve A. thaliana populations are also shown. (XLSX) S6 Table. Mean values of resistance and tolerance to CMV for A. thaliana individuals inoc-
ulated after an eight-week vernalisation period. (XLSX) S6 Table. Mean values of resistance and tolerance to CMV for A. thaliana individuals inoc-
ulated after an eight-week vernalisation period. (XLSX) ulated after an eight-week vernalisation period. (XLSX)
S7 Table. Distribution of genotypes (H) and individuals (lower case three letter codes) in
12 analysed A. thaliana populations. (XLSX)
S8 Table. Values (mean and range of variation) of the environmental variables used in the
study. Values are the averages of 76 population. S7 Table. Distribution of genotypes (H) and individuals (lower case three letter codes) in
12 analysed A. thaliana populations. (XLSX)
S8 Table. Values (mean and range of variation) of the environmental variables used in the S7 Table. Distribution of genotypes (H) and individuals (lower case three letter codes) in
12 analysed A. thaliana populations. (XLSX) S8 Table. Values (mean and range of variation) of the environmental variables used in the
study. Values are the averages of 76 population. (XLSX) PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Supporting information S1 Table. Geographic origin and location of Iberian A. thaliana populations. (XLSX) S2 Table. GLM analysis of values of life history traits of mock-inoculated A. thaliana
plants, of tolerance to CMV, and of virus accumulation, using "host genotype" as a random
factor. For each trait, heritability (h2
b), mean, minimum and maximum values, are shown. (XLSX) S3 Table. Values of climatic variables for 76 Iberian A. thaliana populations, and for the
phenotypic traits of representative individuals. (XLSX) S3 Table. Values of climatic variables for 76 Iberian A. thaliana populations, and for the
phenotypic traits of representative individuals. (XLSX) 18 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations 6. Brown JKM, Tellier A. Plant-Parasite Coevolution: Bridging the Gap between Genetics and Ecology. Annu Rev Phytopathol. 2011; 49: 345–367. https://doi.org/10.1146/annurev-phyto-072910-095301
PMID: 21513455 7. Brown JKM. Durable resistance of crops to disease: a Drawinian perspective. Annu Rev Phytopathol. 2015; 53: 513–539. https://doi.org/10.1146/annurev-phyto-102313-045914 PMID: 26077539 8. Salvaudon L, Giraud T, Shykoff JA. Genetic diversity in natural populations: a fundamental component
of plant–microbe interactions. Curr Opin Plant Biol. 2008; 11: 135–143. https://doi.org/10.1016/j.pbi. 2008.02.002 PMID: 18329329 9. Burdon JJ, Thrall PH. Coevolution of Plants and Their Pathogens in Natural Habitats. Science. 2009;
324: 755–756. https://doi.org/10.1126/science.1171663 PMID: 19423818 10. Roossinck MJ. Symbiosis versus competition in plant virus evolution. Nat Rev Microbiol. 2005; 3:
917–924. https://doi.org/10.1038/nrmicro1285 PMID: 16322740 11. Roossinck MJ, Garcı´a-Arenal F. Ecosystem simplification, biodiversity loss and plant virus emer-
gence. Curr Opin Virol. 2015; 10: 56–62. https://doi.org/10.1016/j.coviro.2015.01.005 PMID:
25638504 12. Fraile A, Garcı´a-Arenal F. Environment and evolution modulate plant virus pathogenesis. Curr Opin
Virol. 2016; 17: 50–56. https://doi.org/10.1016/j.coviro.2016.01.008 PMID: 26827164 13. Xu P, Chen F, Mannas JP, Feldman T, Sumner LW, Roossinck MJ. Virus infection improves drought
tolerance. New Phytol. 2008; 180: 911–921. https://doi.org/10.1111/j.1469-8137.2008.02627.x PMID:
18823313 14. Hily JM, Poulicard N, Mora MA´ , Paga´n I, Garcı´a-Arenal F. Environment and host genotype determine
the outcome of a plant-virus interaction: from antagonism to mutualism. New Phytol. 2016; 209: 812–
822. https://doi.org/10.1111/nph.13631 PMID: 26365599 15. Maskell LC, Raybould AF, Cooper JI, Edwards ML, Gray AJ. Effects of turnip mosaic virus and turnip
yellow mosaic virus on the survival, growth and reproduction of wild cabbage (Brassica oleracea). Ann
Appl Biol. 1999; 135: 401–407. 16. Funayama S, Terashima I, Yahara T. Effects of virus infection and light environment on population
dynamics of Eupatorium makinoi (Asteraceae). Am J Bot. 2001; 88: 616–622. PMID: 11302846 17. Power AG, Mitchell CE. Pathogen spillover in disease epidemics. Am Nat. 2004; 164: S79–S89. https://doi.org/10.1086/424610 PMID: 15540144 18. Malmstrom CM, Hughes CC, Newton LA, Stoner CJ. Virus infection in remnant native bunchgrasses
from invaded California grasslands. New Phytol. 2005; 168: 217–230. https://doi.org/10.1111/j.1469-
8137.2005.01479.x PMID: 16159335 19. Malmstrom CM, Stoner CJ, Brandenburg S, Newton LA. Virus infection and grazing exert counteract-
ing influences on survivorship of native bunchgrass seedlings competing with invasive exotics. J Ecol. 2006; 94: 264–275. https://doi.org/10.1111/j.1365-2745.2006.01101.x PMID: 18784788 20. Prendeville HR, Tenhumberg B. Pilson D. Effects of virus on plant fecundity and population dynamics. New Phytol. 2014; 202: 1346–1356. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 References 1. Woolhouse MEJ, Webster JP, Domingo E, Charlesworth B, Levin BR. Biological and biomedical impli-
cations of the co-evolution of pathogens and their hosts. Nat Genet. 2002; 32: 569–577. https://doi. org/10.1038/ng1202-569 PMID: 12457190 2. Agnew P, Koella JC, Michalakis Y. Host life history responses to parasitism. Microb Infect. 2000; 2:
891–896. 3. Råberg L. How to Live with the Enemy: Understanding Tolerance to Parasites. PLoS Biol. 2014; 12:
e1001989. https://doi.org/10.1371/journal.pbio.1001989 PMID: 25369060 4. Paga´n I, Garcı´a-Arenal F. Tolerance to plant pathogens: theory and experimental evidences. Int J Mol
Sci. 2018; 19: 810. 5. Fraile A, Garcı´a-Arenal F. The coevolution of plants and viruses: Resistance and pathogenicity. Adv
Virus Res. 2010; 76: 1–32. https://doi.org/10.1016/S0065-3527(10)76001-2 PMID: 20965070 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 19 / 24 Virus infection as a selective pressure on Arabidopsis wild populations 28. The 1001 Genomes Consortium. 1,135 Genomes reveal the global pattern of polymorphism in Arabi-
dopsis thaliana. Cell. 2016; 166: 481–491. https://doi.org/10.1016/j.cell.2016.05.063 PMID:
27293186 29. Lee CR, Svardal H, Farlow A, Exposito-Alonso M, Ding W, Novikova P, et al. On the post-glacial
spread of human commensal Arabidopsis thaliana. Nat Commun. 2017; 8: 14458. https://doi.org/10. 1038/ncomms14458 PMID: 28181519 30. Pico´ FX, Mendez-Vigo B, Martinez-Zapater JM, Alonso-Blanco C. Natural genetic variation of Arabi-
dopsis thaliana is geographically structured in the Iberian Peninsula. Genetics. 2008; 180: 1009–
1021. https://doi.org/10.1534/genetics.108.089581 PMID: 18716334 31. Pico´ FX. Demographic fate of Arabidopsis thaliana cohorts of autumn-and spring-germinated plants
along an altitudinal gradient. J Appl Ecol. 2012; 100: 1009–1018. 32. Me´ndez-Vigo B, Gomaa NH, Alonso-Blanco C, Pico´ FX. Among- and within-population variation in
flowering time of Iberian Arabidopsis thaliana estimated in field and glasshouse conditions. New Phy-
tol. 2013; 197: 1332–1343. https://doi.org/10.1111/nph.12082 PMID: 23252608 33. Vidigal DS, Marques AC, Willems LA, Buijs G, Me´ndez-Vigo B, Hilhorst HW, et al. Altitudinal and cli-
matic associations of seed dormancy and flowering traits evidence adaptation of annual life cycle tim-
ing in Arabidopsis thaliana. Plant Cell Environ. 2016; 39: 1737–1748. https://doi.org/10.1111/pce. 12734 PMID: 26991665 34. Mauricio R, Rausher MD. Experimental manipulation of putative selective agents provides evidence
for the role of natural enemies in the evolution of plant defense. Evolution. 1997; 51: 1435–1444. https://doi.org/10.1111/j.1558-5646.1997.tb01467.x PMID: 28568624 35. Jorgensen TH, Emerson BC. Functional variation in a disease resistance gene in populations of Arabi-
dopsis thaliana. Mol Ecol. 2008; 17: 4912–4923. https://doi.org/10.1111/j.1365-294X.2008.03960.x
PMID: 19140981 36. Huard-Chauveau C, Perchepied L, Debieu M, Rivas S, Kroj T, Kars I, et al. An atypical kinase under
balancing selection confers broad-spectrum disease resistance in Arabidopsis. PLoS Genet. 2013; 9:
e1003766. https://doi.org/10.1371/journal.pgen.1003766 PMID: 24068949 37. Karasov TL, Kniskern JM, Gao L, DeYoung BJ, Ding J, Dubiella U, et al. The long-term maintenance
of a resistance polymorphism through diffuse interactions. Nature. 2014; 512: 436–440. https://doi. org/10.1038/nature13439 PMID: 25043057 38. Brachi B, Meyer CG, Villoutreix R, Platt A, Morton TC, Roux F, et al. Coselected genes determine
adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana. Proc
Natl Acad Sci USA. 2015; 112: 4032–4037. https://doi.org/10.1073/pnas.1421416112 PMID:
25775585 39. Jacquemond M. Cucumber mosaic virus. Adv Virus Res. 2012; 84: 439–504. https://doi.org/10.1016/
B978-0-12-394314-9.00013-0 PMID: 22682176 40. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 https://doi.org/10.1111/nph.12730 PMID: 24571200 21. Fraile A, McLeish M, Paga´n I, Gonza´lez-Jara P, Piñero D, Garcı´a-Arenal F. Environmental heteroge-
neity and the evolution of plant-virus interactions: Viruses in wild pepper populations. Virus Res. 2017;
15: 68–76. 22. Rodrı´guez-Nevado C, Montes N, Paga´n I. Ecological Factors Affecting Infection Risk and Population
Genetic Diversity of a Novel Potyvirus in Its Native Wild Ecosystem. Front Plant Sci. 2017; 8: 1958. https://doi.org/10.3389/fpls.2017.01958 PMID: 29184567 23. Pallett DW, Thurston MI, Cortina-Borja M, Edwards ML, Alexander M, Mitchell E, et al. The incidence
of viruses in wild Brassica rapa ssp. sylvestris in southern England. Ann Appl Biol. 2002; 141: 163–
170. 24. Ausin I, Alonso-Blanco C, Martinez-Zapater JM. Environmental regulation of flowering. Int J Dev Biol. 2005; 49: 689–705. https://doi.org/10.1387/ijdb.052022ia PMID: 16096975 25. Hoffmann MH. Biogeography of Arabidopsis thaliana (L.) Heynh. (Brassicaceae) J Biogeogr. 2002;
29: 125–134. 26. Brennan AC, Me´ndez-Vigo B, Haddioui A, Martı´nez-Zapater JM, Pico´ FX, Alonso-Blanco C. The
genetic structure of Arabidopsis thaliana in the south-western Mediterranean range reveals a shared
history between North Africa and southern Europe. BMC Plant Biol. 2014; 14: 17. https://doi.org/10. 1186/1471-2229-14-17 PMID: 24411008 27. Durvasula A, Fulgione A, Gutaker RM, Alacakaptan SI, Flood PJ, Neto C, et al. African genomes illu-
minate the early history and transition to selfing in Arabidopsis thaliana. Proc Natl Acad Sci USA. 2017; 114: 5213–5218. https://doi.org/10.1073/pnas.1616736114 PMID: 28473417 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 20 / 24 Virus infection as a selective pressure on Arabidopsis wild populations 48. Paga´n I, Alonso-Blanco C, Garcı´a-Arenal F. Host Responses in Life-History Traits and Tolerance to
Virus Infection in Arabidopsis thaliana. PLoS Pathog. 2008; 4: e1000124. https://doi.org/10.1371/
journal.ppat.1000124 PMID: 18704166 49. Me´ndez-Vigo B, Pico´ FX, Ramiro M, Martinez-Zapater JM, Alonso-Blanco C. Altitudinal and Climatic
Adaptation Is Mediated by Flowering Traits and FRI, FLC, and PHYC Genes in Arabidopsis. Plant
Physiol. 2011; 157: 1942–1955. https://doi.org/10.1104/pp.111.183426 PMID: 21988878 50. Manzano-Piedras E, Marcer A, Alonso-Blanco C, Pico´ FX. Deciphering the Adjustment between Envi-
ronment and Life History in Annuals: Lessons from a Geographically-Explicit Approach in Arabidopsis
thaliana. PLoS ONE. 2014; 9: e87836. https://doi.org/10.1371/journal.pone.0087836 PMID:
24498381 51. Tabas-Madrid D, Me´ndez-Vigo B, Arteaga N, Marcer A, Pascual-Montano A, Weigel D, et al. Genome-wide signatures of flowering adaptation to climate temperature: regional analyses in a highly
diverse native range of Arabidopsis thaliana. Plant Cell Environ. 2018; 41: 1806–1820. https://doi.org/
10.1111/pce.13189 PMID: 29520809 52. Shukla A, Paga´n I, Garcı´a-Arenal F. Effective tolerance based on resource reallocation is a virus-spe-
cific defence in Arabidopsis thaliana. Mol Plant Pathol. 2018; 19: 1454–1465. https://doi.org/10.1111/
mpp.12629 PMID: 29027740 53. Paga´n I, Alonso-Blanco C, Garcı´a-Arenal F. Differential tolerance to direct and indirect density-depen-
dent costs of viral infection in Arabidopsis thaliana. PLoS Pathog. 2009; 5: e1000531. https://doi.org/
10.1371/journal.ppat.1000531 PMID: 19649316 54. Bjo¨rn HK, Gavin RS, Nienhuis J. Effects of photoperiod and vernalization on the number of leaves at
flowering in 32 Arabidopsis thaliana (Brassicaceae) ecotypes. Am J Bot. 1993; 80: 646–648. 55. Auge GA, Leverett D, Edwards BR, Donohue K. Adjusting phenotypes via within- and across-genera-
tional plasticity. New Phytol. 2017; 216: 343–349. https://doi.org/10.1111/nph.14495 PMID:
28262950 56. Agrawal A, Lively CM. Infection genetics: gene-for-gene versus matching-alleles models and all points
in between. Evol Ecol Res. 2002; 4: 79–90. 57. Agrawal A, Lively CM. Modelling infection as a two-step process combining gene-for-gene and match-
ing-allele genetics. Proc R Sci. 2003; 270: 323–34. 58. Sacrista´n S, Garcı´a-Arenal F. The evolution of virulence and pathogenicity in plant pathogen popula-
tions. Mol Plant Pathol. 2008; 9: 369–384. https://doi.org/10.1111/j.1364-3703.2007.00460.x PMID:
18705877 59. Alexander H, Mauck K, Whitfield A, Garrett K, Malmstrom C. Plant-virus interactions and the agro-eco-
logical interface. Eur J Plant Pathol. 2014; 138: 529–547. 60. Bergelson J, Roux F. Towards identifying genes underlying ecologically relevant traits in Arabidopsis
thaliana. Nat Rev Genet. 2010; 11: 867–879. https://doi.org/10.1038/nrg2896 PMID: 21085205 61. Parker MA. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Hily JM, Garcı´a A, Moreno A, Plaza M, Wilkinson MD, Fereres A, Fraile A, Garcı´a-Arenal F. The Rela-
tionship between Host Lifespan and Pathogen Reservoir Potential: An Analysis in the System Arabi-
dopsis thaliana-Cucumber mosaic virus. PLoS Pathog. 2014; 10: e1004492. https://doi.org/10.1371/
journal.ppat.1004492 PMID: 25375140 41. Paga´n I, Montes N, Milgroom MG, Garcı´a-Arenal F. Vertical Transmission Selects for Reduced Viru-
lence in a Plant Virus and for Increased Resistance in the Host. PLoS Pathog. 2014; 10: e1004293. https://doi.org/10.1371/journal.ppat.1004293 PMID: 25077948 42. Palukaitis P, Garcı´a-Arenal F. Cucumoviruses. Adv Virus Res. 2003; 62: 241–323. PMID: 14719367 43. Paga´n I, Fraile A, Ferna´ndez-Fueyo E, Montes N, Alonso-Blanco C, Garcı´a-Arenal F. Arabidopsis
thaliana as a model for the study of plant-virus co-evolution. Philos Trans R Soc Lond, B, Biol Sci. 2010; 365: 1983–1995. https://doi.org/10.1098/rstb.2010.0062 PMID: 20478893 44. Montes N. Arabidopsis thaliana como modelo de estudio de la coevolucio´n planta-virus Thesis, Uni-
versidad Polite´cnica de Madrid (UPM). 2016; Available from: https://doi.org/10.20868/UPM.thesis. 42853. 45. Fraile A, Alonso-Prados JL, Aranda MA, Bernal JJ, Malpica JM, Garcı´a-Arenal F. Genetic exchange
by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus. J
Virol. 1997; 71: 934–940. PMID: 8995610 46. Bonnet J, Fraile A, Sacrista´n S, Malpica JM, Garcı´a-Arenal F. Role of recombination in the evolution of
natural populations of Cucumber mosaic virus, a tripartite RNA plant virus. Virology. 2005; 332: 359–
368. https://doi.org/10.1016/j.virol.2004.11.017 PMID: 15661167 47. Paga´n I, Alonso-Blanco C, Garcı´a-Arenal F. The Relationship of Within-Host Multiplication and Viru-
lence in a Plant-Virus System. PLoS ONE. 2007; 2: e786. https://doi.org/10.1371/journal.pone. 0000786 PMID: 17726516 21 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations 70. Kaltz O, Shykoff JA. Local adaptation in host–parasite systems. Heredity. 1998; 81: 361–370. 71. de Meaux J, Mitchell-Olds T. Evolution of Plant Resistance at the Molecular Level: Ecological Context
of Species Interactions. Heredity. 2003; 91: 345–52. https://doi.org/10.1038/sj.hdy.6800342 PMID:
14512948 72. Laine AL. Resistance variation within and among host populations in a plant-pathogen metapopula-
tion-implications for regional pathogen dynamics. J. Ecol. 2004; 92: 990–1000. 73. Laine AL. Spatial scale of local adaptation in a plant-pathogen. J Evol Biol. 2005; 18: 930–938. https://
doi.org/10.1111/j.1420-9101.2005.00933.x PMID: 16033565 74. Laine AL. Evolutionary of host resistance: looking for coevolutionry hotspot at small spatial scales. Phi-
los Trans R Soc Lond B Biol Sci. 2006; 273: 267–273. 75. Salvaudon L, He´raudet V, Shykoff JA. Parasite-host fitness trade-offs change with parasite identity:
genotype-specific interactions in a plant-pathogen system. Evolution. 2005; 59: 2518–2524. PMID:
16526500 76. Salvaudon L, Heraudet V, Shykoff JA. Arabidopsis thaliana and the Robin Hood parasite: a chivalrous
oomycete that steals fitness from fecund hosts and benefits the poorest one?. Biol. Lett. 2008; 4:
526–529. https://doi.org/10.1098/rsbl.2008.0332 PMID: 18682359 77. Kniskern JM, Barrett LG, Bergelson J. Maladaptation in wild populations of the generalist plant patho-
gen Pseudomonas syringae. Evolution. 2011; 65: 818–830. https://doi.org/10.1111/j.1558-5646. 2010.01157.x PMID: 21044058 78. Salvaudon L, Shykoff JA. Variation in Arabidopsis developmental responses to oomycete infection:
resilience vs changes in life history traits. New Phytol. 2013; 197: 919–926. https://doi.org/10.1111/
nph.12073 PMID: 23231447 79. Kover PX, Schaal BA. Genetic variation for disease resistance and tolerance among Arabidopsis thali-
ana accessions. Proc Natl Acad Sci USA. 202; 99: 11270–11274. 80. Mitchell-Olds T. Arabidopsis thaliana and its wild relatives: a model system for ecology and evolution. Trends Ecol Evol. 2011; 16: 693–700. 81. Luo Y, Widmer A, Karrenberg. The roles of genetic drift and natural selection in quantitative trait diver-
gence along an altitudinal gradient in Arabidopsis thaliana. Heredity. 2015; 114: 220–228. https://doi. org/10.1038/hdy.2014.89 PMID: 25293874 82. Jakob K, Goss EM, Araki H, Van T, Kreitman M, Bergelson J. Pseudomonas viridiflava and P. syrin-
gae—Natural Pathogens of Arabidopsis thaliana. Mol Plant Microbe Interact. 2002; 15: 1195–1203. https://doi.org/10.1094/MPMI.2002.15.12.1195 PMID: 12481991 83. Goss EM, Bergelson J. Variation in resistance and virulence in the interaction between Arabidopsis
thaliana and a bacterial pathogen. Evolution. 2006; 60: 1562–1573. PMID: 17017057 84. Alonso-Prados JL, Aranda MA, Malpica JM, Garcı´a-Arenal F, Fraile A. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Disease impact and local genetic diversity in the clonal plant Podophyllum peltatum. Evo-
lution. 1989; 43: 540–547. https://doi.org/10.1111/j.1558-5646.1989.tb04250.x PMID: 28568395 62. Jarosz AM, Burdon JJ. Predominance of a single major gene for resistance to Phakopsora pachyrhizi
and Glycine argyrea: Phenotypic and genetic patterns. Heredity. 1990; 64: 347–353. 63. Jarosz AM, Burdon JJ. Host-pathogen interactions in natural populations of Linum marginale and Mel-
ampsora lini. II. Local and regional variation in patterns of resistance and racial structure. Evolution. 1991; 45: 1618–27. https://doi.org/10.1111/j.1558-5646.1991.tb02667.x PMID: 28564135 64. Antonovics J, Thrall PH, Jarosz A, Stratton D. Ecological genetics of metapopulations: the Silene–
Ustilago plant–pathogen system. In: Real L, ed. Ecological genetics. Princeton, US, Princeton Univer-
sity Press; 1994. pp 146–170. 65. Burdon JJ, Jarosz AM. Host–pathogen interactions in natural populations of Linum marginale and Mel-
ampsora lini. I. Patterns of resistance and racial variation in a large host population. Evolution. 1991;
45: 205–217. https://doi.org/10.1111/j.1558-5646.1991.tb05278.x PMID: 28564067 66. Hamilton MG, Williams DR, Tilyard PA, Pinkard EA, Wardlaw TJ, Glen M, et al. A latitudinal cline in dis-
ease resistance of a host tree. Heredity. 2013; 110: 372–379. https://doi.org/10.1038/hdy.2012.106
PMID: 23211794 67. Stahl EA, Dwyer G, Mauricio R, Kreitman M, Bergelson J. Dynamics of disease resistance polymor-
phism at the Rpm1 locus of Arabidopsis. Nature. 1999; 400: 667–671. https://doi.org/10.1038/23260
PMID: 10458161 68. Thrall PH, Burdon JJ, Young A. Variation in resistance and virulence among demes of a plant host-
pathogen metapopulation. J. Ecol. 2001; 89: 736–748. 69. Thrall PH, Burdon JJ. Evolution of gene-for-gene systems in metapopulations: the effect of spatial
scale of host and pathogen dispersal. Plant Pathol. 2002; 51: 169–184. 22 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Virus infection as a selective pressure on Arabidopsis wild populations 94. Carr DE, Murphy JF, Eubanks MD. The susceptibility and response of inbred and outbred Mimulus
guttatus to infection by Cucumber mosaic virus. Evol Ecol. 2003; 17: 85–103. 95. Carr DE, Murphy JF, Eubanks MD. Genetic variation and covariation for resistance and tolerance to
Cucumber mosaic virus in Mimulus guttatus (Phrymaceae): a test for costs and constraints. Heredity. 2006; 96: 29–38. https://doi.org/10.1038/sj.hdy.6800743 PMID: 16189544 96. Gomaa NH, Montesinos-Navarro A, Alonso-Blanco C, Pico´ FX. Temporal variation in genetic diversity
and effective population size of Mediterranean and subalpine Arabidopsis thaliana populations. Mol
Ecol. 2001; 20: 3540–3554. 97. Ninyerola M, Pons X, Roure JM. Atlas Clima´tico Digital de la Penı´nsula Ibe´rica. Metodologı´a y aplica-
ciones en bioclimatologı´a y geobota´nica. Universidad Auto´noma de Barcelona: Bellaterra; 2005. 98. Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RIB. Cucumber Mosaic Virus. Adv Virus Res. 1992;
41: 281–348. PMID: 1575085 99. Lot H, Marrou J, Quiot JB, Esvan C. Contribution à l’e´tude du virus de la mosaïque du concombre
(CMV). Me´thode de purification rapide du virus. Ann Phytopathol. 1975, 4: 25–38. 100. Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Go¨rlach J. Growth stage-
based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell. 2001; 13: 1499–1510. https://doi.org/10.1105/TPC.010011 PMID: 11449047 101. Thompson K, Stewart AJA. The Measurement and Meaning of Reproductive Effort in Plants. Am Nat. 1981; 117: 205–211. 102. Lynch M, Walsh B. Genetics and analysis of quantitative traits. Sunderland, MA, USA: Sinauer Asso-
ciates Inc; 1998. 103. Spitze K. Population structure in Daphnia obtusa: quantitative genetic and allozymic variation. Genet-
ics. 1993; 135: 367–374. PMID: 8244001 104. Bonnin I, Prosperi JM, Olivieri I. Genetic markers and quantitative genetic variation in Medicago trun-
catula (Leguminosae): A comparative analysis of population structure. Genetics. 1996; 143: 1795–
1805. PMID: 8844165 105. Le Corre V. Variation at two flowering time genes within and among populations of Arabidopsis thali-
ana: comparison with markers and traits. Mol Ecol. 2005; 14: 4181–4192. https://doi.org/10.1111/j. 1365-294X.2005.02722.x PMID: 16262868 106. Quinn GP, Keough MJ. Experimental Design and Data Analysis for Biologists. UK: Cambridge Uni-
versity Press; 2002. 107. Weir BS, Cockerham CC. Estimating F-statistics for the analysis of population structure. Evolution. 1984; 38: 1358–1370. https://doi.org/10.1111/j.1558-5646.1984.tb05657.x PMID: 28563791 108. Excoffier L, Lischer HEL. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Satellite RNA of Cucumber
mosaic virus spreads epidemically in natural populations of its helper virus. Phytopathology. 1998; 88:
520–524. https://doi.org/10.1094/PHYTO.1998.88.6.520 PMID: 18944903 85. Antonovics J, Thrall P. The cost of resistance and the maintenance of genetic polymorphism in host-
parasite systems. Philos Trans R Soc Lond B Biol Sci. 1994; 257: 105–110. 86. Herms DA, Mattson WJ. The dilemma of plants: To grow or defend. Q Rev Biol. 1992; 67: 283–335. 87. Manzaneda AJ, Prasad KVSK, Mitchell-Olds T. Variation and fitness costs for tolerance to different
types of herbivore damage in Boechera stricta genotypes with contrasting glucosinolate structures. New Phytol. 2010; 188: 464–477. https://doi.org/10.1111/j.1469-8137.2010.03385.x PMID:
20663059 88. Mauricio R, Rausher MD, Burdick DS. Variation in the defense strategies of plants: are resistance and
tolerance mutually exclusive?. Ecology. 1997; 78: 1301–1311. 89. Boots M, Bowers RG. Three mechanisms of host resistance to microparasites–avoidance, recovery
and tolerance–show different evolutionary dynamics. J Theor Biol. 1999; 201: 13–23. https://doi.org/
10.1006/jtbi.1999.1009 PMID: 10534432 90. Restif O, Koella JC. Shared control of epidemiological traits in a coevolutionary model of host-parasite
interactions. Am Nat. 2003; 161: 827–836. https://doi.org/10.1086/375171 PMID: 12858269 91. Restif O, Koella JC. Concurrent Evolution of Resistance and Tolerance to Pathogens. Am Nat. 2004;
164: E90–E102. https://doi.org/10.1086/423713 PMID: 15459887 92. Roy BA, Kirchner JW. Evolutionary dynamics of pathogen resistance and tolerance. Evolution. 2000;
54: 51–63. PMID: 10937183 93. Miller MR, White A, Boots M. The evolution of host resistance: tolerance and control as distinct strate-
gies. J Theor Biol. 2005; 236: 198–207. https://doi.org/10.1016/j.jtbi.2005.03.005 PMID: 16005309 23 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
May 28, 2019 Arlequin suite ver 3.5: A new series of programs to perform population genet-
ics analyses under Linux and Windows. Mol Ecol Resour. 2010; 10: 564–567. https://doi.org/10.1111/
j.1755-0998.2010.02847.x PMID: 21565059 109. Rosenberg MS, Anderson CD. PASSaGE: pattern analysis, spatial statistics and geographic exege-
sis. Version 2. Methods Ecol Evol. 2011; 2: 229–232. 110. van Berloo R. GGT 2.0: versatile software for visualization and analysis of genetic data. J. Hered. 2008; 99: 232–236. https://doi.org/10.1093/jhered/esm109 PMID: 18222930 111. Sokal RR, Oden NL. Spatial autocorrelation in biology 1. Methodology. Biological Journal of the Lin-
nean Society, 10: 199–228. 112. Moran PAP. Notes on continuous stochastic phenomena. Biometrika. 1950; 37: 17–23. PMID:
15420245 113. Dutilleul P, Clifford P, Richardson S, Hemon D. Modifying the t test for assessing the correlation
between two spatial processes. Biometrics. 1993; 49: 305–314. 114. Rangel TF, Diniz-Filho JAF, Bini LM. SAM: A comprehensive aplication for Spatail Analysis in Macroe-
cology. Ecography. 2010; 33: 1–5. 115. Beale CM, Lennon JJ, Yearsley JM, Brewer MJ, Elston DA. Regression analysis of spatial data. Ecol
Lett. 2010; 13: 246–264. https://doi.org/10.1111/j.1461-0248.2009.01422.x PMID: 20102373 24 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1007810
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Three cases of sporadic meningioangiomatosis with different imaging appearances: case report and review of the literature
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CASE REPORT Open Access * Correspondence: cjr.zhangyunting@vip.163.com
†Equal contributors
Department of Radiology, Tianjin Medical University General Hospital, No.
154, Anshan Dao Road, Heping District, Tianjin 300052, People’s Republic
of China © 2015 Sun et al.; licensee BioMed Central. 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 credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Sun et al. World Journal of Surgical Oncology (2015) 13:89
DOI 10.1186/s12957-015-0477-x Sun et al. World Journal of Surgical Oncology (2015) 13:89
DOI 10.1186/s12957-015-0477-x WORLD JOURNAL OF
SURGICAL ONCOLOGY Abstract Background: Meningioangiomatosis (MA) is a rare meningiovascular malformation or hamartomatous lesion in the
central nervous system. Radiographic findings of MA may show a variety of characteristics according to different
histological components. We present three cases of sporadic MA with different imaging appearances in an attempt
to identify specific imaging characteristics. Case presentation: In case 1, an irregular hyperdense solid mass was localized in the left middle cranial fossa,
demonstrating low and equal signal intensity on T1-weighted imaging (T1WI; TR/TE 2,048.9 ms/26.1 ms), high signal
intensity with multiple flow void effect on T2-weighted imaging (T2WI; TR/TE 4,000 ms/106.4 ms), and significant and
homogeneous enhancement on post-contrast magnetic resonance imaging (MRI). In case 2, the lesion in the right
insular lobe showed a cystic-mural nodule pattern. The cystic content demonstrated similar density or signal intensity as
cerebrospinal fluid, while the mural nodule demonstrated equal density or signal intensity on computed tomography
(CT) and MRI. On post-contrast MRI, the mural nodule showed significant enhancement, but the cystic wall and content
showed no enhancement. In case 3, a remarkably enhanced solid nodule was found in the cortex of the left parietal
lobe with multiple small cysts surrounding it. This nodule showed low signal intensity on T2WI and diffusion-weighted
imaging (DWI; TR/TE 6,000 ms/96.8 ms, b = 1,000 s/mm2). The preoperative diagnoses of the above three cases were
meningioma, hemangioblastoma, and ganglioglioma. However, all were pathologically diagnosed as MA. Conclusion: The presented cases demonstrate that MA may present with solid and cystic imaging patterns, which
may include large cystic-mural nodules and small intra- and extra-cystic patterns. Although MA imaging diagnoses are
difficult, several MRI signs may include specific characteristics, such as a flow void effect on T2WI and separating cysts
in the cystic MA (as shown in our cases), gyriform hyperintensity on T2-fluid attenuated inversion recovery (FLAIR)
sequence, and susceptibility artifacts on T2 gradient echo (GRE) sequences (as found in the literature). Keywords: Meningioangiomatosis, Magnetic resonance imaging, Flow void effect, Cystic meningioangiomatosis and meningovascular proliferation interwoven with bands
of fibrous connective tissue. The radiographic findings of
MA may demonstrate a variety of characteristics accord-
ing to different histological components. In this study, we
present three cases of sporadic MA with different imaging
appearances and attempt to identify specific characteris-
tics to help in preoperative diagnoses. In addition, the
relevant medical literature was reviewed. Three cases of sporadic meningioangiomatosis
with different imaging appearances: case report
and review of the literature hihua Sun†, Fei Jin†, Jing Zhang, Yue Fu, Wei Li, Hong Guo and Yunting Zhang* Zhihua Sun†, Fei Jin†, Jing Zhang, Yue Fu, Wei Li, Hong Guo and Yunting Zhang* Background Meningioangiomatosis (MA) is a rare meningiovascular
malformation or hamartomatous lesion in the central
nervous system, which was first described by Bassoe and
Nuzum [1] and then was named by Worster-Drought
et al. [2]. MA may occur sporadically or in association
with neurofibromatosis (NF) type 2. The pathological
characteristics of MA include leptomeningeal calcification * Correspondence: cjr.zhangyunting@vip.163.com
† Case presentation
Case 1 * Correspondence: cjr.zhangyunting@vip.163.com
†Equal contributors
Department of Radiology, Tianjin Medical University General Hospital, No. 154, Anshan Dao Road, Heping District, Tianjin 300052, People’s Republic
of China A 73-year-old female patient had a history of binocular
diplopia for 1 week. Physical examination showed a Page 2 of 6 Sun et al. World Journal of Surgical Oncology (2015) 13:89 limitation in abduction movement in the left eye. A head
computed tomography (CT) scan showed an irregular
mixed hyperdense mass in the left middle cranial fossa
(Figure 1a). On magnetic resonance imaging (MRI), the
lesion demonstrated low and equal signal intensity on
T1-weighted imaging (T1WI; TR/TE 2,048.9 ms/26.1 ms)
and high signal intensity with multiple flow void effect on
T2-weighted imaging (T2WI; TR/TE 4,000 ms/106.4 ms)
(Figure 1b,c). On post-contrast MRI, the lesion showed
significant and homogeneous enhancement after gado-
linium diethylenetriamine pentaacetate (Gd-DTPA) was
administered. The margin between the lesion and the
adjacent brain cortex was well demarcated, and there
was no obvious mass effect. The preoperative diagnosis
was meningioma. tumor was firm, pink, and enveloped and had a general
blood supply. The base was located in the side wall of
the cavernous sinus and sphenoid crest. Microscopically,
there was extensive fibroblastic proliferation and an in-
creased number of vessels surrounded by meningothelial
cells (Figure 1e,f). Pathological diagnosis was MA. Case 2 A 23-year-old male patient presented with left hemia-
nesthesia for more than 2 years. Physical examination
showed hypesthesia and slight ataxia on the left side,
and a decrease of graphics and two-point discrimination
sensation. A cystic-mural nodule lesion was localized in
the right insular lobe on head plain CT (Figure 2a). The
cystic portion had low density with 9 Hu, and the mural
nodule was isodense with 35 Hu. The cystic content
demonstrated similar signal intensity as cerebrospinal
fluid (CSF), while the mural nodule demonstrated iso-
signal intensity on T1WI, T2WI, and diffusion-weighted
imaging (DWI; TR/TE 6,000 ms/96.8 ms, b = 1,000 s/mm2)
(Figure 2b,c,d). On post-contrast MRI, the mural nodule
showed significant enhancement, but the cystic wall and
content demonstrated no enhancement (Figure 2e). The
adjacent brain parenchyma and sulci were compressed and
deformed, but there was no edema around the tumor. The
preoperative diagnosis was hemangioblastoma. The patient underwent a left temporal craniotomy,
and complete removal of the tumor was performed. The Figure 1 Solid meningioangiomatosis. (a) CT scan showed an
irregular mixed high-density mass in the left middle cranial fossa. (b) On T1WI, the lesion demonstrated low and equal signal intensity. (c) On T2WI, the lesion showed high signal intensity with a multiple
flow void effect. (d) On post-contrast MRI, the lesion showed significant
and homogeneous enhancement. (e, f) Microphotography of specimens
showed extensive fibroblastic proliferation and an increased number of
vessels surrounded by meningothelial cells. The patient underwent a right temporal craniotomy. Upon operation, approximately 50 ml of a yellow trans-
parent cyst fluid was extracted from the cystic tumor. The purple mural nodule was approximately 8 mm in
diameter with fibrous adhesion to the right insular
cortex. Pathological examination demonstrated a classic
MA appearance (Figure 2f). Case 3 (a, b) DWI and T2WI demonstrated a low signal intensity
nodule in the left parietal cortex and multiple small cysts surrounding
it. (c) On post-contrast MRI, the nodule was remarkably enhanced. (d) Microscopically, fibroblast-like spindle cells were arranged in a
spiral shape around multiple vessels, and the cortical neurons were
entrapped within the lesion. accompanied by NF has been shown to mostly occur in
the frontal lobe (35%) [6,7]. Atypical locations have also
been reported [8], including the third ventricle, thalamus,
cerebral peduncle, and brain stem. Multiple MA lesions
accompanied by NF have occurred in 35% of patients, but
the incidence of sporadic MA was only 13% [9]. Figure 2 Cystic meningioangiomatosis with a cystic-mural Figure 2 Cystic meningioangiomatosis with a cystic-mural
nodule pattern. (a) CT scan showed a cystic-mural nodule lesion in
the right insular lobe. (b-d) In non-enhanced MRI, the cystic content
demonstrated similar signal intensity as cerebrospinal fluid (CSF), while
the mural nodule demonstrated iso-signal intensity on T1WI, T2WI,
and DWI. (e) On post-contrast MRI, the mural nodule demonstrated
significant enhancement, while the cystic wall and content showed
no enhancement. (f) Pathological examination showed perivascular
spindle-cell proliferation. Figure 2 Cystic meningioangiomatosis with a cystic-mural
nodule pattern. (a) CT scan showed a cystic-mural nodule lesion in
the right insular lobe. (b-d) In non-enhanced MRI, the cystic content
demonstrated similar signal intensity as cerebrospinal fluid (CSF), while
the mural nodule demonstrated iso-signal intensity on T1WI, T2WI,
and DWI. (e) On post-contrast MRI, the mural nodule demonstrated
significant enhancement, while the cystic wall and content showed
no enhancement. (f) Pathological examination showed perivascular
spindle-cell proliferation. The pathogenesis of MA remains unclear. Proposed
hypotheses include hamartoma with degenerative changes,
leptomeningeal meningioma with invasion in adjacent
brain tissue, and cortical vascular malformation. MA was
formerly classified as a neurocutaneous syndrome, and
some cases associated with NF supported the theory of a
hamartoma [10]. Although not all cases have a meningeal
component and malignancy characteristics are typically
absent, the association between meningioma in rare
cases and molecular aberrations regarding the NF2 gene
in both lesions suggests that MA may be correlated with
meningioma. Kim et al. [11] found that the meningioma-
tosis portions of meningiomatosis-meningioma have loss
of heterozygosity for the 22q12 locus in 28.6% (2/7) of
coexisting cases of meningiomatosis-meningioma, whereas
each pure meningiomatosis harbors one loss of hetero-
zygosity at either 22q12 or 9p21. Case 3 A 9-year-old girl suffered from an involuntary convul-
sion in her right limb for 1 month. Physical examination
showed no positive signs. On non-enhanced MRI, a solid
nodule 11 mm in diameter was found in the cortex of
the left parietal lobe, demonstrating low signal intensity
on DWI and T2WI (Figure 3a,b). Multiple small cysts
were around the nodule. There was a slight mass effect
and surrounding edema. After Gd-DTPA administration,
the nodule was remarkably enhanced (Figure 3c). The
preoperative diagnosis was ganglioglioma. The patient underwent a left parietal craniotomy, and
the tumor was completely removed. The tumor was pur-
ple with an abundant blood supply, and it was unclearly
demarcated from adjacent brain tissue. Microscopically,
fibroblast-like spindle cells were arranged in spiral shape
around multiple vessels, and cortical neurons were en-
trapped within the lesion (Figure 3d). The pathological
diagnosis was MA. Figure 1 Solid meningioangiomatosis. (a) CT scan showed an
irregular mixed high-density mass in the left middle cranial fossa. (b) On T1WI, the lesion demonstrated low and equal signal intensity. (c) On T2WI, the lesion showed high signal intensity with a multiple
flow void effect. (d) On post-contrast MRI, the lesion showed significant
and homogeneous enhancement. (e, f) Microphotography of specimens
showed extensive fibroblastic proliferation and an increased number of
vessels surrounded by meningothelial cells. Page 3 of 6 Sun et al. World Journal of Surgical Oncology (2015) 13:89 Figure 2 Cystic meningioangiomatosis with a cystic-mural
nodule pattern. (a) CT scan showed a cystic-mural nodule lesion in
the right insular lobe. (b-d) In non-enhanced MRI, the cystic content
demonstrated similar signal intensity as cerebrospinal fluid (CSF), while
the mural nodule demonstrated iso-signal intensity on T1WI, T2WI,
and DWI. (e) On post-contrast MRI, the mural nodule demonstrated
significant enhancement, while the cystic wall and content showed
no enhancement. (f) Pathological examination showed perivascular
spindle-cell proliferation. Figure 3 Cystic meningioangiomatosis with a multiple microcystic
pattern. (a, b) DWI and T2WI demonstrated a low signal intensity
nodule in the left parietal cortex and multiple small cysts surrounding
it. (c) On post-contrast MRI, the nodule was remarkably enhanced. (d) Microscopically, fibroblast-like spindle cells were arranged in a
spiral shape around multiple vessels, and the cortical neurons were
entrapped within the lesion. Figure 3 Cystic meningioangiomatosis with a multiple microcystic
pattern. Case 3 The theory of cortical
vascular malformation corresponded to the histopatho-
logical characteristic of MA. Cortical vascular malformation
induced the perivascular meningothelial proliferation of
cells from vessel walls or pluripotent arachnoid cap cells in Discussion MA can be divided into two categories: sporadic or asso-
ciated with NF type 2. Sporadic meningioangiomatosis
usually occurs in young adults or children who present
with seizures or headaches. In contrast, NF2-associated
cases are usually asymptomatic and discovered post-
mortem. MA has a slight male predominance [3], but
according to some authors [4], it has no sex predilection. Meningioma is the most commonly associated neoplasm,
and other associated abnormalities include cerebral hemor-
rhage, oligodendroglioma, arteriovenous malformations,
cerebral softening, and meningeal hemangiopericytoma [5]. MA was mostly localized in the supratentorial region,
particularly in the temporal or frontal lobes. Sporadic
MA has mainly been reported in the temporal lobe
(33%) followed by the frontal lobe (25%), but MA MA was mostly localized in the supratentorial region,
particularly in the temporal or frontal lobes. Sporadic
MA has mainly been reported in the temporal lobe
(33%) followed by the frontal lobe (25%), but MA Sun et al. World Journal of Surgical Oncology (2015) 13:89 Page 4 of 6 Sun et al. World Journal of Surgical Oncology (2015) 13:89 Virchow-Robin spaces. Leptomeninges and arachnoid cap
cells normally surround blood vessels as they penetrate
the cortex. Conceivably, chronic leptomeningeal stimula-
tion by underlying cortical lesions could result in MA
histopathological changes. MR images was iso- to hypointense on T1-weighted im-
ages and hypo- to hyperintense on T2-weighted images. In
case 1, multiple flow void effect could be observed within
MA on T2WI. To our knowledge, this characteristic was
first described for MA. This effect corresponded to a pre-
dominantly vascular histological type, that is, proliferating
vessels. Contrast-enhanced images also showed various
types of enhancement patterns ranging from mild to strong
and even no enhancement, but remarkable enhancement
was the most common pattern with a prevalence of 79.6%
[14]. The mass effect was none or slight. Surrounding
edema was always absent, but adjacent subcortical white
matter was occasionally hyperintense on T2WI resulting
from edema or gliosis [15]. Histopathologically,
MA
has
been
regarded
as
a
hamartomatous, reactive, or neoplasic lesion originating
from multivariate cells, such as meningothelial, fibroblas-
tic, myofibroblastic, smooth muscle, or pluripotent cells
[12]. The characteristics of MA include leptomeningeal
proliferation accompanied by calcification or psammoma
body formation, perivascular cuffs of spindle-shaped
fibroblast-like cells, whorls, or bands of meningothelial
cells in association with sharply demarcated intracortical
plaques of proliferating small vessels. Discussion In our three cases,
all had the specific histological characteristics mentioned
above in the solid portion, but calcification was found only
in case 1. Other histological abnormalities included gliosis,
neuronal dysplasia, cystic degeneration of white matter,
and vascular hyalinization. Some specific signs on MRI may be helpful for MA
imaging diagnoses. Gyriform hyperintense on T2-fluid
attenuated inversion recovery (FLAIR) sequence has
been reported to be the most prominent characteristic
for sporadic MA on MR imaging, which can be attributed
to a thickened cortex with proliferating leptomeningeal
vessels interwoven with bands of fibrous connective tissue
[13]. Calcification may be ignored on routine T1WI and
T2WI, but it produces a susceptibility artifact on T2
gradient echo (GRE) sequences [14]. Chronic hemosiderin
from cavernous malformations also showed susceptibility
artifacts; thus, T2 GRE sequences may only be thought
of as a reference imaging modality for MA diagnoses. Magnetic resonance spectrum (MRS) can aid in the
evaluation of brain lesions by analyzing the spectrum of
metabolites present in the area of study. Rokes et al. [16]
have reported a case of MA with a distinct choline (Cho)
peak and a decreasing N-acetyl aspartate (NAA) peak on
MRS, which suggested a proliferating tumor of presumed
non-neuronal origin. The high Cho peak could have been
a reflection of the proliferation of meningothelial cells
and/or fibroblasts that formed the concentric cuffs sur-
rounding blood vessels in the cortex. However, the high
Cho peak could have also been observed under other
conditions, for example, high-grade brain tumors [17]. Immunohistochemical staining of MA was relatively
unhelpful for diagnosis. Positive vimentin expression
reflected the proliferation of blood vessels and perivas-
cular fibroblast-like cells in almost all cases. However,
the results of other immunohistochemical markers, such
as epithelial membrane antigen (EMA), S-100 protein,
and glial fibrillary acidic protein (GFAP), were variable
in the literature [10]. According to different percentages of perivascular cell
proliferation and cortical vascular proliferation, MA
may be broadly classified into predominantly cellular or
vascular types [13]. Predominantly cellular MA has
moderate to high cellularity such as that found in cases
2 and 3 (Figures 2f and 3d). In the central portion of
the lesion, MA cells emerge from the perivascular location
and infiltrate the cortex. Approximately 90% of reported
cases have cortical invasion, but the proliferating cells
have no significant atypia, mitoses, or necrosis. Cho: choline; CSF: cerebrospinal fluid; CT: computed tomography;
DNET: dysembryoplastic neuroepithelial tumor; DWI: diffusion-weighted
imaging; EMA: epithelial membrane antigen; FLAIR: fluid attenuated inversion Authors’ information Differential diagnoses for MA are not easy due to
variable radiological appearances. Extra-axial tumors,
such as meningiomas, are the most common differential
diagnosis. Intra-axial tumors in the superficial cortex, such
as oligodendroglioma, ganglioglioma, and dysembryoplas-
tic neuroepithelial tumor (DNET), are also frequently
considered [24]. However, for cystic MA, we thought
that differential tumors should include some cystic-mural
tumors with non-enhanced cystic walls, such as heman-
gioblastomas, pilocystic astrocytomas, and pleomorphic
xanthoastrocytomas. The MA cysts are relatively sepa-
rated from solid masses or nodule, which may be helpful
for differential diagnoses. Other non-tumor diseases, that
is, arteriovenous malformation, cavernous hemangioma,
and granuloma, are occasionally misdiagnosed as MA. ZS, FJ, JZ, YF, WL, and YZ are radiologists. HG is an imaging engineer. Competing interests
Th
h
d
l
h p
g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient
for the publication of this case report and any accompany-
ing images. A copy of the written consent is available for
review by the Editor-in-Chief of this journal. 14. Kashlan ON, LaBorde DV, Davison L, Saindane AM, Brat D, Hudgins PA, et al. Meningioangiomatosis: a case report and literature review emphasizing
diverse appearance on different imaging modalities. Case Rep Neurol Med. 2011;2011:361203. 15. Meltzer CC, Liu AY, Perrone AM, Hamilton RL. Meningioangiomatosis: MR
imaging with histopathologic correlation. AJR Am J Roentgenol. 1998;170:804–5. 15. Meltzer CC, Liu AY, Perrone AM, Hamilton RL. Meningioangiomatosis: MR
imaging with histopathologic correlation. AJR Am J Roentgenol. 1998;170:804–5. Conclusions The cases in this study demonstrated that MA may
present with solid or cystic imaging patterns. Imaging
diagnoses for MA were difficult due to non-specific
characteristics. Several signs on MRI may be helpful,
such as gyriform hyperintensity on T2-FLAIR sequences,
susceptibility artifacts on T2 GRE sequence, and flow
void effects on T2WI as in our case. Cysts in cystic MA
demonstrate variable appearances in location, number,
and size, but separation from solid masses or nodules
may be one of its specific characteristics. 8. Kollias SS, Crone KR, Ball Jr WS, Prenger EC, Ballard ET. Meningioangiomatosis of the brain stem. Case report. J Neurosurg. 1994;80:732–5. 9. Kim SH, Yoon SH, Kim JH. A case of infantile meningioangiomatosis with a
separate cyst. J Korean Neurosurg Soc. 2009;46:252–6. 9. Kim SH, Yoon SH, Kim JH. A case of infantile meningioangiomatosis with a
separate cyst. J Korean Neurosurg Soc. 2009;46:252–6. 10. Wiebe S, Munoz DG, Smith S, Lee DH. Meningioangiomatosis. A
comprehensive analysis of clinical and laboratory features. Brain. 1999;122:709–26. 10. Wiebe S, Munoz DG, Smith S, Lee DH. Meningioangiomatosis. A
comprehensive analysis of clinical and laboratory features. Brain. 1999;122:709–26. 11. Kim NR, Cho SJ, Suh YL. Allelic loss on chromosomes 1p32, 9p21, 13q14,
16q22, 17p, and 22q12 in meningiomas associated with
meningioangiomatosis and pure meningioangiomatosis. J Neurooncol. 2009;94:425–30. 12. Kim WY, Kim IO, Kim WS, Cheon JE, Yeon KM. Meningioangiomatosis: MR
imaging and pathological correlation in two cases. Pediatr Radiol. 2002;32:96–8 13. Yao Z, Wang Y, Zee C, Feng X, Sun H. Computed tomography and magnetic
resonance appearance of sporadic meningioangiomatosis correlated with
pathological findings. J Comput Assist Tomogr. 2009;33:799–804. Acknowledgements We thank Sun Cuiyun who provided pathological figures of the cases. This work was supported by National Natural Science Foundation of China
(NSFC, no. 81201152). We thank Sun Cuiyun who provided pathological figures of the cases. This work was supported by National Natural Science Foundation of China
(NSFC, no. 81201152). Received: 1 July 2014 Accepted: 22 January 2015 Received: 1 July 2014 Accepted: 22 January 2015 References 1. Bassoe P, Nuzum F. Report of a case of central and peripheral neurofibromatosis. J Nerv Ment Dis. 1915;42:785–96. 2. Worster-Drought C, Dickson WEC, McMenemy WH. Multiple meningeal and
perineural tumors with analogous changes in the glia and ependyma. Brain. 1937;60:85–117. 3. Arcos A, Serramito R, Santín JM, Prieto A, Gelabert M, Rodriguez-Osorio X,
et al. Meningioangiomatosis: clinical-radiological features and surgical
outcome. Neurocirugía. 2010;21:461–6. Surgical resection is important not only for seizure
control but also for pathological diagnosis. Most MAs
are completely removed without recurrence. However,
long-term seizures disappeared in only 43% of patients
after operation, and antiepileptic drug administration
was required in greater than 70% of patients [10,25]. 4. Tacconi L, Thom M, Symon L. Cerebral meningioangiomatosis: case report. Surg Neurol. 1997;48:255–60. 5. Chen YY, Tiang XY, Li Z, Luo BN, Huang Q. Sporadic meningioangiomatosis-
associated atypical meningioma mimicking parenchymal invasion of brain: a
case report and review of the literature. Diagn Pathol. 2010;5:39–45. 6. Omeis I, Hillard VH, Braun A, Benzil DL, Murali R, Harter DH. Meningioangiomatosis associated with neurofibromatosis: report of 2 cases
in a single family and review of the literature. Surg Neurol. 2006;65:595–603. 7. Abdulazim A, Samis Zella MA, Rapp M, Gierga K, Langen KJ, Steiger HJ, et al. Meningioangiomatosis in a patient with progressive focal neurological
deficient-case report and review of literature. Br J Neurosurg. 2013;27(2):253–5. Authors’ contributions SZ wrote the initial manuscript, and JF corrected grammatical errors in the
text. ZJ, FY, and LW made the imaging diagnoses for the cases, and GH
modified the figures. ZY revised the manuscript, and all authors read and
approved the final manuscript. Discussion Predomin-
antly vascular MA contains thick-walled, hyalinized, and
calcified blood vessels with minimal perivascular cell
proliferation, which is observed in case 1 (Figure 1e,f). Hemorrhage was commonly found in this subtype. We presumed that the MA imaging appearances could
be divided into two patterns: solid and cystic. Solid MA
was more common than cystic MA and demonstrated a
higher probability of calcification and remarkable enhance-
ment in most cases. Less than ten cases with cystic MA
have been reported [18-23], including cases 2 and 3,
although the total number of reported MA cases is
approximately 120. Cysts were localized within or around
solid tumors, one or multiple, and demonstrated micro-
or macrocysts. The exact mechanism underlying MA cyst
development remains controversial. Park et al. [22] specu-
lated that cysts were due to the accumulation of CSF
within lesions in a manner similar to the mechanism of
cyst formation in cystic meningiomas. Supported evidence
included cysts that were eccentric to the solid mass and It has been observed that there are no specific MA
characteristics upon MRI or CT; thus, correct preopera-
tive MA diagnoses were difficult. MA has always been
misdiagnosed as meningioma, oligodendroglioma, gang-
lioglioma, metastasis, or arteriovenous malformation. With
regards to the location of lesions, they may manifest as
intra-axial or extra-axial lesions with well- or ill-defined
margins because of the cortex invasion. CT scans show
that lesions exhibit different densities or even normal
appearance, but calcification may be a specific indication
for MA. The probability of calcification is variable, and the
highest observed was 89.6% [14]. The signal intensity in Sun et al. World Journal of Surgical Oncology (2015) 13:89 Page 5 of 6 Page 5 of 6 adjacent to sulci. However, communication between cysts
and the subarachnoid space has not been confirmed in
MA [20]. Another potential mechanism is enlarged
perivascular spaces in which CSF gradually accumulates,
eventually resulting in the formation of cysts. In our
patient, pathologic examination demonstrated that no
tumor cells were present in the cyst wall. The cyst was
separated from the solid portion of the tumor with no
communication between the cyst and subarachnoid space. Therefore, we assumed that enlarged perivascular space
may be a major reason for forming cystic MA. recovery; GFAP: glial fibrillary acidic protein; GRE: gradient echo;
MA: meningioangiomatosis; MRI: magnetic resonance imaging; MRS: magnetic
resonance spectrum; NAA: N-acetyl aspartate; NF: neurofibromatosis; T1WI:
T1-weighted imaging; T2WI: T2-weighted imaging. 16.
Rokes C, Ketonen LM, Fuller GN, Weinberg J, Slopis JM, Wolff JE. Imaging
and spectroscopic findings in meningioangiomatosis. Pediatr Blood Cancer.
2009;53:672–4. 12.
Kim WY, Kim IO, Kim WS, Cheon JE, Yeon KM. Meningioangiomatosis: MR
imaging and pathological correlation in two cases. Pediatr Radiol. 2002;32:96–8 15.
Meltzer CC, Liu AY, Perrone AM, Hamilton RL. Meningioangiomatosis: MR
imaging with histopathologic correlation. AJR Am J Roentgenol. 1998;170:804–5. Sun et al. World Journal of Surgical Oncology (2015) 13:89 Abbreviations
Ch
h l
CS 16. Rokes C, Ketonen LM, Fuller GN, Weinberg J, Slopis JM, Wolff JE. Imaging
and spectroscopic findings in meningioangiomatosis. Pediatr Blood Cancer. 2009;53:672–4. 16. Rokes C, Ketonen LM, Fuller GN, Weinberg J, Slopis JM, Wolff JE. Imaging
and spectroscopic findings in meningioangiomatosis. Pediatr Blood Cancer. 2009;53:672–4. Page 6 of 6 Page 6 of 6 17. Sibtain NA, Howe FA, Saunders DE. The clinical value of proton magnetic
resonance spectroscopy in adult brain tumours. Clin Radiol. 2007;62:109–19. 18. Fedi M, Kalnins RM, Shuey N, Fitt GJ, Newton M, Mitchell LA. Cystic
meningioangiomatosis in neurofibromatosis type 2: an MRI-pathological
study. Br J Radiol. 2009;82:e129–32. 19. Wang Y, Gao X, Yao ZW, Chen H, Zhu JJ, Wang SX, et al. Histopathological
study of five cases with sporadic meningioangiomatosis. Neuropathology. 2006;26:249–56. 20. Kobayashi H, Ishii N, Murata J, Saito H, Kubota KC, Nagashima K, et al. Cystic
meningioangiomatosis. Pediatr Neurosurg. 2006;42:320–4. 21. Kuchelmeister K, Richter HP, Kepes JJ, Schachenmayr W. Case re 21. Kuchelmeister K, Richter HP, Kepes JJ, Schachenmayr W. Case rep
microcystic meningioma in a 58-year-old man with multicystic
meningioangiomatosis. Neuropathol Appl Neurobiol. 2003;29:170– 22. Park MS, Suh DC, Choi WS, Lee SY, Kang GH. Multifocal
meningioangiomatosis: a report of two cases. AJNR Am J Neuroradiol. 1999;20:677–80. 22. Park MS, Suh DC, Choi WS, Lee SY, Kang GH. Multifocal 23. Li P, Cui G, Wang Y, Geng M, Wang Z. Multicystic meningioangiomatosis. BMC Neurol. 2014;14:32. 24. Feng R, Hu J, Che X, Pan L, Wang Z, Zhang M, et al. Diagnosis and surgical
treatment of sporadic meningioangiomatosis. Clin Neurol Neurosurg. 2013;115(8):1407–14. 25. Seo DW, Park MS, Hong SB, Hong SC, Suh YL. Combined temporal and
frontal epileptogenic foci in meningioangiomatosis. Eur Neurol. 2003;49:184–6. 25. Seo DW, Park MS, Hong SB, Hong SC, Suh YL. Combined temporal and
frontal epileptogenic foci in meningioangiomatosis. Eur Neurol. 2003;49:184–6. Abbreviations
Ch
h l
CS Submit your next manuscript to BioMed Central
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The development and validation of dried blood spots for external quality assurance of syphilis serology
|
BMC infectious diseases
| 2,013
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cc-by
| 5,193
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Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Open Access © 2013 Smit et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. The development and validation of dried blood
spots for external quality assurance of syphilis
serology Pieter W Smit1,2*, Thomas van der Vlis1, David Mabey2, John Changalucha3, Julius Mngara3, Benjamin D Clark2,3,
Aura Andreasen2,4, Jim Todd2,3, Mark Urassa3, Basia Zaba2 and Rosanna W Peeling2 Abstract Background: Syphilis causes up to 1,500,000 congenital syphilis cases annually. These could be prevented if all
pregnant women were screened, and those with syphilis treated with a single dose of penicillin before 28 weeks
gestation. In recent years, rapid point-of-care tests have allowed greater access to syphilis screening, especially in
rural or remote areas, but the lack of quality assurance of rapid testing has been a concern. We determined the
feasibility of using dried blood spots (DBS) as specimens for quality assurance of syphilis serological assays. Methods: We developed DBS extraction protocols for use with Treponema pallidum particle agglutination assay
(TPPA), Treponema pallidum haemagglutination assay (TPHA) and an enzyme immunoassay (EIA) and compared the
results with those using matching plasma samples from the same patient. Results: Since DBS samples showed poor performance with TPHA and EIA (TPHA sensitivity was 50.5% (95%
confidence interval: 39.9–61.2%) and EIA specificity was 50.4% (95% CI: 43.7–57.1%), only the DBS TPPA was used in
the final evaluation. DBS TPPA showed an sensitivity of 95.5% (95% CI: 91.3–98.0%) and a specificity of 99.0% (95%
CI: 98.1–99.5%) compared to TPPA using plasma samples as a reference. Conclusion: DBS samples can be recommended for use with TPPA, and may be of value for external quality
assurance of point-of-care syphilis testing. Keywords: Dried blood spots, Syphilis, Treponema pallidum, DBS, Sensitivity, Evaluation therefore less suitable than point-of-care tests (POCT) for
use in rural or remote locations. POCT screening tests for
syphilis that are sensitive and specific in detecting trepo-
nemal
antibodies
are
now
available
[3]. The
main
advantages of POCTs are that they are easy to use, can be
stored at room temperature, and can be used with whole
blood, collected with a finger prick. The Global Report on
Preterm Birth and Stillbirth and modelling studies have
identified syphilis POCTs testing and treatment as an ur-
gent priority for reducing perinatal morbidity and mortality
[4-6]. Many countries have therefore started to scale up
the use of POCTs in prenatal screening programmes for
syphilis, but the lack of suitable methods for external qual-
ity assurance (EQA) is a serious concern. * Correspondence: Pieter.smit@lshtm.ac.uk
1Leiden Cytology and Pathology Laboratory, Leiden, The Netherlands
2London School of Hygiene & Tropical Medicine, Keppel Street, London
WC1E 7HT, UK
Full list of author information is available at the end of the article Background It is estimated that the burden of congenital syphilis is
large, with 1.5 million cases per year worldwide [1]. These
could be prevented if all pregnant women were screened
and treated with a single dose of benzathine penicillin be-
fore 28 weeks gestation [2]. Syphilis testing is usually done
using laboratory based assays such as Treponema pallidum
haemagglutination assay (TPHA), Treponema pallidum
particle agglutination assay (TPPA), rapid plasma reagin
(RPR), or enzyme Immunoassay (EIA). These tests need to
be used with serum or plasma samples and require a cen-
trifuge, shaker, and refrigeration for the reagents. They are Most QA methods have been developed to monitor
the quality of tests performed in the laboratory, and are
not designed for monitoring POCT usage by healthcare Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Page 2 of 6 Page 2 of 6 workers at remote locations [7]. Dried Blood Spots
(DBS) have been suggested to be a suitable EQA meth-
odology for HIV POCTs, since they are easily collected,
require minimal training and can be sent at ambient
temperature for retesting at a centralised laboratory [8]. DBS samples have been used in prevalence studies for
syphilis serology, but without prior validation of the
methodology [9-11]. DBS samples have been evaluated
with TPPA [12], TPHA [13], and an in-house EIA which
is not commercially available [14]. The TPHA used in
the study by Backhouse et al. is no longer commercially
available. The fourteen year old TPPA protocol used by
Coates et al. did not include a control for biologically re-
active samples, and the final testing concentration of
DBS eluate was more diluted than with plasma, poten-
tially leading to reduced sensitivity for samples with low
antibody titres [12]. The objectives of this study were to
develop and validate DBS protocols for use with com-
mercially available syphilis diagnostic assays, and to de-
termine their performance in syphilis serological assays
using plasma samples as a reference. clinicians and transported to the NIMR laboratory in
Mwanza. EDTA blood samples were bar-coded in the
field to ensure anonymous testing. Within 24 hours, the
blood samples were centrifuged and plasma was stored
in 3 aliquots (1 mL each) at −20°C. From all subjects
participating in the serosurvey who opted for VCT,
1,645 samples were randomly selected for this study. All syphilis serology assays were performed according
to manufacturer’s directions. Procedures For the community based HIV study, DBS samples
(Proteinsaver 903 filter paper, Whatman, GE healthcare,
USA) were collected by finger prick, air dried at ambient
temperature for at least 3 hours and stored with
desiccants in individual ziplock bags. DBS samples were
transported at ambient temperature and upon arrival at
the
laboratory
stored
at
−20°C. After
participants
donated a DBS sample, they were invited to opt in for
VCT. Whole
blood
samples
were
collected
from
consenting participants who opted in for VCT by trained Background The positive and negative
controls supplied with the kits were used with every run. The technicians were blinded to other test results and
TPHA and TPPA results were read by two trained la-
boratory technicians. All tests were performed in at the
NIMR laboratory, which participates in the WHO EQA
programs for TPPA and Rapid plasma Reagin (RPR)
tests. An active syphilis case is defined as RPR positive,
confirmed by a treponemal test. The results were
entered using the laboratory information management
system. The protocol development and evaluation was divided
into two phases. In the first phase, we determined the
feasibility of using DBS with TPHA, TPPA and EIA
using 464 samples. During DBS protocol development,
care was taken to ensure an equivalent DBS sample in-
put was used for each serological method, compared to
plasma. In the second phase, the serological tests that
work best with DBS were selected and validated with
1,181 samples (Figure 1). Plasma samples were used as
the reference standard. The RPR test was performed on
plasma samples that gave discrepant results between
DBS and plasma samples to determine if any active
syphilis cases might have been missed. Active syphilis in-
fection was defined as a positive TPPA and RPR test. Samples were prospectively collected from July through
September 2010 and were tested until March 2011. Dur-
ing phase 1, the preliminary evaluation of different syph-
ilis serological methods with DBS samples, TPPA, TPHA
and EIA were all performed from the same DBS eluate
as only one DBS spot was available. As a reference
method, TPPA, TPHA and EIA were performed on
matching plasma samples in parallel. To ensure blinded
reading in the laboratory, plasma samples were tested
before DBS samples. Additionally, the three tests on
DBS were performed separately from each other, to
maintain blindness to other test results. Research setting The Kisesa open cohort is a well-established on-going
community-based study covering six villages in northern
Tanzania. The cohort study conducts regular demographic
surveillance on HIV prevalence and incidence [15]. Sexual
behaviour data and HIV status are collected by surveys for
which all adults aged 15 years or older are eligible to par-
ticipate. Study participants that opted for voluntary coun-
selling and testing (VCT) were offered HIV and syphilis
POCT (SD Bioline, USA) performed by trained and
experienced technicians. If the VCT participant was trepo-
nemal antibody positive by POCT, free medical treatment
was provided according to the Tanzanian government
recommendations, and all those positive for HIV were re-
ferred to Tanzanian care-and treatment centres. The study
was approved by the Medical Research Coordinating
Committee of the National Institute for Medical Research
in Tanzania (NIMR) and the ethical committee of the
London School of Hygiene and Tropical Medicine. RPR Quantitative
RPR
(BD
Macro-vue
RPR,
Beckton
Dickinson, Sparks MD, USA) was performed according
to manufacturer’s protocols using plasma samples by
trained laboratory technicians. Data analysis The sensitivity, specificity and confidence intervals were
calculated according to standard methods. The agree-
ment between various methods was tabulated. Microsoft
Excel (Microsoft, USA) and the statistical software Stata
11 (StataCorp LP, Texas, USA) were used for analysis of
the results. EIA Plasma aliquots were brought to room temperature and
50 μl were used to test for treponemal antibodies by En-
zyme Immuno Assay (EIA) (Lab21 Syphilis Total Anti-
body EIA, Lab21 healthcare, Kentford, UK). EIA plasma
tests
were
performed
according
to
manufacturer’s
protocols by trained laboratory technicians. A DBS EIA protocol was developed in collaboration
with the developer of the assay. 40 μl DBS eluate was
added and incubated for two hours at 37°C. Plates were
washed five times using an automated washer, 50 μl con-
jugate was added, shaken and incubated for 30 minutes
at 37°C, washed five times, 50 μl substrate was added
and incubated for 30 minutes at room temperature while
kept in the dark. 50 μl stop solution was added and the
wells were read as Optical Density (OD) 450/620 nm
using an automated reader (DTX 800, Beckman Coulter,
USA) with cut-off limits calculated according to the in-
struction manual. The results were then entered directly
into the laboratory information management system. Figure 1 Study design diagram. The number of matching plasma
and DBS samples used for each phase of the study are given in the
boxes on the left and the number of matching DBS and plasma
samples tested for each of the assays are given in the boxes on the
right. RPR has been performed on plasma, not on DBS. in 100 μl Phosphate buffered Saline (PBS) with 0.05%
Tween80 in a clean 96 flat wells plate, shaken for 2 -
minutes and eluted overnight at 4°C. Upon the next day,
the plate was shaken 2 minutes and brought to room
temperature. 25 μl sample dilution buffer was added to
the first column of a clean 96 U-shaped plate, 25 μl DBS
eluate was added and mixed thoroughly. 25 μl of the
mixture was transferred to a second column and 25 μl
sensitized particles were added to column one, 25 μl
unsensitized
particles
to
column
two. Plates
were
covered and incubated for at least two hours at room
temperature on a vibration free surface, before result in-
terpretation. Discordant
results
between
the
two
technicians were recorded as indeterminate. This proto-
col allows TPPA and HIV serological tests to be
performed from one DBS spot. TPPA A total of 1,645 plasma aliquots were brought to room
temperature and 25 μl were used to test for treponemal
antibodies by TPPA (Fujirebio, Tokyo, Japan), according
to manufacturer’s protocols, by laboratory technicians
who routinely perform TPPA on plasma samples. For DBS TPPA testing, the protocol was adjusted as
follows. A 6 mm disk was manually punched and eluted Page 3 of 6 Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 1645
matching plasma
and DBS samples
Phase 1
464 matching
samples
TPPA-464 samples tested
EIA-282 samples tested
TPHA-445 samples tested
Phase 2
1181 matching
samples
TPPA
RPR–1171 plasma samples
tested
–1181 samples
Figure 1 Study design diagram. The number of matching plasma
and DBS samples used for each phase of the study are given in the
boxes on the left and the number of matching DBS and plasma
samples tested for each of the assays are given in the boxes on the
right. RPR has been performed on plasma, not on DBS. For DBS TPHA testing, the following protocol was
developed; 25 μl of DBS eluate (obtained as described
above) was added to 25 μl sample diluent, mixed and
divided over two wells (25 μl each). 75 μl test or control
cells were added and incubated for 1 hour. For both
TPHA and TPPA protocols, the final DBS sample elu-
tion volume was kept comparable with plasma sample
volume. Discordant results between the two technicians
were recorded as indeterminate. matching plasma
and DBS samples Phase 1
464 matching
samples TPHA During the first phase of the project, protocols for DBS
samples were developed for TPPA, (DBS TPPA) TPHA
(DBS TPHA) and EIA (DBS EIA). 464 DBS samples
were tested with TPPA, TPHA, and EIA. Table 1 shows
the sensitivity and specificity of all three syphilis serology
tests using DBS samples compared to plasma samples. Plasma aliquots were brought to room temperature and
25 μl were used to test for treponemal antibodies by TPHA
(Lab21 syphilis TPHA, Lab21 healthcare, Kentford, UK). TPHA
plasma
tests
were
performed
according
to
manufacturer’s protocols by trained laboratory technicians. Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Page 4 of 6 Table 1 Preliminary evaluation: performance of three syphilis serological assays using Dried Blood Spots compared to
plasma
Plasma as reference (using the same assay)
Positive samples
detected
Negative samples
detected
Sensitivity (95% CI) *
Specificity (95% CI) *
DBS TPPA† (n=463)
82/96
363/367
85.4%
98.9%
(76.7–91.8%)
(97.2–99.7%)
DBS EIA (n=282)
53/56
114/226
94.6%
50.4%
(85.1–98.9%)
(43.7–57.1%)
DBS TPHA‡ (n=445)
46/91
353/354
50.5%
99.7%
(39.9–61.2%)
(98.4–100%)
n= samples. * 95% confidence interval. † 1 TPPA DBS indeterminate excluded. ‡ 19 TPHA indeterminate results excluded. Table 1 Preliminary evaluation: performance of three syphilis serological assays using Dried Blood Spots compared to
plasma
*
* performance of three syphilis serological assays using Dried Blood Spots compared to the 34 indeterminate results for DBS TPPA, 30 (88.3%)
were negative and 4 (11.7%) were positive for TPPA using
plasma samples. Out of the 34 indeterminate results, 15
samples were deemed indeterminate because of discord-
ant readings by two technicians and 16 samples were
called indeterminate by both technicians. Of the 179
TPPA positive plasma samples, 66 were RPR positive
(Table 3). DBS detected 66 out of 67 samples that were
plasma TPPA and RPR positive. The one discordant result
was DBS TPPA indeterminate. Unfortunately it was not
possible to retest the 10 false positive DBS samples since
insufficient sample eluate was available by the time plasma
and DBS results were compared. To obtain the titration of
the eight false negative DBS samples, matching plasma
samples were retested with quantitative TPPA and quanti-
tative RPR, as shown in Table 4. Four plasma samples
were negative when retested with TPPA, suggesting a bor-
derline sample or false positive reading when initially
tested. TPHA The DBS EIA was discontinued before the end of phase
1 because of the many false positive results (specificity
50.4%). The TPHA was also excluded from further
testing because of low sensitivity (50.6%). To improve sensitivity of DBS TPPA, technicians were
trained to use a lower cut-off for interpretation of the DBS
TPPA test results, based on the agglutination patterns
seen in phase 1. Reading was adjusted by altering the ag-
glutination positive and negative thresholds applied for
DBS samples because of the higher background compared
to plasma samples (Figure 2). Phase 2: Evaluation of DBS samples in comparison to
matching plasma samples For the final evaluation of DBS TPPA, 1,181 matching
DBS and plasma samples were included. The average age
was 31.9 years (range 15–84) and 760 participants were fe-
male (64%). For 8 persons, no data on gender or age was
recorded and for another 3 persons no data on gender
was available. Out of the 1,181 samples, 179 (15.2%)
plasma samples tested positive by TPPA (Table 2). Exclud-
ing 34 indeterminate results, DBS TPPA showed a sensi-
tivity of 95.5% (95% CI: 91.3–98.0%) and a specificity of
99.0% (95% CI: 98.1–99.5%) compared to TPPA plasma as
the
reference
method. The
DBS
TPPA
reading
adjustments incorporated in phase 2 resulted in a 10.1%
increase in sensitivity without compromising specificity. An overall agreement of 98.7% between the two readers
was found. No non-specific reactivity (positive with
unsensitized particles) with DBS samples was detected. Of Discussion This potentially makes DBS a suitable sample for EQA
of POCTs in remote settings. DBS samples showed excellent sensitivity for the detec-
tion of active syphilis It should be noted the study took place in Tanzania. Although tests were performed and stored according to
the manufacturer’s recommendations, potential environ-
mental effects by transporting and using the kits under
tropical conditions could not be completely ruled out. Additionally, only one DBS spot was available per sam-
ple which restricted the ability to retest discordant test
results or develop appropriate procedures for indeter-
minate samples. Unfortunately, we were not able to obtain acceptable
performance for the use of DBS samples with the Syph-
ilis Total Antibody EIA and Lab21 Syphilis TPHA. The
EIA false positive results were primarily caused by a high
background, possibly due to substances eluted from the
filter
paper
and
whole
blood
that
adhered
non-
specifically to the wells. The TPHA false negative results
were most likely caused by a reduced sensitivity when
using DBS samples. DBS samples tested with TPPA gave
a sensitivity of 95.5% and specificity of 99.0% compared
to plasma samples. Because TPPA is an agglutination assay, experience in
reading results is essential and therefore training is ne-
cessary. 34 DBS samples (3%) were marked as indeter-
minate due to difficulty in interpreting the results or
because of discordant reading by two technicians. DBS
samples can potentially be used for quantitation with
TPPA, although it would require evaluation against titers
obtained with plasma samples. Because of the subjectiv-
ity, it is recommended that TPPA should be read by two
readers. Of the eight samples that were false negative by DBS
TPPA, four were negative when retested with quantita-
tive TPPA on plasma samples and four were false
negatives, of which two had relatively high TPPA titres
(1/320 and 1/640). We tested all plasma samples using
the RPR assay to determine if any of the false negative
samples were from women with active syphilis, defined
as being RPR and TPPA positive. Since only one false
negative DBS sample was positive for RPR, TPPA using DBS TPPA has been used as a surveillance tool in a
few studies [9,11,12] that used a protocol developed by
Coates et al. [12]. The DBS TPPA protocol developed in
this study is an improvement to the protocol developed
by Coates et al. Discussion We developed and validated protocols for the use of
DBS samples with various syphilis serological assays. DBS samples can be recommended for use with TPPA,
and may be of value for external quality assurance of
point-of-care syphilis testing. When finger-prick blood
has been obtained to perform syphilis POC testing, DBS
can be used to collect blood for EQA purposes directly
afterwards. Finger-prick blood spotted onto filter paper
can
be
stored
and
shipped
at
room
temperature, Figure 2 DBS TPPA test result of seven patients. Test outcome (−= negative, + −= indeterminate, and + = positive) was used in phase 2 of
this study. Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Page 5 of 6 Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Page 5 of 6 Table 2 Correlation between detection of Treponema
pallidum antibodies by plasma TPPA and DBS TPPA
(n=1147)
TPPA plasma
Positive
Negative
Total
DBS TPPA
Positive
169
10
179
Negative
8
960
968
Total
177
970
1147*
sensitivity of DBS against plasma 95.5% (95% CI: 91.3–98,0%). specificity of DBS against plasma 99.0% (95% CI: 98.1–99.5%). * excluding 34 indeterminate results. Table 2 Correlation between detection of Treponema
pallidum antibodies by plasma TPPA and DBS TPPA Table 4 Rapid Plasma Reagin (RPR) results on 8 plasma
samples with false negative DBS TPPA results
Sample
DBS
TPPA*
RPR†
BBI99P
N
N
N
BBI792
N
N
N
BBI6GG
N
N
N
BBI5YI
N
N
N
BBI8ZC
N
1/80
N
BBI6LP
N
1/160
N
BBI6K6
N
1/320
N
BBI6NZ
N
>1/640
1/64
N= negative. * Quantitative TPPA. † Quantitative RPR. Table 4 Rapid Plasma Reagin (RPR) results on 8 plasma
samples with false negative DBS TPPA results sensitivity of DBS against plasma 95.5% (95% CI: 91.3–98,0%). specificity of DBS against plasma 99.0% (95% CI: 98.1–99.5%). * excluding 34 indeterminate results. sensitivity of DBS against plasma 95.5% (95% CI: 91.3–98,0%). specificity of DBS against plasma 99.0% (95% CI: 98.1–99.5%). * excluding 34 indeterminate results. allowing the samples to be transported to a central la-
boratory where retesting can be done. The reduction in
required materials, no cold chain requirements and min-
imal training of personnel at clinic level, decreases costs
considerably in comparison to standard blood collection
by venepuncture [16]. Additionally, the stability of
human antibodies stored on DBS has been shown to be
resilient to ambient temperatures for months [17-19]. Discussion as we adjusted the elution so that the
sample input into the TPPA assays from DBS and
plasma are comparable. We also included unsensitized
particles in the procedure to control for biologically re-
active samples. Table 3 RPR titres of TPPA DBS positive samples Table 3 RPR titres of TPPA DBS positive samples Table 3 RPR titres of TPPA DBS positive samples
RPR titre
N=179
Negative
113
1/1
13
1/2
19
1/4
13
1/8
7
1/16
4
1/32
6
1/64
1
>1/128
3 Author details
1 1Leiden Cytology and Pathology Laboratory, Leiden, The Netherlands. 2London School of Hygiene & Tropical Medicine, Keppel Street, London
WC1E 7HT, UK. 3National Institute for Medical Research, NIMR, Mwanza,
Tanzania. 4Mwanza Intervention Trials Unit, Mwanza, Tanzania. doi:10.1186/1471-2334-13-102
Cite this article as: Smit et al.: The development and validation of dried
blood spots for external quality assurance of syphilis serology. BMC
Infectious Diseases 2013 13:102. Received: 6 August 2012 Accepted: 21 February 2013
Published: 26 February 2013 Received: 6 August 2012 Accepted: 21 February 2013
Published: 26 February 2013 Competing interest
The authors declare that they have no competing interests. Competing interest
The authors declare that they have no competing interests. Acknowledgements We are grateful to the study participants, the staff of Kisesa cohort study, the
laboratory staff of the National Institute for Medical Research, Mwanza, and
Mathilde E. Boon for their participation in this project. This study was funded
by a grant to the UNICEF/UNDP/World Bank/WHO Special Programme on
Research and Training in Tropical Diseases from the Bill & Melinda Gates
Foundation (OPP 47697). The EIA and TPHA kits were kindly donated by the
manufacturer for this evaluation. 18. Ferraz AS, Belo EF, Coutinho LM, Oliveira AP, Carmo AM, Franco DL, Ferreira
T, Yto AY, Machado MS, Scola MC, et al: Storage and stability of IgG and
IgM monoclonal antibodies dried on filter paper and utility in Neisseria
meningitidis serotyping by Dot-blot ELISA. BMC Infect Dis 2008, 8:30. 18. Ferraz AS, Belo EF, Coutinho LM, Oliveira AP, Carmo AM, Franco DL, Ferreira
T, Yto AY, Machado MS, Scola MC, et al: Storage and stability of IgG and
IgM monoclonal antibodies dried on filter paper and utility in Neisseria
meningitidis serotyping by Dot-blot ELISA. BMC Infect Dis 2008, 8:30. Foundation (OPP 47697). The EIA and TPHA kits were kindly donated by the
manufacturer for this evaluation. 19. Behets F, Kashamuka M, Pappaioanou M, Green TA, Ryder RW, Batter V,
George JR, Hannon WH, Quinn TC: Stability of human immunodeficiency
virus type 1 antibodies in whole blood dried on filter paper and stored
under various tropical conditions in Kinshasa, Zaire. J Clin Microbiol 1992,
30(5):1179–1182. Authors’ contribution 13. Backhouse JL, Lee MH, Nesteroff SI, Hudson BJ, Hamilton PA: Modified
indirect hemagglutination test for detection of treponemal antibodies in
finger-prick blood. J Clin Microbiol 1992, 30(3):561–563. PWS initiated the study, developed laboratory protocols and drafted the
manuscript. TV developed protocols and drafted the manuscript. DM and
RWP provided supervision throughout the study and made major
contributions to editing the manuscript. JT was responsible for sample
collection, sample process and revision of the manuscript before submission. JM and JC provided supervision for laboratory work, handling and extraction
of the data. BDC provided guidance on the data analysis and participated in
the interpretation of results. AA, BZ and MU planned sample collection and
collaborated in writing of the manuscript. All authors read and approved the
final manuscript. 14. Stevens R, Pass K, Fuller S, Wiznia A, Noble L, Duva S, Neal M: Blood spot
screening and confirmatory tests for syphilis antibody. J Clin Microbiol
1992, 30(9):2353–2358. 15. Tazama project. http://www.tazamaproject.org. 16. De Castro Toledo Jr AC, Januario JN, Rezende RMS, Siqueira AL, De Mello
BF, Fialho EL, Ribeiro RA, Da Silva HL, Pires EC, Simoes TC, et al: Dried blood
spots as a practical and inexpensive source for human
immunodeficiency virus and hepatitis C virus surveillance. Memorias do
Instituto Oswaldo Cruz 2005, 100(4):365–370. 16. De Castro Toledo Jr AC, Januario JN, Rezende RMS, Siqueira AL, De Mello
BF, Fialho EL, Ribeiro RA, Da Silva HL, Pires EC, Simoes TC, et al: Dried blood
spots as a practical and inexpensive source for human
immunodeficiency virus and hepatitis C virus surveillance. Memorias do
Instituto Oswaldo Cruz 2005, 100(4):365–370. 17. Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J,
Abeku T, Bousema T, Ghani AC, et al: Dried blood spots as a source of
anti-malarial antibodies for epidemiological studies. Malar J 2008, 7:195. doi:10.1186/1475-2875-7-195. 17. Corran PH, Cook J, Lynch C, Leendertse H, Manjurano A, Griffin J, Cox J,
Abeku T, Bousema T, Ghani AC, et al: Dried blood spots as a source of
anti-malarial antibodies for epidemiological studies. Malar J 2008, 7:195. doi:10.1186/1475-2875-7-195. Conclusions As prenatal screening for syphilis using POCTs becomes
widely implemented, an EQA method appropriate for
use with blood collected by a finger prick must be
developed to assure the proficiency of POC testing in
rural or remote areas. The objectives of this study were
to develop and validate DBS protocols for use with com-
mercially available syphilis diagnostic assays, and to Smit et al. BMC Infectious Diseases 2013, 13:102
http://www.biomedcentral.com/1471-2334/13/102 Page 6 of 6 Page 6 of 6 determine their performance in syphilis serological assays
using plasma samples as a reference. Based on the high
sensitivity and specificity of DBS TPPA compared to
plasma TPPA, DBS can be recommended for use with
TPPA. Our study also showed the importance of training
laboratory technicians in performing and reading the DBS
TPPA, even when they are already trained in plasma TPPA. We
obtained
a
10.1%
increase
in
sensitivity
when
technicians were more experienced in interpreting DBS
TPPA agglutinations. rapid syphilis tests into an antenatal syphilis screening programme in
Mwanza, Tanzania. Sex Transm Infect 2006, 82(Suppl 5):v38–v43. 6. Aledort JE, Ronald A, Rafael ME, Girosi F, Vickerman P, Le Blancq SM, Landay
A, Holmes K, Ridzon R, Hellmann N, et al: Reducing the burden of sexually
transmitted infections in resource-limited settings: the role of improved
diagnostics. Nature 2006, 444(Suppl 1):59–72. g
pp
7. Plate DK: Evaluation and implementation of rapid HIV tests: the
experience in 11 African countries. AIDS Res Hum Retroviruses 2007,
23(12):1491–1498. 8. Chaillet P, Zachariah R, Harries K, Rusanganwa E, Harries AD: Dried blood
spots are a useful tool for quality assurance of rapid HIV testing in Kigali,
Rwanda. Trans R Soc Trop Med Hyg 2009, 103(6):634–637. 9. Dada Y, Milord F, Frost E, Manshande JP, Kamuragiye A, Youssouf J, Khelifa
M, Pepin J: The Indian Ocean paradox revisited: HIV and sexually
transmitted infections in the Comoros. Int J STD AIDS 2007, 18(9):596–600. References 1. Schmid GP, Stoner BP, Hawkes S, Broutet N: The need and plan for global
elimination of congenital syphilis. Sex Transm Dis 2007, 34(7 Suppl):S5–S10. 1. Schmid GP, Stoner BP, Hawkes S, Broutet N: The need and plan for global
elimination of congenital syphilis. Sex Transm Dis 2007, 34(7 Suppl):S5–S10. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Abbreviations
DBS D i d bl
d 10. Gregson S, Mason PR, Garnett GP, Zhuwau T, Nyamukapa CA, Anderson RM,
Chandiwana SK: A rural HIV epidemic in Zimbabwe? Findings from a
population-based survey. Int J STD AIDS 2001, 12(3):189–196. DBS: Dried blood spots; TPPA: Treponema pallidum particle agglutionation;
TPHA: Treponema pallidum haemagglutination assay; EIA: Enzyme
immunoassay; RPR: Rapid plasma reagin; VCT: Voluntary counselling and
testing; OD: Optical density; POCT: Point of care test; QA: Quality assurance. 11. Hesketh T, Li L, Ye X, Wang H, Jiang M, Tomkins A: HIV and syphilis in
migrant workers in eastern China. Sex Transm Infect 2006, 82(1):11–14. 11. Hesketh T, Li L, Ye X, Wang H, Jiang M, Tomkins A: HIV and syphilis in
migrant workers in eastern China. Sex Transm Infect 2006, 82(1):11–14. 12. Coates GL, Guarenti L, Parker SP, Willumsen JF, Tomkins AM: Evaluation of
the sensitivity and specificity of a Treponema pallidum dried blood spot
technique for use in the detection of syphilis. Trans R Soc Trop Med Hyg
1998, 92(1):44. Submit your next manuscript to BioMed Central
and take full advantage of: 2. Watson-Jones D, Gumodoka B, Weiss H, Changalucha J, Todd J, Mugeye K,
Buve A, Kanga Z, Ndeki L, Rusizoka M, et al: Syphilis in pregnancy in
Tanzania. II. The effectiveness of antenatal syphilis screening and single-
dose benzathine penicillin treatment for the prevention of adverse
pregnancy outcomes. J Infect Dis 2002, 186(7):948–957. y
p
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit • Convenient online submission • Thorough peer review 3. Mabey D, Peeling RW, Ballard R, Benzaken AS, Galban E, Changalucha J,
Everett D, Balira R, Fitzgerald D, Joseph P, et al: Prospective, multi-centre
clinic-based evaluation of four rapid diagnostic tests for syphilis. Sex
Transm Infect 2006, 82(Suppl 5):v13–v16. 4. Victora CG, Rubens CE, Group GR: Global report on preterm birth and
stillbirth (4 of 7): delivery of interventions. BMC Pregnancy Childbirth 2010,
10(Suppl 1):S4. 5. Vickerman P, Peeling RW, Terris-Prestholt F, Changalucha J, Mabey D,
Watson-Jones D, Watts C: Modelling the cost-effectiveness of introducing
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https://openalex.org/W2811465081
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https://dergipark.org.tr/en/download/article-file/484528
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English
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Effect of Tilia tomentosa methanolic extract on growth performance, digestive enzyme activity, immune response and haematological indices of common carp (Cyprinus carpio)
|
Marine science and technology bulletin
| 2,018
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cc-by
| 6,749
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Ahmed Alhadi Almabrok1,2, Iman Daw Amhamed1,2, Gamaia Ali Mohamed1,2,
Soner Bilen2, Tarek Abdalsalam Salem Altief1,2* Ahmed Alhadi Almabrok1,2, Iman Daw Amhamed1,2, Gamaia Ali Mohamed1,2,
Soner Bilen2, Tarek Abdalsalam Salem Altief1,2* 1 Kastamonu University, Institute of Science, Department of Aquaculture, Kastamonu, Turkey
2 Kastamonu University, Faculty of Fisheries and Aquaculture, Department of Aquaculture Kastamonu, Turkey Keywords: Tilia tomentosa
Common carp
Growth
Haematology
Digestive enzyme activity
Immune indices Please cite this paper as follows: Almabrok, A.A., Amhamed, I.D., Mohamed, G.A., Bilen, S., Altief, T.A.S. (2018). Effect of Tilia tomentosa methanolic extract on
growth performance, digestive enzyme activity, immune response and haematological indices of common carp (Cyprinus carpio). Marine Science and Technology Bulletin, 7(1): 12-20. Also it considered as the most growing food produce industry
with an average growth rate more than 7.7% per year through
the last decades, the majority of aquaculture production are
come from Asia (Gjedrem et al., 2012). Approximately 600
aquatic species are raised in captivity in around 190 countries
for produce in fish culture system of varying input intensity * Corresponding author
E-mail address: telhasy@yahoo.com (T.A.S. Altief) Mar. Sci. Tech. Bull. (2018) 7(1): 12-20
e-ISSN: 2147-9666
info@masteb.com Mar. Sci. Tech. Bull. (2018) 7(1): 12-20
e-ISSN: 2147-9666
info@masteb.com http://dergipark.gov.tr/masteb
http://www.masteb.com/ A B S T R A C T This study was conducted to determine the effect of dietary supplementation with
Tilia tomentosa on the growth performance, digestive enzyme activity, haematological
indices and nonspecific immune indices of juvenile common carp (Cyprinus carpio). Fish
with an average weight of 4.35 ± 0.16 g were fed a diet supplemented with an aqueous
methanolic extract of T. tomentosa at a dose of 0% (control), 0.01%, 0.05% or 0.1% for
45 days. The final weight, weight gain and specific growth rate were observed to be
significantly higher for the 00.1% and 0.1% groups compared with the control group (P <
0.05). The feed conversion ratio was significantly decreased in the 0.05% and 0.1%
groups compared with the control (P < 0.05). The activities of various digestive enzymes
(amylase, lipase and trypsin) were also measured and no significant differences were
observed compared to the control (P > 0.05). The mean cell volume of the 0.01% group
was significantly increased compared to the control (P < 0.05) and increased lysozyme
activity was observed in the 0.05% and 0.1% groups. Respiratory burst activity was
significantly increased (P < 0.05) on days 15 and 30 for the 0.1% and 0.05% groups,
respectively. No differences were observed for myeloperoxidase activity among the four
groups. These results suggest that aqueous methanolic extract of T. tomentosa has a
growth-promoting and immunostimulatory effect on common carp. Received: 04.05.2018
Received in revised form: 23.05.2018
Accepted: 23.05.2018
Available online: 30.06.2018 Preparation of T. tomentosa Extract The plants were collected from the Kastamonu province in
the north of Turkey and extracted using a methanol
extraction method (Pakravan et al., 2012) with some
modifications (Bilen et al., 2016) as follows: All ripe parts of
the fruit except the seeds were ground in a mechanical
grinder to a fine powder. Every 50 g of the ground plant were
mixed with 1 L of 40% methanol (Sigma-Aldrich) and the
mixture was allowed to stand at room temperature for three
days with brief shaking once a day to mix. The extract was
then filtered through filter paper (Whatman filter paper No. 1) and the filtrate was collected and evaporated in a rotary
evaporator at 55–65 °C to remove the alcohol from the fruit
extract. The final crude product was dissolved in distilled
water and kept in a flask at 4 °C for later experiments. 𝑊𝐺(%) = 100 × [
(𝐹𝑖𝑛𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡−𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡)
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡
]
𝑆𝐺𝑅 (𝑤𝑒𝑖𝑔ℎ𝑡 % 𝑑−1) = 100 × [𝑙𝑛(𝐹𝑖𝑛𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡) −𝑙𝑛(𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝐹𝑖𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡)
𝐸𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝐷𝑎𝑦𝑠
]
𝐹𝐶𝑅= 𝐹𝑒𝑒𝑑 𝐼𝑛𝑡𝑎𝑘𝑒 (𝑔)
𝑊𝐺 (𝑔)
𝑆𝑅(%) = 100 × [ 𝐹𝑖𝑛𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ] 𝐹𝐶𝑅= 𝐹𝑒𝑒𝑑 𝐼𝑛𝑡𝑎𝑘𝑒 (𝑔)
𝑊𝐺 (𝑔)
𝑆𝑅(%) = 100 × [ 𝐹𝑖𝑛𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ
𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐹𝑖𝑠ℎ] In these formulae, WG indicates weight gain, SGR
indicates specific growth rate, FCR indicates feed conversion
ratio, and SR indicates survival rate. and technological sophistication (Aklakur et al., 2015). 4.35 ± 0.16 g were obtained from a commercial fish farm in
Antalya, Turkey. The fish were transported to the Faculty of
Fisheries and Aquaculture, Kastamonu University, Turkey. The 480 carp were randomly divided into four groups of three
replicates each (i.e. four groups of three aquariums, 12
aquariums in total) with 40 fish per aquarium. The fish were
acclimatised for two weeks before the experiment started. During acclimation, the fish were fed with a commercial diet
twice a day. During the experimental period, the fish were
fed the commercial diet supplemented with T. tomentosa
extract at the following percentages (w/w): 0%, 0.01%,
0.05%, or 0.1%. The fish were fed by hand with the
experimental diet for 45 days to satiation twice a day (at 9
am and 4 pm). The fish were maintained under a natural
photoperiod (12-h dark/12-h light). The water quality
parameters were checked daily and were within the accepted
margins throughout the experiment (dissolved oxygen, 6.8–
7.2 mg/L; pH, 7.7–8.5; water temperature, 25–28 °C). During last decades there has been a continuous growth of
aquaculture industries all over the world and such intensive
production would experience disease problems. Infectious
diseases that occur as sporadic events in wild fish populations
may cause high mortalities when appearing in intensive fish
farming (Gudding et al., 1999). Enhancing the immune system
of cultured fish appears to be the most promising method for
preventing disease. Fish typically depend on nonspecific
immune mechanisms to a much greater extent than most
animals (Chakrabarti and Vasudeva, 2006). Medicinal plants contain phytochemicals, which exhibit
biological activities such as the prevention of chronic
degenerative diseases (Fukumoto and Mazza, 2000). Herbal
medicines have been used in aquaculture to promote growth,
improve the immune system, and stimulate the appetite and
fight against microbes and other stressors, due to the
presence of various active compounds like flavanoids,
alkaloids, phenolics, pigments, steroids, terpenoids and
essential oils (Citarasu, 2010). Recently, immunostimulants of
herbal origin have been shown to boost the disease resistance
of fish towards a number of diseases by improving their
nonspecific and specific defence mechanisms (Harikrishnan et
al. 2011). Indeed, many herbs have been reported to enhance
the immune response of fish (Khondoker et al, 2016). Sample Collection Every 15 days of the feeding trial (i.e. on days 15, 30 and
45), three fish per aquarium (a total of nine carp from each
experimental group) were randomly chosen, anaesthetised by
phenoxyethanol at 0.01 mL/L and individually weighed and
sampled. The kidney tissues were collected and transferred
individually to 1.5 mL RPMI-1640 medium (Invitrogen,
Carlsbad, CA, USA) for direct assay of the immunological
parameters. Blood was collected on day 45 of the feeding
trials from the caudal vein using heparinised syringes in
EDTA-tubes, and later used for the direct assay of the
haematological parameters. Samples of the intestine were
also collected on day 45 of the feeding trials. These samples
were cleaned to remove waste and all visible fat and then
stored at −80 °C for the digestive enzyme assay. The
Tilia
tomentosa
extracts
contained
flavonols
(quercetin, kaempferol, apigenin derivatives) as principal
components with the exception of a single commercial
extract with hydroxycinnamic acids as the most abundant
metabolites (İIeri et al., 2015). T. tomentosa is used as a
medicinal plant in Turkish folk medicine (Baser et al., 2005). The present study was carried out to determine whether the
T. tomentosa extract influences the digestive enzyme
activity, growth, haematological parameters, or activity of
the nonspecific immune response of the common carp
(Cyprinus carpio). Material and Methods The weight of each fish was individually measured at the
beginning and end of the study. The growth performance was
calculated according to the following equations (Tekinay and
Davies, 2001): Introduction *Aquaculture is the most important sector in the world. Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Digestive Enzymes The
digestive
enzyme
activity
levels
for
each
experimental group are shown in Table 2. No significant
differences in the activity of amylase, lipase or trypsin were
observed for fish supplemented with 0.01%, 0.05%, or 0.1% T. tomentosa extract compared with the control group. Table 2. Digestive enzyme activity in the intestines of
Cyprinus carpio supplemented with a methanolic extract of
T. tomentosa for 45 days. Groups
Enzymes
Amylase
Lipase
Trypsin
Control
1.84 ± 0.54
0.009a ± 0.003
0.11 ± 0.01
0.01%
1.58 ± 0.51
0.009 ± 0.001
0.08 ± 0.01
0.05%
3.95 ± 1.26
0.004 ± 0.002
0.12 ± 0.01
0.10%
3.96 ± 1.58
0.006 ± 0.015
0.08 ± 0.05
Note: Values represent the mean ± SE. Haematological Profiles White blood cell counts (WBC × 107 mm−3), red blood cell
counts (RBC × 106 mm−3), haemoglobin levels (Hb, g/dL) and
haematocrit measurements (Hct, %) were measured according
to the methods described by Blaxhall and Daisley (1973). Blood indices included the mean cell volume (MCV, fL), mean
cell Hb (MCH, pg) and mean cell Hb concentration (MCHC, %),
which were calculated according to the formulae of Lewis et
al. (2001). Note: Values represent the mean ± SE; different superscript letters in
a column indicate significant differences between groups (P < .05). IW: initial weight, FW: final weight, WG: weight gain, FCR: feed
conversion ratio, SGR: specific growth rate, SR: survival rate. Nonspecific Immune Parameters Significant increases (P < 0.05) in FW, WG and SGR were
observed for fish supplemented with 0.01% and 0.1% extract
compared to the control. In addition, the FCR was
significantly (P < 0.05) lower in the 0.05% and 0.1%
supplemented groups compared with the control group. No
significant differences were observed between the FW, FCR,
SGR and SR of the other treated groups compared with the
control group. Head kidney cells were isolated from euthanised C. carpio
according to the method of Kono et al. (2012) with slight
modifications as follows. Briefly, the head kidney tissue was
carefully removed and gently pushed through a 100-µm nylon
mesh (John Stanier & Co., Whitefield, Manchester, UK) into
RPMI-1640 medium (Invitrogen) supplemented with 5% foetal
bovine serum (Invitrogen) and a 1% solution of 10,000 g/mL
streptomycin plus 10,000 U/mL penicillin (Invitrogen). This
mixture was then pushed through a 40-µm nylon mesh cell
strainer (Becton, Dickinson & Co., Franklin Lakes, NJ, USA). The final homogenate was placed in a 3-mL Falcon tube. Head kidney cell suspensions were pelleted at 1,800 rpm for
3 min at 4 °C. After centrifugation, the supernatant was
collected to measure myeloperoxidase activity using 3,3,5,5-
tetramethyl benzidine hydrochloride (Sigma-Aldrich) as the
substrate (Sahoo et al., 2005). The lysozyme activity was
measured using a lyophilised Micrococcus lysodeikticus
bacterial cell solution (Sigma-Aldrich) as the substrate (Bilen
et al., 2014). Each pellet was resuspended in 1 mL of the
same medium to directly assay nitroblue tetrazolium (Sigma-
Aldrich) reduction, according to the method described by
Biswas et al. (2013). Statistical Analysis The results were analysed with SPSS software. One-way
ANOVA and Duncan’s multiple range test were used to
determine the significant differences between groups. All
results were expressed as the mean ± SE and P < 0.05 was
considered statistically significant. Results supernatant was removed and stored at −80 °C to test for
digestive enzyme activity as follows. The amylase activity
was determined using 2% starch (Sigma-Aldrich) as a substrate
according to the Worthington (1991) method. The lipase
activity was determined using hydrolysis of 4-nitrophenyl
myristate (Sigma-Aldrich) according to the method described
by Gawlicka et al. (2000). The trypsin activity was
determined using the method of Erlanger et al. (1961) and
benzoyl-DL-arginine p-nitroanilide (Sigma-Aldrich) as the
substrate. The protein concentrations were evaluated with
Bradford (1976) method. The final weight (FW), FCR, SGR, WG and SR of common
carp fed on the experimental diets for 45 days were
determined and presented in Table 1. Table 1. Growth indices of Cyprinus carpio supplemented
with a methanolic extract of T. tomentosa for 45 days. Groups IW (g)
FW (g)
WG (%)
FCR
SGR (%/day)
Control 4.09 ± 0.10a 6.88 ± 0.08a 168.07 ± 5.06a 1.68 ± 0.01a 1.15 ± 0.04a
0.01%
4.06 ± 0.05a 7.71 ± 0.09b 189.87 ± 6.28b 1.78 ± 0.02a 1.42 ± 0.02b
0.05%
4.23 ± 0.06a 7.25 ± 0.07a 171.23 ± 4.57c 1.24 ± 0.01b 1.20 ± 0.02a
0.10%
4.12 ± 0.14a 7.56 ± 0.21b 183.22 ± 6.19d 1.56 ± 0.01c 1.34 ± 0.03b
Note: Values represent the mean ± SE; different superscript letters in
a column indicate significant differences between groups (P < .05). IW: initial weight, FW: final weight, WG: weight gain, FCR: feed
conversion ratio, SGR: specific growth rate, SR: survival rate. Groups IW (g)
FW (g)
WG (%)
FCR
SGR (%/day)
Control 4.09 ± 0.10a 6.88 ± 0.08a 168.07 ± 5.06a 1.68 ± 0.01a 1.15 ± 0.04a
0.01%
4.06 ± 0.05a 7.71 ± 0.09b 189.87 ± 6.28b 1.78 ± 0.02a 1.42 ± 0.02b
0.05%
4.23 ± 0.06a 7.25 ± 0.07a 171.23 ± 4.57c 1.24 ± 0.01b 1.20 ± 0.02a
0.10%
4.12 ± 0.14a 7.56 ± 0.21b 183.22 ± 6.19d 1.56 ± 0.01c 1.34 ± 0.03b
Note: Values represent the mean ± SE; different superscript letters in
a column indicate significant differences between groups (P < .05). IW: initial weight, FW: final weight, WG: weight gain, FCR: feed
conversion ratio, SGR: specific growth rate, SR: survival rate. Experimental Design The intestine samples were homogenised with a Potter
Elvehjem homogeniser in cold double-distilled water (0.1
g/mL) and centrifuged at 9,000 rpm for 20 min at 4 °C. The Common carp (C. carpio) with an average body weight of 13 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Nonspecific Immune Parameters The immunostimulatory effects of the T. tomentosa
extract are shown in Figures 1, 2 and 3. The lysozyme activity
of fish supplemented with different concentrations of T. tomentosa extract was increased on day 30 of the
experiment; this increase was found to be significant (P ˂
0.05) only for the 0.1% supplementation compared with the
control (Figure 1). On the other hand, the lysozyme activity
decreased significantly on day 45 of the experiment for the
0.01% supplementation compared with the control (P ˂ 0.05). The lysozyme activity levels of the other supplementation
groups did not change significantly over time. Figure 1. Lysozyme activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Different
symbols above the bars indicate significant differences
between groups (P ˂ 0.05). Figure 1. Lysozyme activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Different
symbols above the bars indicate significant differences
between groups (P ˂ 0.05). The myeloperoxidase activity of the treated fish did not
significantly differ from that of the control fish during the g
y
g
Table 3. Haematological profiles of Cyprinus carpio supplem Table 3. Haematological profiles of Cyprinus carpio supplemented with a methanolic extract of T. tomentosa for 45 days. Nonspecific Immune Parameters Groups
WBC
(×107cells mm−3)
RBC
(×106cells mm−3)
Hb (g dl−1)
Hct (%)
MCV (fl)
MCH (pg)
MCHC (%)
15th day
Control
24.47 ± 0.18
1.98 ± 0.04
8.22 ± 0.07
27.82 ± 0.84
141.11 ± 7.12
41.57 ± 1.05
296.47 ± 7.63
0.01%
25.63 ± 1.26
1.86 ± 0.09
7.77 ± 0.25
27.68 ± 0.55
148.92 ± 3.73
41.76 ± 0.78
281.13 ± 4.32
0.05%
22.57 ± 1.48
1.97 ± 0.06
8.07 ± 0.10
27.88 ± 0.53
142.44 ± 6.20
41.14 ± 0.89
289.85 ± 8.25
0.10%
26.03 ± 0.97
1.98 ± 0.05
8.27 ± 0.11
28.09 ± 0.87
143.29 ± 8.22
42.02 ± 1.08
295.31 ± 10.34
30th day
Control
22.61 ± 1.67
1.97± 0.08
8.37 ± 0.17
25.99 ± 0.47
132.72 ± 2.71
42.69 ± 0.89
321.95 ± 2.56*
0.01%
26.84 ± 0.91
1.76 ± 0.09
7.72 ± 0.15
26.72 ± 0.33
153.71 ± 6.98*
44.23 ± 1.42
289.10 ± 4.66
0.05%
26.34 ± 1.00
1.81 ± 0.03
7.68 ± 0.36
25.59 ± 0.73
141.85 ± 2.26
42.51 ± 1.21
300.12 ± 7.38
0.01%
26.35 ± 0.45
1.93 ± 0.07
8.08 ± 0.10
26.84 ± 0.09
140.89 ± 5.95
42.1 2± 1.32
301.50 ± 4.48
45th day
Control
23.76 ± 1.85
1.90 ± 0.09
7.31 ± 0.33
25.59 ± 0.62
134.97 ± 4.07
38.45 ± 0.55
285.78 ± 7.19
0.10%
24.16 ± 1.73
2.02 ± 0.05
7.81 ± 0.19
26.35 ± 0.64
131.08 ± 4.68
38.70 ± 1.29
269.27 ± 1.26
0.50%
26.74 ± 1.13
1.97 ± 0.07
7.80 ± 0.10
26.18 ± 0.27
133.49 ± 5.52
39.65 ± 1.08
298.42 ± 6.54
0.01%
27.41 ± 0.15
1.92 ± 0.06
7.70 ± 0.23
26.87 ± 0.48
140.42 ± 4.23
40.14 ± 0.30
286.51 ± 7.17 The myeloperoxidase activity of the treated fish did not
significantly differ from that of the control fish during the
study period (Figure 2). treated groups on day 15 compared to the control group. On
day 30 of the experiment, significantly higher levels of
respiratory burst were observed for the 0.05% and 0.1%
groups, while a significantly lower level was observed for the
0.01% group compared with the control (P ˂ 0.05). The same
pattern was exhibited on day 45 of the experiment (P ˂
0.05). The respiratory burst level of the 1% group was
significantly lower (P ˂ 0.05) than that of the control on day
15 of the experiment (Figure 3). Haematological Profiles Fish supplemented with T. tomentosa extract at different
concentrations had the same haematological parameters as 14 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 study period (Figure 2). Figure 1. Lysozyme activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Different
symbols above the bars indicate significant differences
between groups (P ˂ 0.05). the control group throughout the experimental periods (Table
3), with the exception of the MCV of the 0.01% group on day
30 of the experiment (P ˂ 0.05) and the MCHC of the control
on day 30 of the experiment (highest recorded result
compared with the other groups; P ˂ 0.05). the control group throughout the experimental periods (Table
3), with the exception of the MCV of the 0.01% group on day
30 of the experiment (P ˂ 0.05) and the MCHC of the control
on day 30 of the experiment (highest recorded result
compared with the other groups; P ˂ 0.05). Nonspecific Immune Parameters No significant differences in
the respiratory burst level were observed among the other 15 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Aloe vera did not promote the growth performance of
rainbow trout (Oncorhynchus mykiss). Yılmaz et al. (2012)
reported that the WG, FCR and SGR of sea bass
(Dicentrarchus labrax) were unaffected by 1000 mg/kg
dietary rosemary (Rosmarinus officinalis) and fenugreek. Also, the growth rate of koi carp (Cyprinus carpio) was
unaffected by dietary supplementation with tetra (Cotinus
coggygria) (Bilen et al., 2013). Figure 2. Myeloperoxidase activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Figure 2. Myeloperoxidase activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45 Digestive enzymes play a significant role in the hydrolysis
of proteins, lipids and carbohydrates, thereby assisting with
the assimilation of nutrients. These nutrients are transported
into the tissues and incorporated into cellular materials or
used as an energy source for growth and reproduction (Furne
et al., 2005). The digestion of foods begins with the digestive
enzymes in the stomach and continues in the intestine with
the digestive enzymes secreted by the pancreas, such as
trypsin, chymotrypsin, amylase and lipase (Cockson and
Bourne, 1972; Moriarty, 1973; Fang and Chiou, 1989). Figure 2. Myeloperoxidase activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Figure 2. Myeloperoxidase activity in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Figure 3. Respiratory burst level in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Different
letters above the bars indicate significant differences
between groups (P ˂ 0.05). Our study did not find any significant differences in the
amylase, lipase and trypsin activity levels in any of the
experimental groups compared with the control group. This
result is in line with a study done by Iqbal et al. (2016), who
found no significant differences in the digestive enzyme
activity and haematology of juvenile Labeo rohita following
supplementation with different plant and animal origin feeds
(fishmeal). In contrast, a study by Kawai and Ikeda (1973)
reported an increase in amylase activity in O. Nonspecific Immune Parameters mykiss when
fed a diet containing increased amounts of plant protein. Many
fish
physiologists
have
concentrated
on
haematological studies as this has proved a valuable
diagnostic approach for evaluating fish quality (Kori-Siakpre
et al., 2005; Oluyemi et al, 2008; Patra et al., 2014). Variations in the haematological parameters of fish are due
to environmental stress (Hickey, 1982), malnutrition (Casillas
and Smith, 1977), gender (Siddique and Naseem, 1979;
Collazos et al., 1998), fish size (Garcia et al., 1992), seasonal
differences and breeding efficiency (Cech and Wohlschlang,
1981). The blood characteristics of fish are therefore an
effective and sensitive index for monitoring physiological and
pathological changes (Iwama et al., 1976; Chakrabarti and
Banerjee, 1988; Orun et al., 2003; Patra et al., 2014). The
present study indicated no significant effect on the blood
parameters of C. carpio supplemented with 0.01%, 0.05%, or
0.1% T. tomentosa extract over the study period. This result
suggested that the extract tested here did not stress the fish
physiologically. Figure 3. Respiratory burst level in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Different
letters above the bars indicate significant differences
between groups (P ˂ 0.05). Figure 3. Respiratory burst level in kidney leucocytes of
Cyprinus carpio fed with different experimental diets for 45
days. Values are expressed as the mean ± SE. Different
letters above the bars indicate significant differences
between groups (P ˂ 0.05). Discussion In the present study, T. tomentosa was shown to promote
the growth of common carp based on observations of
increased WG, SGR and efficiency of feed conversion. These
results agree with those of previous studies demonstrating
that medicinal plants promote the growth of various aquatic
animals (Kour et al., 2004; Kaleeswaran et al., 2011; Ojha et
al., 2014). The WG was significantly improved when Japanese
flounder (Paralichthys olivaceus) were supplemented with an
herbal mixture to 500 mg/kg (Seung-Cheol et al., 2007). Most
probably fat was used for energy, and protein was used for
growth in the herbal-supplemented diet (Yılmaz et al., 2012). Nile tilapia fingerlings fed with a basal diet containing 0, 0.5,
1 and 1.5 g/100 g fenugreek (Trigonella foenum-graecum)
seed meal for three months (Mostafa et al. 2009) and they
found that the use of 1 g/100 g fenugreek seed meal
improved the fish growth performance. However, although
some herbs have positive effects on the fish growth (Xie et
al., 2008; Mahdavi et al., 2013), other herbal supplements
have not been observed to have any effects. For example,
Farahi et al. (2012) found that dietary supplementation with Lysozyme is an important enzyme in the humeral
nonspecific defence mechanism that provides defence against
microbial invasion (Evelyn, 2002). The bactericidal action of
this enzyme involves the hydrolysation of the peptidoglycan
layers of the bacterial cell wall, which lyses the cells and
prevents colonisation by microorganisms (Saurabh and Sahoo,
2008). Lysozyme also induces antibacterial activity in the
presence of a complement (Harikrishnan et al., 2011). The
present study recorded significantly increased lysozyme
activity in the 1% supplemented group compared with the
control. Increasing lysozyme activity is in agreement with
several reports on herbal immunostimulants (Rao et al., 2006; 16 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Choi et al., 2008; Bilen et al., 2011). Similar results were
observed for common carp when fed a diet supplemented
with methanolic extracts of Cotinus coggygria (Bilen et al.,
2014) or various Chinese herbal extracts (Jian and Wu, 2004). enhanced the non-specific immune mechanisms and
disease resistance in Oreochromis mossambicus. Fish &
Shellfish Immunology, 29(5): 765-772. Alishahi, M., Ranjbar, M., Ghorbanpoor, M., Peyghan, R. &
Mesbah, M. (2010). Effects of dietary Aloe vera on
some specific and nonspecific immunity in the
common carp (Cyprinus carpio). Iranian Journal of
Veterinary Medicine, 4(3): 189–195. Discussion Neutrophils contain myeloperoxidase in their cytoplasmic
granules (Rodriguez et al., 2003). Myeloperoxidase is an
important enzyme with microbiocidal properties as it utilises
a reactive oxygen species (H2O2) to produce hypochlorous
acid (Dalmo et al., 1997). This process is believed to be key
in killing microorganisms (Johnston, 1978). Although some
studies have shown increased myeloperoxidase activity in fish
following supplementation with, for example, quercetin or
black cumin seed oil (Awad et al., 2013; Alexander et al.,
2010; Bilen et al. 2013), our study showed no significant
effect
on
myeloperoxidase
activity
following
supplementation with T. tomentosa extract at different
concentrations. Awad, E., Austin, D. & Lyndon, A.R. (2013). Effect of black
cumin seed oil (Nigella sativa) and nettle extract
(Quercetin) on enhancement of immunity in rainbow
trout, Oncorhynchus mykiss (Walbaum). Aquaculture,
388: 193-197. Baser, K.H.C., Tümen, G., Malyer, H. & Kirimer,N. (2006). Plants used for common cold in Turkey. In Proceedings
of the IVth International Congress of Ethnobotany
(ICEB 2005), 133, p. 137. Bilen, S., Biswas, G., Otsuyama, S., Kono, T., Sakai, M. &
Hikima, J.I. (2014). Inflammatory responses in the
Japanese pufferfish (Takifugu rubripes) head kidney
cells stimulated with an inflammasome-inducing
agent,
nigericin. Developmental
&
Comparative
Immunology, 46(2): 222-230. Phagocytosis and the respiratory burst response by
phagocytes in blood and tissues present a major antibacterial
defense mechanism in fish (Secombes, 1996). Respiratory
burst activity measured by nitroblue tetrazolium (NBT) is one
of the most important bactericidal mechanisms in fish
(Secombes and Fletcher, 1992). In this study, the respiratory
burst levels of common carp were significantly decreased on
days 15, 30 and 45 of supplementation with 0.1%, 0.05% and
0.01% T. tomentosa extract, respectively, compared with the
control group. For the groups supplemented with 0.05% and
0.1% extract, on days 30 and 45 of the experiment we found
a significant increase in this parameter compared with the
control. This was similar to results observed by Bilen et al. (2011) when O. mykiss was supplemented with Cotinus
coggygria leaves. Harikrishnan et al. (2010) reported a
significant increase in respiratory bursts for olive flounder
supplemented with three different Korean plants. Haghighi
and Rohani (2013) reported a significantly higher respiratory
burst level in rainbow trout fed a commercial diet containing
Zingiber officinale. Our results are also in agreement with
several studies on the use of dietary immunostimulants in
various fish species (Yin et al., 2009; Bilen and Bulut, 2010). Bilen, S., Ünal, S. Acknowledgement Bilen, S., Yılmaz, S., Bilen, A.M. & Biswas, G. (2014). Effects
of Dietary Incorporation of Tetra (Cotinus coggygria)
Extract on Immune Response and Resistance to
Aeromonas hydrophila in Koi Carp (Cyprinus carpio). The Israeli Journal of Aquaculture-Bamidgeh, 66: 1-6. An earlier version of this paper has been presented at
International Congress on Engineering and Life Science ICELIS
2018, Kastamonu, Turkey. Biswas, G., Korenaga, H., Nagamine, R., Kawahara, S.,
Takeda, S., Kikuchi, Y., Dashnyam, B., Yoshida, T. &
Sakai,
M. (2013). Cytokine
mediated
immune
responses in the Japanese pufferfish (Takifugu
rubripes) administered with heat-killed Lactobacillus
paracasei spp. paracasei (06TCa22) isolated from the
Mongolian
dairy
product. International
Immunopharmacology, 17(2): 358-365. Discussion & Güvensoy, H. (2016). Effects of oyster
mushroom (Pleurotus ostreatus) and nettle (Urtica
dioica) methanolic extracts on immune responses and
resistance to Aeromonas hydrophila in rainbow trout
(Oncorhynchus mykiss). Aquaculture, 454: 90-94. Bilen, S. & Bulut, M. (2010). Effects of laurel (Laurus nobilis)
on the non-specific immune responses of rainbow trout
(Oncorhynchus mykiss, Walbaum). Journal of Animal
and Veterinary Advances, 9(8): 1275-1279. Bilen, S., Bulut, M. & Bilen, A.M. (2011). Immunostimulant
effects
of
Cotinus
coggyria
on
rainbow
trout
(Oncorhynchus mykiss). Fish & Shellfish Immunology,
30(2): 451-455. Bilen, S., Yılmaz, S., & Bilen, A.M. (2013). Influence of tetra
(Cotinus coggygria) extract against Vibrio anguillarum
infection in koi carp, Cyprinus carpio with reference
to haematological and immunological changes. Turkish
Journal of Fisheries and Aquatic Sciences, 13(3): 517–
522. Conflict of Interest The authors declare that there is no conflict of interest. References The effects of powdered
ginger (Zingiber officinale) on the haematological and
immunological
parameters
of
rainbow
trout
Oncorhynchus mykiss. Journal of medicinal Plant and
Herbal Therapy Research, 1(1): 8-12. Citarasu, T. (2010). Herbal biomedicines: a new opportunity
for aquaculture industry. Aquaculture International,
18(3): 403-414. Haghighi, M., Sharif Rohani, M., Samadi, M., Tavoli, M.,
Eslami, M. & Yusefi, R. (2014). Study of effects Aloe
vera extract supplemented feed on hematological and
immunological indices of rainbow trout (Oncorhynchus
mykiss). International Journal of Advanced Biological
and Biomedical Research, 2(6): 2143-2154. Cockson, A. & Bourne, D. (1972). Enzymes in the digestive
tract of two species of euryhaline fish. Comparative
Biochemistry and Physiology--Part A: Physiology,
41(4): 715-718. Collazos, M.E., Ortega, E., Barriga, C. & Rodrìguez, A.B. (1998). Seasonal
variation
in
haematological
parameters in male and female Tinca tinca. Molecular
and Cellular Biochemistry, 183(1-2): 165-168. Harikrishnan, R., Balasundaram, C. & Heo, M.S. (2011). Impact of plant products on innate and adaptive
immune system of cultured finfish and shellfish. Aquaculture, 317(1-4): 1-15. Dalmo, R.A., Ingebrigtsen, K. & Bøgwald, J. (1997). Non‐
specific defence mechanisms in fish, with particular
reference to the reticuloendothelial system (RES). Journal of Fish Diseases, 20(4), 241-273. Harikrishnan,
R.,
Jin,
C.N.,
Kim,
M.C.,
Kim,
J.S.,
Balasundaram, C. & Heo, M.S. (2010). Effectiveness
and
immunomodulation
of
chemotherapeutants
against scuticociliate Philasterides dicentrarchi in
olive flounder. Experimental Parasitology, 124(3):
306-314. Erlanger, B.F., Kokowsky, N. & Cohen, W. (1961). The
preparation and properties of two new chromogenic
substrates of trypsin. Archives of Biochemistry and
Biophysics, 95(2): 271-278. Hickey, C.R. (1976). Fish hematology, its uses and
significance. NY Fish Game J, 23(2): 170-175. Evelyn, T.P.T. (2002). Finfish immunology and its use in
preventing infectious diseases in cultured finfish. Diseases in Asian Aquaculture IV, (CR Lavilla-Pitogo &
ER Cruz-Lacierda ed.), 303-324. Ieri, F., Innocenti, M., Possieri, L., Gallori, S. & Mulinacci, N. (2015). Phenolic composition of “bud extracts” of
Ribes nigrum L., Rosa canina L. and Tilia tomentosa
M. Journal of Pharmaceutical and Biomedical Analysis,
115: 1-9. Farahi, A., Kasiri, M., Sudagar, M., Soleimani Iraei, M. &
Zorriehzahra,
S.M.J. (2012). Effect
of
dietary
supplementation of Melissa officinalis and Aloe vera
on hematological traits, lipid oxidation of carcass and
performance in rainbow trout (Oncorhynchus mykiss). Online Journal of Animal and Feed Research, 1: 1-5. Iqbal, K.J., Ashraf, M., Javid, A., Khan, N., Abbas, F.,
Hafeez-ur-Rehman, M., Rafique, M.K., Rasool, F.,
Azmat, H. & Altaf, M. (2016). References Blaxhall, P.C. & Daisley, K.W. (1973). Routine haematological
methods for use with fish blood. Journal of Fish
Biology, 5(6): 771-781. Aklakur, M., Rather, M.A. & Kumar, N. (2015). Nano delivery:
an emerging avenue for nutraceuticals and drug
delivery. Critical reviews in Food Science and
Nutrition, 56(14): 2352-2361. Aklakur, M., Rather, M.A. & Kumar, N. (2015). Nano delivery:
an emerging avenue for nutraceuticals and drug
delivery. Critical reviews in Food Science and
Nutrition, 56(14): 2352-2361. Bradford, M.M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein
utilizing
the
principle
of
protein-dye
binding. Analytical Biochemistry, 72(1-2): 248-254. Alexander, C.P., Kirubakaran, C.J.W. & Michael, R.D. (2010). Water soluble fraction of Tinospora cordifolia leaves 17 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 haematocrit value of two teleosts: rainbow trout
(Oncorhynchus
mykiss)
and
European
sea
bass
(Dicentrarchus labrax L.). Comparative biochemistry
and physiology. Comparative Physiology, 101(4): 733-
736. Casillas, E. & Smith, L.S. (1977). Effect of stress on blood
coagulation and haematology in rainbow trout (Salmo
gairdneri). Journal of Fish Biology, 10(5): 481-491. Casillas, E. & Smith, L.S. (1977). Effect of stress on blood
coagulation and haematology in rainbow trout (Salmo
gairdneri). Journal of Fish Biology, 10(5): 481-491. Cech Jr, J.J. & Wohlschlag, D.E. (1982). Seasonal patterns of
respiration,
gill
ventilation,
and
hematological
characteristics in the striped mullet, Mugil cephalus L. Bulletin of Marine Science, 32(1): 130-138. Gawlicka, A., Parent, B., Horn, M.H., Ross, N., Opstad, I. &
Torrissen, O.J. (2000). Activity of digestive enzymes in
yolk-sac larvae of Atlantic halibut (Hippoglossus
hippoglossus): indication of readiness for first feeding. Aquaculture, 184(3-4): 303-314. Chakrabarty,
P. &
Banerjee,
V. (1988). Effect
of
organophosphorus
pesticides
on
the
peripheral
hemogram of the fish Channa punctatus. Environment
and Ecology, 6(2): 390-394. Gjedrem, T., Robinson, N. & Rye, M. (2012). The importance
of selective breeding in aquaculture to meet future
demands for animal protein: a review. Aquaculture,
350: 117-129. Chakrabarti, R. & Vasudeva, R.Y. (2006). Achyranthes aspera
stimulates the immunity and enhances the antigen
clearance
in
Catla
catla. International
Immunopharmacology, 6(5): 782-790. Gudding, R., Lillehaug, A. & Evensen, Ø. (1999). Recent
developments
in
fish
vaccinology. Veterinary
Immunology and Immunopathology, 72(1-2): 203-212. Choi, S.H., Park, K.H., Yoon, T.J., Kim, J.B., Jang, Y.S. &
Choe, C.H. (2008). Dietary Korean mistletoe enhances
cellular non-specific immune responses and survival of
Japanese eel (Anguilla japonica). Fish & Shellfish
Immunology, 24(1): 67-73. Haghighi, M. & Rohani, M.S. (2013). References Effect of Different Plant
and Animal Origin (Fishmeal) Feeds on Digestive
Enzyme Activity and Haematology of Juvenile Labeo
rohita. Pakistan Journal of Zoology, 48(1): 201-207. Fukumoto, L. R. & Mazza, G. (2000). Assessing antioxidant
and prooxidant activities of phenolic compounds. Journal of Agricultural and Food Chemistry, 48(8):
3597-3604. Iwama, G.K., Greer, G.L. & Larkin, P.A. (1976). Changes in
some hematological characteristics of coho salmon
(Oncorhynchus kisutch) in response to acute exposure
to dehydroabietic acid (DHAA) at different exercise
levels. Journal of the Fisheries Board of Canada,
33(2): 285-289. Furne, M., Hidalgo, M.C., Lopez, A., Garcia-Gallego, M.,
Morales, A.E., Domezain, A., Domezaine, J. & Sanz, A. (2005). Digestive
enzyme
activities
in
Adriatic
sturgeon
Acipenser
naccarii
and
rainbow
trout
Oncorhynchus
mykiss. A
comparative
study. Aquaculture, 250(1-2): 391-398. Jian, J. & Wu, Z. (2004). Influences of traditional Chinese
medicine on non-specific immunity of Jian carp
(Cyprinus
carpio
var. Jian). Fish
&
Shellfish
Immunology, 16(2): 185-191. Garcia, M.P., Echevarria, G., Martinez, F.J. & Zamora, S. (1992). Influence of blood sample collection on the 18 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 Johnston,
J.R. (1978). Oxygen
metabolism
and
the
microbicidal activity of macrophages. Federation
Proceedings, 37(13): 2759-2764. Chandraprakash, S.P. (2014). Effect of ethanolic
extract of Mucuna pruriens on growth, metabolism
and immunity of Labeo rohita (Hamilton, 1822)
fingerlings. International
Journal
of
Fauna
&
Biological Studies, 1(5): 1-9. Johnston,
J.R. (1978). Oxygen
metabolism
and
the
microbicidal activity of macrophages. Federation
Proceedings, 37(13): 2759-2764. Kaleeswaran, B., Ilavenil, S. & Ravikumar, S. (2011). Dietary
supplementation with Cynodon dactylon (L.) enhances
innate immunity and disease resistance of Indian
major carp, Catla catla (Ham.). Fish & Shellfish
Immunology, 31(6): 953-962. Oluyemi, K.G., Adecprusi, E.A. & Olanrewage, J. (2008). Basic hematological parameters in African cat fish,
Clarias gariepinus (Burchell, 1822) FED ascribe acid
supplemented diets. Research Journal of Animal
Sciences, 2(1): 17-21. Kawai, S. & Ikeda, S. (1973). Studies in digestive enzymes of
fishes. III. Development of the digestive enzymes of
rainbow trout after hatching and the effect of dietary
change on the activities of digestive enzymes in the
juvenile stage. Bulletin of the Japanese Society for
the Science of Fish, 38: 265-270. Örün, I., Dörücü, M. & Yazlak, H. (2003). Haematological
parameters of three cyprinid fish species from
Karakaya Dam Lake, Turkey. OnLine Journal of
Biological Sciences, 3: 320-328. Pakravan, S., Hajimoradloo, A. & Ghorbani, R. (2012). References Effect
of dietary willow herb, Epilobium hirsutum extract on
growth
performance,
body
composition,
haematological parameters and Aeromonas hydrophila
challenge
on
common
carp,
Cyprinus
carpio. Aquaculture Research, 43(6): 861-869. Khondoker, S., Hossain, M.M.M., Hasan-Uj-Jaman, M., Alam,
M.E., Zaman, M.F.U. & Tabassum, N. (2016). Effect of
Nigella sativa (Black Cumin Seed) to Enhance the
Immunity of Common Carp (Cyprinus carpio) Against
Pseudomonas fluorescens. American Journal of Life
Sciences, 4(3): 87-92. Patra, B.B., Panda, R.P. & Patra, A.K. (2014). Seasonal
Variations in Certain Hematological Factors of Catla
catla (Hamilton 1822). Journal of Applied Physics, 6:
1-7. Kono, T., Hamasuna, S., Korenaga, H., Iizasa, T., Nagamine,
R., Ida, T. & Sakai, M. (2012). The role of neuromedin
U during inflammatory response in the common carp. Fish & Shellfish Immunology, 32(1): 151-160. Kori-Siakpere,
O.,
Ake,
J.E.G. &
Idoge,
E. (2005). Haematological
characteristics
of
the
African
snakehead, Parachanna obscura. African Journal of
Biotechnology, 4(6): 527-530. Rao, Y.V., Das, B.K., Jyotyrmayee, P. & Chakrabarti, R. (2006). Effect of Achyranthes aspera on the immunity
and survival of Labeo rohita infected with Aeromonas
hydrophila. Fish & Shellfish Immunology, 20(3):, 263-
273. Kour, D., Sharma, L.L. & Sharma, B.K. (2004). Use of herb,
Bala (Sida cordifolia Linn.) as growth promoter in the
supplementary feed of Cirrhinus mrigala (Ham.). Indian Journal of Fisheries, 51(4): 501-504. Rodríguez, A., Esteban, M. & Meseguer, J. (2003). Phagocytosis and peroxidase release by seabream
(Sparus aurata L.) leucocytes in response to yeast
cells. The Anatomical Record, 272(1): 415-423. Kumar, I.V., Chelladurai, G., Veni, T., Peeran, S.S.H. &
Mohanraj,
J. (2014). Medicinal
plants
as
immunostimulants for health management in Indian
cat fish. Journal of Coastal Life Medicine, 2(6): 426-
430. Sahoo, P.K., Kumari, J. & Mishra, B.K. (2005). Non‐specific
immune responses in juveniles of Indian major carps. Journal of Applied Ichthyology, 21(2): 151-155. Saurabh, S. & Sahoo, P.K. (2008). Lysozyme: an important
defence molecule of fish innate immune system. Aquaculture Research, 39(3): 223-239. Lee-Shing, F. & Shu-Fen, C. (1989). Effect of salinity on the
activities of digestive proteases from the tilapia fish,
Oreochromis
niloticus
in
different
culture
environments. Comparative
Biochemistry
and
Physiology Part A: Physiology, 93(2): 439-443. Secombes, C.J. (1996). The nonspecific immune system:
cellular defenses. The Fish Immune System: Organism,
Pathogen and Environment, 15: 63-103. Lewis, S.M., Bain, B.J., and Bates, I. (2001). Dacie and Lewis
Practical Haematology, 9th ed. London: Churchill
Livingstone; p 633. Secombes, C.J. & Fletcher, T.C. (1992). Xie, J., Liu, B., Zhou, Q., Su, Y., He, Y., Pan, L. Ge, X. & Xu,
P., (2008). Effects of anthraquinone extract from
rhubarb Rheum officinale Bail on the crowding stress
response and growth of common carp Cyprinus carpio
var. Jian. Aquaculture, 281: 5–11. Yin, G., Ardó, L., Thompson, K.D., Adams, A., Jeney, Z. &
Jeney, G. (2009). Chinese herbs (Astragalus radix and
Ganoderma lucidum) enhance immune response of Yılmaz, S., Ergün, S. & Çelik, E.Ş. (2012). Effects of herbal
supplements on growth performance of sea bass
(Dicentrarchus labrax): Change in body composition
and some blood parameters. Journal of BioScience and
Biotechnology, 1(3): 217-222. References The role of
phagocytes in the protective mechanisms of fish. Annual Review of Fish Diseases, 2: 53-71. Mahdavi, M., Hajimoradloo, A. & Ghorbani, R., (2013). Effect
of Aloe vera extract on growth parameters of common
carp (Cyprinus carpio). World Journal of Medical
Sciences, 9: 55–60. Seung-Cheol, J.I., Jeong, G., Gwang-Soon, I.M., Lee, S., Yoo,
J. & Takii, K. (2007). Dietary medicinal herbs improve
growth performance, fatty acid utilization, and stress
recovery of Japanese flounder. Fisheries Science,
73(1): 70-76. Moriarty, D.J.W. (1973). The physiology of digestion of blue‐
green algae in the cichlid fish, Tilapia nilotica. Journal of Zoology, 171(1): 25-39. Siddiqui, A.Q. & Naseem, S.M. (1979). The haematology of
Rohu, Labeo rohita. Journal of Fish Biology, 14(1): 67-
72. Mostafa, A.A.Z.M., Ahmad, M.H., Mousallamy, A. & Samir, A. (2009). Effect of using dried Fenugreek seeds as
natural feed additives on growth performance, feed
utilization, whole-body coMyeloperoxidase activity
sition and entropathogenic Aeromonas hydrophila-
challinge of monsex Nile tilapia O. niloticus (L)
fingerlings. Australian Journal of Basic and Applied
Sciences, 3(2): 1234-1245. Tekinay, A.A. & Davies, S.J. (2001). Dietary carbohydrate
level influencing feed intake, nutrient utilisation and
plasma glucose concentration in the rainbow trout,
Oncorhynchus mykiss. Journal of Veterinary and
Animal Sciences, 25(5): 657-666. Worthington, C.S (1991). Worthington enzyme manual related
Biochemical. Freehold, New Jersey, USA. Ojha, M.L., Chadha, N.K., Saini, V.P., Damroy, S. & Ojha, 19 Almabrok et al. (2018) Marine Science and Technology Bulletin 7(1): 12-20 carp,
Cyprinus
carpio,
and
protection
against
Aeromonas hydrophila. Fish & Shellfish Immunology,
26(1): 140-145. carp,
Cyprinus
carpio,
and
protection
against
Aeromonas hydrophila. Fish & Shellfish Immunology,
26(1): 140-145. Yılmaz, S., Ergün, S. & Çelik, E.Ş. (2012). Effects of herbal
supplements on growth performance of sea bass
(Dicentrarchus labrax): Change in body composition
and some blood parameters. Journal of BioScience and
Biotechnology, 1(3): 217-222. 20
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Snake bite on scrotum – a case report
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Abstract A 22-year old man was bitten by a snake on his scrotum. This interesting and unusual case occurred in the rural area of District Aligarh, India. The
uniqueness of the case lies in the fact that scrotum is an extremely rare and unusual site for snake bite. Further, with negligible local signs of
envenoming the patient presented with classical signs of neurotoxicity. Due to numerous superstitions associated with snake bite, the patient was
treated with traditional home made ointment before coming to hospital. The authors realized that the case may be brought to the notice of the
readers because the scrotal bite by the snake with no local signs of envenomation is the first reported case. Received: 21/08/2011 - Accepted: 07/09/2011 - Published: 20/10/2011 Received: 21/08/2011 - Accepted: 07/09/2011 - Published: 20/10/2011 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com)
Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Anjum Arshad1,&, Mateen Azfar1, Husain Munawwar1, Usmani A Jawed1 1Department of Forensic medicine and Toxicology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Department of Forensic medicine and Toxicology, Jawaharlal Nehru Medical College, Aligarh Muslim University, A &Corresponding author: Anjum Arshad, Department of Forensic medicine and Toxicology, Jawaharlal Nehru Medical College, Aligarh Muslim
University, Aligarh, Uttar Pradesh, India Key words: Snake bite, scrotum, India Case presentation The patient Mr ABC (patient details kept anonymous), 22 years old male, is a resident of Iglas, a small town which is about 25 km from the main
city Aligarh. In the city, adequate facilities are available for treatment of all sorts of snake bite, including critical care management in the worst
eventuality. However, due to ritual-based customs and dogma, the victim was treated by home-made ointment (Figure 1). The patient was bitten
by the snake at about 12.30 am and hence precious six hours were lost while the patient was shunted from local quack to “tantrik” who generally
claim to have cure for all sorts of illnesses. Finally, the patient was brought to the Emergency Section of Jawaharlal Nehru Medical College Hospital,
Aligarh, at 06:30 am. According to the history given by the relatives of the patient, and which was substantiated and authenticated by the patient himself upon recovery,
he was sleeping on the roof-top of his mud constructed house wearing a “lungi” (the loin cloth). At 12:30 am he felt something crawling up his
thighs. By the time his reflexes worked the snake bit him on the scrotum and he felt immediate pain followed by little ooze of blood from the bite
site. Immediately, driven by fright he caught the snake and threw it violently on the roof. It was killed by stamping over. No effort was made to
identify it whether it was the poisonous or non poisonous variety. In India, cutting across all distinction between rich or poor, educated or illiterate,
it is an ingrained belief that belligerent snake should be burnt after killing as the picture of its killer gets recorded in the eyes of it. Later, people
believe the female snake particularly distinguish the killer by seeing the eyes of the killed snake and take revenge. Hence the snake was not
available to identify its species. Pan African Medical Journal. 2011; 10:25 This article is available online at: http://www.panafrican-med-journal.com/content/article/10/25/full/ © Anjum Arshad et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited. Page number not for citation purposes
1 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com)
Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 1 Background Since the dawn of civilization, snakes have inspired a mystic feeling of good and evil in human mind. In India popular folklores and deep rooted
superstitions have put the asp in some places at the height of God and somewhere at the depth of hell as the evil incarnate. In India, snakes are
found everywhere from 12,000 feet altitude of the Himalayas down to Cape Comorin, but different areas have different species preponderance. India is inhabited by more than 60 species of venomous snakes out of which only four have been popularly known to be dangerously poisonous to
man; cobra, common krait, Russell viper and Saw Scaled Viper [1]. In India each year approximately 200,000 number of cases of snake bite are
reported, out of which 45,000 to 50,000 succumb to death [2]. The problem is so under-rated that it was only added to WHO´s list of neglected
tropical diseases in April 2009 [2]. Many cases are non poisonous in nature, but emotional calamity and fright render them disastrous to the
victims and their families. The most frequent site of bites is the lower extremity [3]. However, till date snake bite on the scrotum with negligible
local signs has not been reported in scientific journals. Signs and symptoms The patient presented with a classic sings of neurotoxicity. He had ptosis, drooling of saliva, sluggishness, apathy, disorientation, slurring of
speech, inability to hold neck and difficulty in respiration (Figure 2). On examination of bite site, there was slight redness on inferior aspect of
scrotum without any features of swelling, bruising, blistering, local bleeding, etc. The patient was shifted to ICU for support of mechanical
ventilation and was promptly administered the Anti Snake Venom (ASV). The adjunct therapy included atropine, neostigmine, antibiotic, i.v. fluids
etc. This standard treatment continued, and he was weaned away from the ventilator after 36 hours and later shifted to ward for observation. The patient made a remarkable recovery and was discharged without any sequelae after ten days of hospital admission. He is asked to report after
1 week for further evaluation. Page number not for citation purposes 2 Conclusions In Indian subcontinent, people particularly in rural areas sleep on roof tops, which make them prone to Snake bites. Scrotum is a very rare and
unusual site for snake bite. There are numerous superstitions associated with snakes and people still rely on traditional measures as first line of
treatment, and thus valuable time is lost before patient is brought to hospital. Author’s contributions AA collected the data and did analysis and interpretation of literature and made the case report; AM have been involved in collecting pictures and
review articles; MH drafted the manuscript and did proof reading and JAU have given final approval of the version to be published. All authors read
and approved the final manuscript. Proposal The patient was a married man who tied the nuptial knot three months before the unfortunate incident. His wife was not pregnant at the time of
the tragic occurrence. It is justifiably proposed that the patient would be followed up periodically to assess his virility and fertility. Regular
examination of sperm count and normalcy would be done with the consent of the patient. A second reporting shall be done after adequate follow-
up of the case. Figures Figure 1: Homemade ointment rubbed over the scrotum and on bitten part (arrow) as a measure of first line of treatment in a patient victim of
snake bite on scrotum Figure 2: Patient victim of a snake bite on scrotum, admitted in ICU with typical neurotoxic symptoms (ptosis and inability to hold neck) Consent Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written
consent is available for review by the Editor-in-Chief of this journal. Author’s contributions 2.
WHO SEARO (2010): Guidelines on management of snake-bites. New Delhi: WHO Regional Office for South-East Asia, Available at:
http://www.searo.who.int/LinkFiles/BCT_snake_bite_guidelines.pdf. Accessed 1 September 2011 1.
Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, Rodriguez PS, Mishra K, Whitaker R, Jha P; Million Death Study
Collaborators. Snake bite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis. 2011 Apr 12;5(4):e1018.
This article on PubMed 3.
Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of Burdwan, West Bengal. J Indian Med
Assoc. 1992 Jun;90(6):145-7. This article on PubMed Page number not for citation purposes
4 Page number not for citation purposes References References 1. Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, Rodriguez PS, Mishra K, Whitaker R, Jha P; Million Death Study
Collaborators. Snake bite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis. 2011 Apr 12;5(4):e1018. This article on PubMed 1. Mohapatra B, Warrell DA, Suraweera W, Bhatia P, Dhingra N, Jotkar RM, Rodriguez PS, Mishra K, Whitaker R, Jha P; Million Death Study
Collaborators. Snake bite mortality in India: A nationally representative mortality survey. PLoS Negl Trop Dis. 2011 Apr 12;5(4):e1018. This article on PubMed 2. WHO SEARO (2010): Guidelines on management of snake-bites. New Delhi: WHO Regional Office for South-East Asia, Available at:
http://www.searo.who.int/LinkFiles/BCT_snake_bite_guidelines.pdf. Accessed 1 September 2011 2. WHO SEARO (2010): Guidelines on management of snake-bites. New Delhi: WHO Regional Office for South-East Asia, Available at:
http://www.searo.who.int/LinkFiles/BCT_snake_bite_guidelines.pdf. Accessed 1 September 2011 Page number not for citation purposes 3. Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of Burdwan, West Bengal. J Indian Med
Assoc. 1992 Jun;90(6):145-7. This article on PubMed 3. Hati AK, Mandal M, De MK, Mukherjee H, Hati RN. Epidemiology of snake bite in the district of Burdwan, West Bengal. J Indian Med
Assoc. 1992 Jun;90(6):145-7. This article on PubMed Page number not for citation purposes
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Rituximab to treat prolidase deficiency due to a novel pathogenic copy number variation in<i>PEPD</i>
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RMD open
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on October 23, 2024 by guest. Protected by copyrig
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Autoimmunity RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 Decem CLINICAL CASE WHAT THIS STUDY ADDS ⇒We report the second large scale deletion in PEPD
gene, expanding the genetic spectrum of PD. ⇒We report the second large scale deletion in PEPD
gene, expanding the genetic spectrum of PD.i ►Additional supplemental
material is published online only. To view, please visit the journal
online (http://dx.doi.org/10.
1136/rmdopen-2023-003507). ⇒It suggests the therapeutic efficacy of rituximab in
treating PD-associated autoimmunity. To cite: Atschekzei F,
Fedchenko M, Elsayed A, et al.
Rituximab to treat prolidase
deficiency due to a novel
pathogenic copy number
variation in PEPD. RMD Open
2023;9:e003507. doi:10.1136/
rmdopen-2023-003507 WHAT IS ALREADY KNOWN ON THIS TOPIC Prolidase deficiency (PD) is a rare autosomal recessive
inborn error of immunity caused by biallelic homozygous
or compound heterozygous loss-of-function mutations
in PEPD, the gene that encodes prolidase. PD typically
manifests with variable dysmorphic features, chronic
cutaneous ulcers, recurrent infections and autoimmune
features, including systemic lupus erythematosus. So far,
there is no consensus regarding treatment of PD and its
autoimmune manifestations. Here, we present a 28-year-
old female patient with PD due to a novel homozygous
intragenic deletion in PEPD, diagnosed at the age of 6
years and 7 months with an undifferentiated connective
tissue disease that, apart from its very early onset, would
be consistent with the diagnosis of Sjögren’s syndrome. Steroids and diverse conventional synthetic disease-
modifying antirheumatic drugs failed to control PD-
associated vasculitis and mucocutaneous ulcerations and
led to infectious complications, including cytomegalovirus
colitis. Introduction of rituximab (RTX) treatment in this
patient led to sustained recession of mucocutaneous
ulceration, enabling tapering of steroids. High interleukin-
1β (IL-1β) production by this patient’s monocytes, together
with the detection of both IL-1β and interleukin-18
(IL-18) in her serum, suggest enhanced inflammasome
activation in PD, whereas the therapeutic efficacy of RTX
implies a role for CD20 positive B cells in the complex
immunopathogenesis of PD. ⇒Prolidase deficiency (PD) is an inborn error of immu-
nity associated with autoimmunity, especially with
systemic lupus erythematosus (SLE). ⇒Prolidase deficiency (PD) is an inborn error of immu-
nity associated with autoimmunity, especially with
systemic lupus erythematosus (SLE). ⇒Prolidase deficiency (PD) is an inborn error of immu-
nity associated with autoimmunity, especially with
systemic lupus erythematosus (SLE). HOW THIS STUDY MIGHT AFFECT RESEARCH,
PRACTICE OR POLICY ⇒This study may raise awareness among rheumatol-
ogists of PD and lead to its consideration in case
of patients with dysmorphic features and early-
onset autoimmunity mimicking SLE or Sjögren’s
syndrome. ⇒This study may raise awareness among rheumatol-
ogists of PD and lead to its consideration in case
of patients with dysmorphic features and early-
onset autoimmunity mimicking SLE or Sjögren’s
syndrome. Received 17 July 2023
Accepted 23 November 2023 Received 17 July 2023
Accepted 23 November 2023 ⇒Early consideration of rituximab treatment in pa-
tients with PD may spare infectious and autoim-
mune complications. Biallelic homozygous or compound hetero-
zygous loss-of-function mutations in PEPD,
the gene encoding prolidase, cause an auto-
somal recessive inborn error of immunity
(IEI), prolidase deficiency (PD), falling
under diseases of immune dysregulation and
in particular, under the subgroup of auto-
immunity.3 4 Typical manifestations of PD
include dysmorphic features, chronic skin
ulcers, recurrent infections and features of
autoimmune connective tissue diseases. Asso-
ciated laboratory findings include throm-
bocytopaenia,
hypergammaglobulinaemia,
detection of diverse autoantibodies and hypo-
complementaemia. So far, 92 cases and 35
pathogenic mutant alleles have been reported
worldwide.3 5 Diagnosis of PD is based on the
identification of high excretion of imidodi-
peptides in urine or the reduced enzymatic
activity of prolidase in erythrocytes and ►Additional supplemental
material is published online only.
To view, please visit the journal
online (http://dx.doi.org/10.
1136/rmdopen-2023-003507). Rituximab to treat prolidase deficiency
due to a novel pathogenic copy number
variation in PEPD on October 23, 2024 by guest. Protected by copyright. http://rmdopen.bmj.com/
st published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from http://rmdo
blished as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Faranaz Atschekzei,1,2 Mykola Fedchenko,3 Abdulwahab Elsayed,1,2
Natalia Dubrowinskaja,1 Theresa Graalmann,1,4,5 Felix C Ringshausen,5,6,7
Torsten Witte,1,2 Georgios Sogkas1,2 Faranaz Atschekzei,1,2 Mykola Fedchenko,3 Abdulwahab Elsayed,1,2
Natalia Dubrowinskaja,1 Theresa Graalmann,1,4,5 Felix C Ringshausen,5,6,7
Torsten Witte,1,2 Georgios Sogkas1,2 To cite: Atschekzei F,
Fedchenko M, Elsayed A, et al. Rituximab to treat prolidase
deficiency due to a novel
pathogenic copy number
variation in PEPD. RMD Open
2023;9:e003507. doi:10.1136/
rmdopen-2023-003507 INTRODUCTION Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course
of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior
and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with
rituximab (RTX). Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line
highlights upper limit of reference range). on Octob
http://rmdopen.bmj.com/
36/rmdopen-2023-003507 on 7 December 2023. Downloaded from 36/rmdopen-2023-003507 on 7 December 20 tt
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0 3
o
oaded
o Figure 1 Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency
(PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis
(B). Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course
of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior
and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with
rituximab (RTX). Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line
highlights upper limit of reference range). leukocytes in patients with characteristic clinical features,
which leads to genetic testing for PEPD mutations. parents of Turkish descent. She displayed dysmorphic
features, including a low hairline, a depressed nasal
root and micrognathia and telangiectasia predomi-
nantly at her hands and feet. Since the age of 3 years
and 7 months, she displayed painful mucocutaneous
ulcers in her mouth, nose and feet. In addition, she
displayed focal painful parchment-like skin lesions at
both her feet. She had a history of recurrent bron-
chitis since the age of 2 years. At the age of 6.7 years,
she was diagnosed with an undifferentiated connec-
tive tissue disease, whose diagnostic workup revealed Here, we report a 28-year-old female with PD, displaying
features of early-onset Sjögren’s syndrome (SjS) and
vasculitis, due to a novel homozygous large deletion
in PEPD. Rituximab (RTX) treatment in this patient
was successful in controlling vasculitis and cutaneous
ulcerations. uest. Protected by copyright. INTRODUCTION Prolidase or peptidase D (PEPD) is a cytosolic
metalloproteinase
hydrolysing
dipeptides
with a C-terminal proline or hydroxyproline.1
Proline and hydroxyproline are ubiquitous
collagen amino acids, constituting more than
10% of the residues of collagen proteins.2
Hence, dipeptides produced during the
catabolism of collagen are major prolidase
substrates.1 Given its involvement in collagen
catabolism, prolidase plays an essential role in
extracellular matrix remodelling and conse-
quently in wound healing and inflammation. Correspondence to
Dr Georgios Sogkas;
sogkas.georgios@mh-hannover.
de Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 1 RMD Open
RMD Open
RMD Open
Figure 1 Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency
(PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis
(B). Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course
of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior
and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with
rituximab (RTX) Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line
on Oc
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from on October 23,
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from RMD Open
RMD Open
RMD Open Figure 1 Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency
(PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis
(B). Timeline depicting the course of PD in studied patient together with employed immunomodulatory treatments (C). Course
of European Alliance of Associations for Rheumatology (EULAR) Sjögren’s syndrome (SjS) Disease Activity Index (ESSDAI) prior
and during treatment with rituximab (RTX) (D). Course of C reactive protein (CRP) serum values prior and during treatment with
rituximab (RTX). Arrows indicate peaks associating with the diagnosis of bronchitis due to Haemophilus influenza (E; red line
highlights upper limit of reference range). Figure 1 Low hairline, micrognathia and symmetric telangiectasias at fingers and hands of a patient with prolidase deficiency
(PD) (A). CT findings. CT of the lungs showing thymic hyperplasia (indicated with arrows) and bilateral basilar bronchiectasis
(B). Case presentation The index patient (figure 1A) is the older one of two
female siblings born to healthy non-consanguineous Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 on October 23, 2024 by guest. Protected by c
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Autoimmunity
Autoimmunity
Autoimmunity
Figure 2 Histopathological findings from a patient with prolidase deficiency. Biopsy of nasal septal mucosa revealing vascular
occlusion with leukocytoclasia and fibrinoid vessel wall necrosis (marked with *) (A) and immunohistochemistry of same
biopsy revealing substantial C1q, C3 and IgM deposition at the wall of inflamed arterioles (B–D, respectively). Rectum biopsy
(E), revealing chronic active ulcerating colitis. Splenic section after splenectomy, revealing an infarction of unknown aetiology
(marked with *) (F). Liver biopsy (G), showing ectasia of small portal vein branches and adjoining sinusoids, and minimal lobular
and portal hepatitis. on October 23, 2
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded f Figure 2 Histopathological findings from a patient with prolidase deficiency. Biopsy of nasal septal mucosa revealing vascular
occlusion with leukocytoclasia and fibrinoid vessel wall necrosis (marked with *) (A) and immunohistochemistry of same
biopsy revealing substantial C1q, C3 and IgM deposition at the wall of inflamed arterioles (B–D, respectively). Rectum biopsy
(E), revealing chronic active ulcerating colitis. Splenic section after splenectomy, revealing an infarction of unknown aetiology
(marked with *) (F). Liver biopsy (G), showing ectasia of small portal vein branches and adjoining sinusoids, and minimal lobular
and portal hepatitis. the lungs revealed bilateral basilar bronchiectasis,
when she was 20 years old (figure 1B). Same CT scan
showed a thymus hyperplasia. An infection history
and the latter finding led to immunological investi-
gations that revealed reduced T-cells and especially
CD4+-T cell counts (online supplemental table S1),
a finding that led to cotrimoxazole prophylaxis. At
the age of 23 years, she displayed ascites and sple-
nomegaly and was diagnosed with portal hyperten-
sion, whose aetiology remains unknown. Shortly
thereafter, she developed pancytopenia attributed to
hypersplenism, which led to splenectomy (figure 2F)
and consequently improvement of pancytopenia. Given the refractory course of cutaneous ulcerations
and pathological evidence suggesting their vasculitic
aetiology, an RTX treatment was initiated at the age
of 25 years. This included initially two intravenous
1 g infusions separated by 2 weeks, followed by single
1 g infusions every 6 months. Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 Case presentation Since the introduction
of RTX, chronic mucocutaneous ulcerations resolved
completely and C3 as well as C4 complement values
remained normal. These findings associated with a
sustained reduction in the European Alliance of Asso-
ciations for Rheumatology SjS Disease Activity Index
(figure 1D).8 Within the 44-month follow-up period
since the introduction of RTX no new mucocuta-
neous ulcerations appeared. She further displayed no
additional opportunistic or other severe infections. During the aforementioned follow-up period, only
three upper respiratory tract infections (detection of
Haemophilus influenzae in sputum tests in two of those)
have been documented (figure 1E). A chronological fulfilled classification criteria of SjS (SjS), though
very early disease onset was not typical for primary
SjS.6 7 This diagnosis was made on the basis of clin-
ical findings, including bilateral keratoconjuncti-
vitis sicca with positive Schirmer’s test, Raynaud’s
phenomenon and cutaneous vasculitis, histopatho-
logical findings confirming vasculitic aetiology of
mucosal ulcerations (figure 2A–D) as well as labora-
tory findings and in particular, polyclonal hypergam-
maglobulinaemia, hypocomplementeamia and anti-
nuclear antibodies with positive Ro (SS-A) and La
(SS-B) antibodies. Due to aforementioned diagnosis,
prednisolone treatment was started, initially as intra-
venous pulse treatment with a dose of 10 mg/kg and
combined with a variety of conventional synthetic
disease-modifying antirheumatic drugs (csDMARDs)
(methotrexate, azathioprine, ciclosporin). Aggrava-
tion of ulcerations, especially at patient’s feet, led
also to high-dose immunoglobulin treatment. All
aforementioned treatments failed to control chronic
ulcers, which led to a bilateral Syme’s amputation at
the age of 11 years and 6 months, a transtibial ampu-
tation at the age of 14 years and repetitive wound
debridement thereafter. At the age of 13 years, the
patient presented with chronic diarrhoea and was
diagnosed with colitis ulcerosa. Besides infected skin
ulcers and surgical wounds, the patient displayed
no discernible infections during her middle and
late childhood. However, at the age of 19 years she
was diagnosed with cytomegalovirus colitis that was
attributed to the immunosuppressive effect of ciclo-
sporin and prednisolone treatment. A CT scan of on October 23, 2024 by guest. Protected by copyright. mdopen.bmj.com/ ober 23, 2024 by guest. Protected by copyright. 3 Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 on October 23, 2
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Case presentation Downloaded from p
Figure 3 Integrative genomics viewer (IGV) screenshot from whole genome sequencing (WGS) analysis of the studied patient,
showing the large 3 kb deletion in PEPD, spanning exon 4 (A). Western blotting performed with peripheral blood mononuclear
cell (PBMC)-derived protein showing the loss of prolidase expression (B). Urine proline and hydroxyproline levels measured in
a 24-hour urine collection (C; red line highlights upper limit of reference range). High serum levels of interleukin-1β (IL-1β) and
interleukin-18 (IL-18) in studied PD patient compared with a healthy blood donor (HD). IL-1β and IL-18 were measured with
standard ELISAs (D). Higher IL-1β secretion by monocytes from studied PD-patient, compared with an HD, stimulated with
lipopolysaccharide (LPS; 500 ng/mL) and (ATP; 1 mM) (E). Finally, enhanced production of tumour necrosis factor α (TNF-α) by
monocytes from studied PD-patient, compared with an HD, stimulated with ultrapure lipopolysaccharide (LPS; 500 ng/mL) (F). Figure 3 Integrative genomics viewer (IGV) screenshot from whole genome sequencing (WGS) analysis of the studied patient,
showing the large 3 kb deletion in PEPD, spanning exon 4 (A). Western blotting performed with peripheral blood mononuclear
cell (PBMC)-derived protein showing the loss of prolidase expression (B). Urine proline and hydroxyproline levels measured in
a 24-hour urine collection (C; red line highlights upper limit of reference range). High serum levels of interleukin-1β (IL-1β) and
interleukin-18 (IL-18) in studied PD patient compared with a healthy blood donor (HD). IL-1β and IL-18 were measured with
standard ELISAs (D). Higher IL-1β secretion by monocytes from studied PD-patient, compared with an HD, stimulated with
lipopolysaccharide (LPS; 500 ng/mL) and (ATP; 1 mM) (E). Finally, enhanced production of tumour necrosis factor α (TNF-α) by
monocytes from studied PD-patient, compared with an HD, stimulated with ultrapure lipopolysaccharide (LPS; 500 ng/mL) (F). summary of the disease course and immunomodula-
tory medication of the patient is shown in figure 1C. patient-derived monocytes displayed higher IL-1β
secretion as measured by ELISA (figure 3E), that was
already present after stimulation with LPS only. tory medication of the patient is shown in figure 1C. Dysmorphic features, the early onset of autoimmu-
nity and the multiple affected organs suggested an
underlying IEI. Therefore, we initiated genetic testing,
by means of targeted next-generation sequencing,
aiming at evaluating the diagnosis of an autoinflam-
matory disorder and in particular, a type I interfer-
onopathy, as performed previously,9 which yielded no
pathogenic variant. Case presentation Thereafter, we performed whole
genome sequencing (WGS), which revealed a homo-
zygous intragenic deletion of approximately 3 kb
in PEPD gene, spanning exon 4 (NC_000019.10:g. (33989982_33992982del); (33989982_33992982del))
(figure 3). To confirm the diagnosis of PD we initiated
a 24-hour urine collection, which led to detection
of high urinary excretion of proline and hydroxy-
proline, consistent with a PD-associated imidopepti-
duria. Western blotting of protein from the patient’s
peripheral blood mononuclear cell (PBMC) revealed
the absence of prolidase expression. Consistent
with previous reports, the present patient displayed
elevated serum levels of IL-18. In addition, we
detected elevated serum levels of IL-1β. Evaluation of
IL-1β- and IL-18 in serum has been only performed
at last follow-up visit, after introduction of RTX treat-
ment and was suggestive of enhanced inflammasome
activation, that however, did not correlate with clin-
ically evident disease activity (figure 1D). To eval-
uate the latter, monocytes were isolated from the
patient’s PBMC and stimulated with LPS-ATP. Indeed, DISCUSSION Here, we report a case of PD due to a novel large copy
number variation (CNV), spanning exon 4 of PEPD, iden-
tified through WGS. With the exception of a previously
reported large deletion in PEPD,10 all so far reported
pathogenic variants in PEPD were small scale ones.1
Considering aforementioned novel variant, 36 patho-
genic variants have been reported to cause PD. Those
include 16 missense/nonsense variants, 9 indels, 9 splice
variants and 2 CNVs. Autoimmunity in PD can manifest as systemic lupus
erythematosus (SLE)-like disease in approximately 10%
of patients ranging from typical serological evidence
of SLE to severe manifestations such as nephritis and
vasculitis.1 3 5 Additional immune-related manifesta-
tions include vasculitis, AIHA, dermatitis and arthritis. Very early SjS or fulfilment of the relevant diagnostic
criteria6 in the present patient, expands the pheno-
typic spectrum of PD. The exact mechanism of auto-
immunity in PD remains unclear.1 Thymic hyperplasia
in the present patient may indicate a defect in central
tolerance.11 Consistent with previous reports,3 5 the
present patient displayed elevated serum levels of IL-18,
suggesting increased inflammasome activation, which
was confirmed by the higher LPS and ATP-induced IL-1β
secretion by patient-derived monocytes. The efficacy of ober 23, 2024 by guest. Protected by copyright. est. Protected by copyright. Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 4 Autoimmunity
Autoimmunity
Autoimmunity RTX in controlling PD-associated autoimmunity suggests
the pathogenic relevance of autoantibodies and new
plasma cell differentiation (as targeted CD20+ B cells
are required intermediary cells) or the pathogenicity of
alternative B cell functions other than the production of
autoantibodies, such as their antigen-presenting role.12 13 6Department of Respiratory Medicine and Infectious Diseases, Hannover Medical
School, Hannover, Germany 7European Reference Network on Rare and Complex Respiratory Diseases (ERN-
LUNG), Frankfurt, Germany Acknowledgements We thank all nurses and physicians of the outpatient clinics
of the department of Rheumatology and Immunology of the Hannover Medical
School for collecting blood samples and documenting patient’s course and
medications. DISCUSSION g
p
g
Efforts to treat PD with replacement of prolidase
activity included blood transfusions, gene therapy with
an adenoviral vector and enzyme replacement with
liposome-coated prolidase.3 5 All those approaches were
of limited efficacy.3 Allogenic hematopoietic stem cell
transplantation (HSCT) has been tried in a single patient,
who despite reconstitution of prolidase activity died
3 months after HSCT of an invasive fungal infection.14
In this case, steroids, diverse csDMARDs and high-dose
intravenous immunologlobulin treatment were unsuc-
cessful in treating PD and associated immune dysregu-
lation. Remission of vasculitis and consequently steroid
tapering were only possible after the introduction of RTX
treatment. Consistent with a previous report by Sato et
al, reporting the efficacy of RTX as an induction treat-
ment for lupus nephritis and skin ulcers in a 16-year-old
male with PD,15 here we report sustained regression of
vasculitis and mucocutaneous ulcers in a patient with
PD. In case of presented patients, csDMARDs were prior-
itised and repetitive surgical interventions and infectious
complications led to a relatively late introduction of RTX. This case together with the report by Sato et al suggest the
early consideration of RTX treatment in patients with PD
displaying autoimmunity. http://rmdope
hed as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from Contributors GS and FA conceived and planned the study. GS took the lead in
writing the manuscript. FA, MF, TG, FCR and TW significantly contributed to drafting
and revision of the paper. FA and ND performed GS. AE performed western blotting
and ELISAs. GS, TG, FCR and TW were main treating physicians of studied patient. All authors approved the final version. Funding This project was primarily funded by the Rosemarie-Germscheid
foundation. It was additionally supported by the Deutsche Forschungsgemeinschaft
(DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC
2155 'RESIST'—Project ID 39087428 and the German network for multiorgan
autoimmune diseases (GAIN_01GM2206B) and the German Center for Infection
Research (DZIF TTU 01.801). Competing interests None declared. Patient consent for publication Consent obtained directly from patient(s). Ethics approval This study involves human participants and this study was
conducted in accordance with the Declaration of Helsinki and was also approved
from the Ethical committee of the Hannover Medical School (approval number:
5582; 8875_BO_K_2020). All patients signed an informed consent form. Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. DISCUSSION Supplemental material This content has been supplied by the author(s). It has
not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been
peer-reviewed. Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise. on October 23, 2024 by guest. Protected by
http://rmdopen.bmj.com/
23. Downloaded from p y
g
y
Identification of IEIs among patients with well-
classifiable rheumatic disorders can be a clinically rele-
vant diagnostic challenge for rheumatologists.16 Red
flags suggesting an underlying IEI in rheumatic patients
include infectious complications, persistent secondary
hypogammaglobulinaemia and secondary haemophago-
cytic lymphohistiocytosis or macrophage activation
syndrome.16–19 In the present case and overall in PD,
early-onset treatment-refractory connective tissue disease
associating with dysmorphic features should lead to the
diagnostic consideration of PD. In addition, chronic typi-
cally very painful ulcers and atrophic stellate scars, espe-
cially on the feet (atrophie blanche) are very suggestive
for this rare disorder.5 20 Raising awareness of PD and
other rare IEIs, especially disorders of immune dysreg-
ulation whose clinical spectrum overlaps with rheumatic
conditions, may lead to timely genetic diagnosis and
improved clinical outcomes. Open access This is an open access article distributed in accordance with the
Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits
others to copy, redistribute, remix, transform and build upon this work for any
purpose, provided the original work is properly cited, a link to the licence is given,
and indication of whether changes were made. See: https://creativecommons.org/
licenses/by/4.0/. REFERENCES 1 Eni-Aganga I, Lanaghan ZM, Balasubramaniam M, et al. PROLIDASE: a review from discovery to its role in health and
disease. Front Mol Biosci 2021;8:723003. 1 Eni-Aganga I, Lanaghan ZM, Balasubramaniam M, et al. PROLIDASE: a review from discovery to its role in health and
disease. Front Mol Biosci 2021;8:723003. 2 Krane SM. The importance of proline residues in the structure,
stability and susceptibility to proteolytic degradation of collagens
Amino Acids 2008;35:703–10. 2 Krane SM. The importance of proline residues in the structure,
stability and susceptibility to proteolytic degradation of collagens. Amino Acids 2008;35:703–10. 3 Spodenkiewicz M, Spodenkiewicz M, Cleary M, et al. Clinical
genetics of prolidase deficiency: an updated review. Biology (Basel)
2020;9:108. i 4 Tangye SG, Al-Herz W, Bousfiha A, et al. Human inborn errors of
immunity: 2022 update on the classification from the International
Union of immunological societies expert committee. J Clin Immunol
2022;42:1473–507. 5 Alrumayyan N, Slauenwhite D, McAlpine SM, et al. Prolidase
deficiency, a rare inborn error of immunity, clinical phenotypes,
immunological features, and proposed treatments in twins. Allergy
Asthma Clin Immunol 2022;18:17. ;
6 Shiboski CH, Shiboski SC, Seror R, et al. American college of
rheumatology/European league against rheumatism classification
criteria for primary Sjögren’s syndrome: a consensus and data-
driven methodology involving three international patient cohorts. Arthritis Rheumatol 2017;69:35–45. Author affiliations
1 1Rheumatology and Immunology, Hannover Medical School, Hannover, Germany
2Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover,
Germany
3 1Rheumatology and Immunology, Hannover Medical School, Hannover, Germany
2Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover,
Germany 7 Yayla ME, Karaman Z, Torgutalp M, et al. Early onset primary Sjögren
syndrome, clinical and laboratory characteristics. Clin Rheumatol
2020;39:2689–96. 3Institute of Pathology, Hannover Medical School, Hannover, Germany
4Junior Research Group for Translational Immunology, TWINCORE, Center for
Infection Research and the Hannover Medical School, Hannover, Germany
5Biomedical Research in End-Stage and Obstructive Lung Disease Hannover
(BREATH), German Center for Lung Research (DZL), Hannover, Germany 8 Seror R, Bowman SJ, Brito-Zeron P, et al. EULAR Sjögren’s
syndrome disease activity index (ESSDAI): a user guide. RMD Open
2015;1:e000022. Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507 5 on October 23, 2024 by guest. Protected by c
http://rmdopen.bmj.com/
RMD Open: first published as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from RMD Open
RMD Open
RMD Open 20 Dunn R, Varigos G, Winship I. A photographic essay of prolidase
deficiency. Clin Dysmorphol 2011;20:194–9. RMD Open
RMD Open
RMD Open 15 Sato S, Ohnishi T, Uejima Y, et al. Induction therapy with rituximab
for lupus nephritis due to prolidase deficiency. Rheumatology
(Oxford) 2020;59:e57–9. 9 Atschekzei F, Dubrowinskaja N, Anim M, et al. Identification of
variants in genes associated with autoinflammatory disorders
in a cohort of patients with psoriatic arthritis. RMD Open
2022;8:e002561. 15 Sato S, Ohnishi T, Uejima Y, et al. Induction therapy with rituximab
for lupus nephritis due to prolidase deficiency. Rheumatology
(Oxford) 2020;59:e57–9. (
)
16 Sogkas G, Witte T. The link between rheumatic disorders and inborn
errors of immunity. EBioMedicine 2023;90:104501. 10 Hintze JP, Kirby A, Torti E, et al. Prolidase deficiency in a Mexican-
American patient identified by array CGH reveals a novel and the
largest PEPD gene deletion. Mol Syndromol 2016;7:80–6. 17 Sogkas G, Adriawan IR, Ringshausen FC, et al. A novel NFKBIA
variant substituting serine 36 of IκBα causes immunodeficiency
with warts, bronchiectasis and juvenile rheumatoid arthritis in the
absence of ectodermal dysplasia. Clin Immunol 2020;210:108269. 11 Sogkas G, Atschekzei F, Adriawan IR, et al. Cellular and molecular
mechanisms breaking immune tolerance in inborn errors of
immunity. Cell Mol Immunol 2021;18:1122–40. http://rmdopen
shed as 10.1136/rmdopen-2023-003507 on 7 December 2023. Downloaded from y p
18 Zhang M, Behrens EM, Atkinson TP, et al. Genetic defects in
cytolysis in macrophage activation syndrome. Curr Rheumatol Rep
2014;16:439. 12 Randall KL. Rituximab in autoimmune diseases. Aust Prescr
2016;39:131–4. i 19 Sogkas G, Dubrowinskaja N, Adriawan IR, et al. High
frequency of variants in genes associated with primary
immunodeficiencies in patients with rheumatic diseases
with secondary hypogammaglobulinaemia. Ann Rheum Dis
2021;80:392–9. 13 Kurien BT, D’Sousa A, Bruner BF, et al. Prolidase deficiency
breaks tolerance to lupus-associated antigens. Int J Rheum Dis
2013;16:674–80. 19 Sogkas G, Dubrowinskaja N, Adriawan IR, et al. High
frequency of variants in genes associated with primary
immunodeficiencies in patients with rheumatic diseases
with secondary hypogammaglobulinaemia. Ann Rheum Dis
2021;80:392–9. 14 Caselli D, Cimaz R, Besio R, et al. Partial rescue of biochemical
parameters after hematopoietic stem cell transplantation in a patient
with prolidase deficiency due to two novel PEPD mutations. JIMD
Rep 2012;3:71–7. 14 Caselli D, Cimaz R, Besio R, et al. Partial rescue of biochemical
parameters after hematopoietic stem cell transplantation in a patient
with prolidase deficiency due to two novel PEPD mutations. JIMD
Rep 2012;3:71–7. 20 Dunn R, Varigos G, Winship I. A photographic essay of prolidase
deficiency. Clin Dysmorphol 2011;20:194–9. RMD Open
RMD Open
RMD Open 6 Atschekzei F, et al. RMD Open 2023;9:e003507. doi:10.1136/rmdopen-2023-003507
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FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,022
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cc-by
| 2,564
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«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya
va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya “In fact, the "fire of war" was imposed on the Afghan people from the outside, this
is not their choice. Over the years, hundreds of thousands of civilians have become
victims of the confrontation, and millions of people were forced to leave their
homes and seek refuge in other countries. The involvement of more and more
forces in the conflict led to its unprecedented escalation. It has ceased to be purely
Afghan, becoming more and more of a complex international problem. The
expansion of the presence of international terrorist groups in Afghanistan, the
ongoing violence and bloodshed, the drug business - all this indicate the
inadmissibility of the world community ignoring the situation in this country. Most importantly, an entire generation has grown up in Afghanistan in conditions
of armed confrontation and violence. But, nevertheless, this is not the "lost
generation", as some experts cynically claim. These are just people who are tired
of war, deprivation and hardships, who want and strive to put an end to hostility,
return to peaceful life and creation for the benefit of their country. I am firmly
convinced that the Afghan people have the strength, wisdom, courage and
resilience to start a new, peaceful life, build it for the well-being of their children
and future generations...” [1] These words were voiced by the President of
Uzbekistan during his speech at the Tashkent conference. Where in this way he
opened a new page in the relationship between Afghanistan and Uzbekistan, and
not only. Now the Republic of Uzbekistan has determined for itself new priorities in foreign
policy: economic openness and attractiveness of the country for investment and
business; focus on solving all problematic issues on the basis of consensus, mutual
respect and understanding; balanced and mutually beneficial relations with
world powers and other states; the desire for interaction on the basis of political
trust and respect for international law - this is how the country's current foreign
policy is trying to be built. Before turning to specific areas of foreign policy, I
would like to say a few words about how the country's foreign policy priorities
are formed in principle. It is known that, speaking of priorities, many people have in mind bilateral
relations with certain countries, especially singling out the world's leading states
with significant political, economic, military and other potential. `
«Zamon
va ama `
«Zamon
va ama «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 This is of course
obvious and even necessary. 354 354 «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya
va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya
However, it is also necessary to proceed from what the main tasks in general
hould be solved by foreign policy. The most important of them is the creation of
avorable external conditions for the successful internal development of the state
and society, the implementation of reforms, ensuring the growth of the welfare of
he population, and improving the quality of life. The second task is to form a belt of peace stability and security around `
«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy
va amaliyot» nomli ilmiy, masofaviy, onlayn konferensi
However, it is also necessary to proceed from what the main tasks in general
should be solved by foreign policy. The most important of them is the creation of
favorable external conditions for the successful internal development of the state
and society, the implementation of reforms, ensuring the growth of the welfare of
the population, and improving the quality of life. The second task is to form a belt of peace, stability and security around
Uzbekistan. It is security in the region where the Republic of Uzbekistan is located
that is the main condition for sustainable development. From which we can see that Afghanistan occupies a special place in the foreign
policy of the Republic of Uzbekistan. Today, Uzbekistan and Afghanistan are
developing close political, trade, economic, cultural and humanitarian relations. The security of Afghanistan is perceived as the security of Uzbekistan, a guarantee
of stability and prosperity for the entire vast region. Uzbekistan's policy towards
Afghanistan is based on the understanding that the prospects for stable
development in Central Asia are closely linked to the achievement of peace in
neighboring Afghanistan. `
y
y
q q
y
va amaliyot» nomli ilmiy, masofaviy, onlayn konferens
However, it is also necessary to proceed from what the main tasks in general
should be solved by foreign policy. The most important of them is the creation of
favorable external conditions for the successful internal development of the state
and society, the implementation of reforms, ensuring the growth of the welfare of
the population, and improving the quality of life. However, it is also necessary to proceed from what the main tasks in general
should be solved by foreign policy. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 On the other hand, Uzbekistan is trying to pursue its own economic
policy in relation to Afghanistan, and the northern regions of the country, in
particular.[2] 355 355 `
«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy
va amaliyot» nomli ilmiy, masofaviy, onlayn konferens
It should be noted that the participation of Uzbekistan in the Afghan processes
remains hardly noticeable in the international information environment. This is
largely due to the weak information policy of Tashkent and the lack of a PR
component of foreign policy. At the same time, Uzbekistan remains one of the
most active actors in Afghanistan. After declaring independence in 1991, Uzbekistan found itself in a rather difficult
situation. Internal problems associated with the need to reform the public
administration system, the transition to a market economy, the implementation
of social reforms, and the prevention of interethnic conflicts put serious pressure
on the stability of the state. At the same time, external factors related to the
destabilization of neighboring Afghanistan and Tajikistan also raised concerns. In
this regard, the Afghan issue has firmly taken its place as one of the highest
priorities on the foreign policy agenda of Uzbekistan. Tashkent tried to make the
most of the stands of international organizations to draw the attention of the
international community to the aggravation of the situation in the neighboring
country. In the 90s. the process of forming the position of Uzbekistan on the
events in Afghanistan took place. Tashkent identified the destabilization of
Afghanistan as the main threat to the security of all of Central Asia. At the same
time, Uzbekistan advocated a peaceful settlement of the conflict through
negotiations. In addition, great importance, in the opinion of the Uzbek side,
should have been given to ensuring conditions for disarming the parties and
preventing the use of force. In particular, during the 50th session of the UN
General Assembly in 1995, Tashkent put forward an initiative to impose an
embargo on the supply of weapons to Afghanistan.[3] `
«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
It should be noted that the participation of Uzbekistan in the Afghan processes
remains hardly noticeable in the international information environment. This is
largely due to the weak information policy of Tashkent and the lack of a PR
component of foreign policy. At the same time, Uzbekistan remains one of the
most active actors in Afghanistan. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 The most important of them is the creation of
favorable external conditions for the successful internal development of the state
and society, the implementation of reforms, ensuring the growth of the welfare of
the population, and improving the quality of life. From which we can see that Afghanistan occupies a special place in the foreign
policy of the Republic of Uzbekistan. Today, Uzbekistan and Afghanistan are
developing close political, trade, economic, cultural and humanitarian relations. The security of Afghanistan is perceived as the security of Uzbekistan, a guarantee
of stability and prosperity for the entire vast region. Uzbekistan's policy towards
Afghanistan is based on the understanding that the prospects for stable
development in Central Asia are closely linked to the achievement of peace in
neighboring Afghanistan. According to the Concept of Foreign Policy of Uzbekistan, adopted in 2012,
ensuring a peaceful settlement of the conflict, stabilizing Afghanistan and building
pragmatic relations with the southern neighbor are among the main priorities of
the Uzbek foreign policy. The Uzbek strategy towards Afghanistan began to take shape as early as AD. 90s
The position of Uzbekistan on the Afghan crisis, announced during the 48th and
50th sessions of the UN General Assembly in 1993 and 1995 hasn't changed much
in the last 20 years. Tashkent believes that the settlement of the conflict is
possible only through diplomatic means, without the use of force. In addition,
special attention is expected to be paid to building a strong vertical of power,
economic recovery, and creating conditions for the observance of the rights of all
ethnic and national minorities in Afghanistan. The Uzbek strategy is being
implemented in two directions. On the one hand, Tashkent participates in the
process of resolving the Afghan crisis at the political and diplomatic level: the
initiative to create the Contact Group "6 + 2" (1997), proposals to impose an
embargo on arms supplies to Afghanistan (1995) and the demilitarization of the
country (2001). .), calls to resume the activities of the Contact Group (2008), the
provision of territory for the transportation of goods of the international
coalition. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 The achievements of Uzbekistan in this matter is that on March 26-27, 2018,
Tashkent hosted a High-Level International Conference on Afghanistan, which
was attended by the President of the IRA A. Ghani, the High Representative of the
European Union for Foreign Affairs and Security Policy F. Mogherini and
representatives member states of the UN Security Council. It was the first time
that such a large-scale forum dedicated to solving the problem of Afghanistan was
held at such a high level. The main result and success of the Tashkent conference
is that it was possible to develop and approve a consolidated international
approach to the issue of settling the Afghan crisis. All this is reflected in the
Tashkent Declaration, which was recognized by the absolute majority of the states
involved in the Afghan dialogue. The Tashkent conference and, in general, Uzbekistan's policy towards
Afghanistan led to the intensification of international efforts to resolve the Afghan 356 356 `
«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariy
va amaliyot» nomli ilmiy, masofaviy, onlayn konferens
conflict and involve this country in regional trade, economic and infrastructure
projects that should help advance the peace process. Uzbekistan began economic cooperation with Afghanistan in 2002. This
cooperation affects several aspects: infrastructure construction, energy and
trade. The opening of the Hairatan bridge on the Uzbek-Afghan border in 2002
was the first step towards the implementation of the strategy. Already the
following year, the Airitom customs post began to function in Termez. These steps
were aimed at ensuring the delivery of humanitarian aid to Afghanistan.[4] In
addition, at the request of the Afghan government, Uzbekistan restored 11
bridges between Mazar-i-Sharif and Kabul that were destroyed during the civil
war. This was of strategic importance, because. these bridges link the northern
and eastern regions of Afghanistan and are an important component for economic
recovery in the region.[5] Much attention is paid to the development of transport communications. In 2008,
the Asian Development Bank (ADB), Uzbekistan and Afghanistan signed a
protocol of understanding to expand cooperation in the field of railway
construction. In the period 2008-2010. Uzbekistan Railways (Uzbek Railways)
implemented the ADB-financed Hairatan-Mazari-Sharif railway construction
project with a total cost of $170 million. «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 Источник:
Пресс-служба
Президента
Республики
Узбекистанhttp://isrs.uz/ru/page/pdf/o-mezdunarodnoj-konferenci-po-
afganistanu afganistanu
[2] Скромный актор в афганском урегулировании: стратегия Узбекистана
в Афганистане 21 августа, 2015 Юрий Саруханян
[3] http://www.press-service.uz/ru/document/4825/
[4] Akmalov, Shoislam, 2009 « Uzbekistan’s Role in Stability and Development
of Afghanistan », in Policy Perspectives http://www.ips.org.pk/pakistan-and-its-
neighbours/1048-uzbekistans-role-in-stability-and-development-of-
afghanistan.html
[5] Эргашев, Бахтиер, 2012 «Узбекистан и Афганистан: на пути к
региональной
безопасности
в
Центральной
Азии»
http://www.12news.uz/news/2012/12/uzbekistan-i-afganistan-na-puti-k-
regi/
[6] http://www.railway-technology.com/projects/hairatanuzbekistanra/
[7] «Узбекистан в 2013 году приступит к строительству ж/д Мазари-
Шариф-Андхой», 2013 http://www.12news.uz/news/2013/02/uzbekistan-v-
2013-godu-pristupit-k-stroit/ [7] «Узбекистан в 2013 году приступит к строительству ж/д Мазари-
Шариф-Андхой», 2013 http://www.12news.uz/news/2013/02/uzbekistan-v-
2013-godu-pristupit-k-stroit/ «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazari
va amaliyot» nomli ilmiy, masofaviy, onlayn konferen
FOREIGN POLICY INITIATIVES TO REGULATE THE AFGHAN CRISIS
Meliyev Noʻmonxon Ibodulla oʻgʻli
Toshkent state university of oriental studies
Assistant teacher
https://doi.org/10.5281/zenodo.7192713 Its length is 75 km, the volume of cargo
transportation is 7 million tons with a possible increase to 20 million tons.[6]
According to official data, in 2013 Tashkent began construction of a 230-
kilometer section of the railway between Mazar-i-Sharif and Andkhoy, which is
part of the Sherkhan-Bandar-Kunduz-Khulm-Naybabad-Andkhoy-Herat railway
project, implemented under the program of the Central -Asian Regional Economic
Cooperation (CAREC).[7] It should be noted that such projects are important for
strengthening the Afghan economy by uniting the Afghan provinces through the
transport network. At the same time, the effective functioning of transport
corridors is an opportunity for Uzbekistan to reduce the negative impact of the
country's isolation and gain access to South Asian markets and the Persian Gulf. Despite the difficult situation, the long-term systemic crisis in Afghanistan, the
foreign policy initiative of Uzbekistan calls on the countries of the region to
consider Afghanistan as a friendly partner and neighbor. Analyzing the priority
directions of the foreign policy of the Republic of Uzbekistan, which pursue
different goals, one can come to the conclusion that this state is taking all
measures to resolve the Afghan conflict, namely political and economic measures. The foreign policy strategy of Uzbekistan is capable of ensuring a sufficiently
effective participation in the processes of settling the Afghan conflict, both at the
regional level and within the framework of bilateral relations. Despite the 357 `
«Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya
va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya
scenario of further development of the situation in the country, Uzbekistan
remains interested in minimizing the negative impact of Afghanistan on the `
«Zamon
va amal «Zamonaviy dunyoda innovatsion tadqiqotlar: Nazariya
va amaliyot» nomli ilmiy, masofaviy, onlayn konferensiya scenario of further development of the situation in the country, Uzbekistan
remains interested in minimizing the negative impact of Afghanistan on the
countries of the region and continues to play an important role in resolving the
Afghan military-political crisis. [1] Выступление Президента Республики Узбекистан Шавката Мирзиёева
на международной конференции по Афганистану: "Мирный процесс,
сотрудничество в сфере безопасности и региональное взаимодействие" 27
мар. 2018
г.
|
https://openalex.org/W2131746445
|
https://zenodo.org/record/2312946/files/article.pdf
|
English
| null |
LXXVII.—Steric influence: static and dynamic. Part II
|
Journal of the Chemical Society. Transactions
| 1,915
|
public-domain
| 2,628
|
View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue 728 DAVIS AND RIXON : STERIC lNFLUENCE : By OLIVER CHARLES MINTY DAVIS arid
FREDERIC
WILLIAM
RIXON. IN a previous communication (Davis, ZeitscA. physikal. Chern.,
1912, 78, 353) the results of an investigation on the interaction
of formic acid with aniline and its congeners were described, the
reaction being expressed by the general equation : H*CO,H + R*NH,
R*NH*COH + H,O. View Article Online View Article Online 729 STATIC AND DYNAMIC. rmr 11. I n such a system it is possible to measure under suitable con-
ditions : d on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 915. Downloaded by University of Chicago on 29/10/2014 17:47:52. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. (1) The velocity of formation of the anilide. (1) The velocity of formation of the anilide. ( )
y
(2) The velocity of decomposition of the anilide. y
(2) The velocity of decomposition of the anilide. ( )
y
p
(3) The position of the point of equilibrium obtained by a direct
method. (3) The position of the point of equilibrium obtained by a direct
method. The research described dealt almost exclusively with the third
possibility above mentioned, and numerous equilibrium points were
determined by a static method. The main object of the present
iiivestigatioii was to obtain three independent sets of data by a
study of the measurements enumerated above, with a view of Published on 01 January 1915. Downloaded by University of Chicago on 29/10 Published on 01 January 1915. Downloaded by University of Chicag Frc. 1. Decomposition of for Inn 11 ilicle. Published on 01 January 1915. Downloaded by Univ l6 I
.4
P 12
5
8
4
/
/
/
/
/*
/
/
/
/
1
/
/
#
/
#
&#*
AM’
4
8
12
16
20
/
/
/
/
/*
/
/
/
/
1
/
/
#
/
#
&#*
AM’
4
8
12
16
20
Hozrrs. cornparing their relative value, as applied particularly to steric
influence. I n order to enable such a comparison to be made, the
three setis of data have been determined, working under conditions
as similar as possible in all cases. By OLIVER CHARLES MINTY DAVIS arid
FREDERIC
WILLIAM
RIXON. l6 I
.4
P 12
5
8
4
/
/
/
/
/*
/
/
/
/
1
/
/
#
/
#
&#*
AM’
4
8
12
16
20
/
/
/
/
/*
/
/
/
/
1
/
/
#
/
#
&#*
AM’
4
8
12
16
20
Hozrrs. .4
P 5
8 Hozrrs. cornparing their relative value, as applied particularly to steric
influence. I n order to enable such a comparison to be made, the
three setis of data have been determined, working under conditions
as similar as possible in all cases. Experimental Conditio?ts.-A solvent was employed consisting
of two volumes of pyridine and one of water, this solvent giving
homogeneous solutdons. The temperatlure of reaction was that of
boiling water, variations due to barometric changes being of 110
importance. Preliminary experiments were made with solutions of the follow-
iiig concentrations : View Article Online 730 DAVIS AND RIXON : STERIC INFLUENCE : Rate of Forrnafion .-One
gram-molecule of formic acid and
1 gram-molecule of aniline per litre. 915. Downloaded by University of Chicago on 29/10/2014 17:47:52. shed on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52 Rate of Decomposition.-One
gram-molecule of the anilide and
1 gram-molecule of water per litre." The measurement of rates of formation and equilibria in such
solutions could be carried out satisfactorily, but the determination
of the rates of decomposition were not completely successful. The
following table and curve show the results obtained for the decom-
position of formanilide. Published on 01 January 1915. Downloaded by University of Chicago on Time in hours. Per cent. decomposed. 2
0.26
3
0.51
5
1-09
6
1-34
16
9.1
20
14.83 Time in hours. Per cent. decomposed. 2
0.26
3
0.51
5
1-09
6
1-34
16
9.1
20
14.83 The reaction appears to- be catalysed by one of the reaction
products, and the addition of formic acid produces a considerable
increase in t,he initial rate of decomposition, thus: Per cent. decomposed. A
r
7
After the addition
Molecular
of 0.1 gram-mol. Substance. Hours. solution. of
formic
acid. Formanilide
... ... 1 ;
Formo-o-toIuidide ... ... * This water is in addition to that already present in the solvent. By OLIVER CHARLES MINTY DAVIS arid
FREDERIC
WILLIAM
RIXON. {
0.26
0.51
0-53
1-14
3.45
4.77
3-8
6.2 1 This effect is confined to the decomposition measurements, a
similar excess of acid in the other determinations pro'ducing altera-
tions which could be attributed normally to the increase in the
mass of the formic acid; thus in the reaction between aniline and
formic acid 59.2 per cent. of the anilide was formed in molecular
solution, whilst with an excess of the acid as stated, the formation
was 62.38 per cent. The solutions used throughout the following experiments were
made up as follows:
i
ili
d
i
i
1
l
li Rate of Formation-Aniline
or derivative, 1 gram-mol. per litre,
and formic acid, 1.1 gram-mol. per litre. Rate of D1ecomposition.-Formanilide or derivative, 1 gram-mol. per litre; formic acid, 0.1 gram-mol. per litre; and water, 1 gram-
mol. per litre (in addition to water present in the solvent). View Article Online STATIC AND DYNAMIC. PART 11. 731 t A slight variation was introduced, the formic acid solution and the water
being measured into the flask instead of being weighed. * The loss due to volatilisation was found to be negligible.
t Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 15. Downloaded by University of Chicago on 29/10/2014 17:47:52. Eate of Fornintion.-The
aniline or derivative was weighed into
a stoppered measuring flask, a weighed quantity of standardised
formic acid solution in the pyridine-water solvent was added, the
required volume made up with the solvent, and the flask weighed. Of this solution one portion was weighed in a stoppered flask and
titrated with N / 10-sodium hydroxide, and six other portions were
placed in tubes of Jena-glass, previously steamed. These tubes,
closed by corks having narrow grooves cut in them,%- were lieatecl
on a water-bath, being supported in the lid of the bath by enlarge-
ments on the tubes. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 915. Downloaded by University of Chicago on 29/10/2014 17:47:52. shed on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52 E'quili 6 riuin : a
Kw=-
b+c' where a, 6, c are the concentrations of the anilide or derivative,
aniline or derivative, and formic acid respectively. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 Published on 01 January 1915. Downloaded by University of Chicag At intervals of iift’eer. minutes a tube was taken out and rapidly
cooled, and the contents were poured into a stoppered flask,
weighed, and titrated. Rate of Decomposition.-The
method adopted for making the
solution was that described above. A portion of the solution
having been titrated, the remainder was heated in a Jena flask
fitted with a ground-in reflux condenser. Portions of the solution
were removed by a pipette every hour, rapidly cooled, weighed, and
i5trated.t Equilibrium.-Portions
of the solutions prepared for the above
determinations were sealed in glass tubes, the method used being
that described by Davis (Zoc. cit.). Treatment of Data.-Empirical
formula: have been used to cal-
culate the constants, giving fairly consistent results, as will be
seen from the table which follows. Water being present in com-
paratively large quantity, a practical constancy
been assumed in calculating the decomposition
equilibrium constant. in its amount has
constant and the The formulae used are: Formation : where t =time in minutes, a= concentration of the aniline or deriv-
ative = 1, b =concentration of the formic acid = 1.1, and x = amount
of the anilide formed. where t =time in minutes, a= concentration of the aniline or deriv-
ative = 1, b =concentration of the formic acid = 1.1, and x = amount
of the anilide formed. D e co m Position : 1
a(b+x)
k - -log-
’ - t(u - 6)
6(a - x)’ where a =concentration of the anilide or derivative = 1, b = concen-
* * The loss due to volatilisation was found to be negligible. t t A slight variation was introduced, the formic acid solution and the water
being measured into the flask instead of being weighed. View Article Online View Article Online 732 DAVIS AKD RIXON : STERIC INFLUENCE : tratioii of the formic acid = 0.1, ~t: =ainouiit of the anilide decoiii-
posed in t minutes. tratioii of the formic acid = 0.1, ~t: =ainouiit of the anilide decoiii-
posed in t minutes. Discussion of R eszdts. Published on 01 January 1915. Downloaded by University of Chica It is possible1 t o compare the results in two distinct ways; either
the effect of the position of a single subst.ituent group may be FIG. 2. FIG. 2. 11
10
4
0
g
9
-J 0 000
considered, or the result of substituting different groups in the
same position may be taken as a basis of comparison. FIG. 2. 11
10
4
0
g
9
-J 0 000 4
0
g 4
0
g considered, or the result of substituting different groups in the
same position may be taken as a basis of comparison. It will be seen by consulting the table already given or the
one following that the rates of formation and the percentage of
substance formed run approximately parallel, and that the values
in the case of ortho-, meta-, and para-isomerides increase in the
order mentioned, the increase being particularly pronounced for
0- and m-halogen-substituted derivatives. g
No such regularity is found in thel rates of decomposition. Refelrring to the effects of the various groups when substituted
in similar pwitions, it may be said that it is difficult to arrange d on 01 January 1915. D de .............................. oluidide
..................... toluidide ..................... oluidide ........................ nisidide
..................... isidide ..................... ormanilide ..................... ormanilide ..................... rmanilide ..................... ormanilide ..................... rmanilide ..................... naphthalide .................. naphthalide .................. ormanilide ...................... Average
4
0.0045
0.00108
0.0053
0.0067
0.0018
0.0099
0.00028
0.00 14
0.0021
0.00013
0.00154
0.0025
0.00072
0-0036
Maximum
difference
of any
constant
from
average. 0.0004
0.00005
0.0007
0.0003
0*0001
0*0004
0-0001
0.00007
0~0001
0.00008
0.00004
0~0002
0~00002
0.0005
rather large maximum difference from the average i
the catalytic action whic View Article Online View Article Online DAVIS AND RIXON : STEHIC INFLUENCE : 734 them in order of influence. I n the orthepositions they may be
ordered thus: ,@-ring, O*CH,, CH,, a-ring, Br, Cl.* them in order of influence. I n the orthepositions they may be
ordered thus: ,@-ring, O*CH,, CH,, a-ring, Br, Cl.* 5. Downloaded by University of Chicago on 29/10/2014 17:47:52. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. I n the meta- and para-positions the effect of the halogen atoms
is in tho reverse order. Stress cannot be laid on this owing to the
slight differences in the numbers obt'ained for these particular
substances, especially in the case of
the ortho- and meta-
isonierides. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 The rates of decomposition do not lend themselves to any ordered
comparison. Furtlier comparisons may be readily made by the aid of the
table on p. 735. Conclusion. Equilibrium measurements applied to a reaction or series of
reactions give more reliable results than do the determinations of
either the rates of formation or decomposition, and should be, if
possible, selected for the investigation of a reaction provided it is
only intended to make one type of observation. On the other
hand, the comparison of velocity measurements with equilibrium
data gives, with regard t o the mechanism of a reaction, much more
complete and valuable information, which may probably be used
to discriminate between the various factors in the reaction. Published on 01 January 1915. Downloaded by Unive Published on 01 January 1915. Down Factors of the Reactions. I n the formation and the decomposition of the anilides the steric
influence is one factor, the chemical effect of the substituent may
be regarded as a second, and the action of the formic acid as a
third; there may be other factors, but these three are9 obvious. y
I f H represents a factor compounded of the firs6 and second,
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made in which the a- and 8-rings are involved. kaaFH. By combining these two relations, the factor F, which would be
supposed to have equal accelerative effect on both reactions, can
be eliminated, and
1 H2k,aL2 or H2a k, 1 k,. * The a- and &rings are included, although the inclusion can hardly
be justified; this remark applies equally to other comparisons that are
made in which the a- and 8-rings are involved. View Article Online STATIC AND DYNAMIC. PART 11. 735 s
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L cvn. Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. [Received, February 20th, 19 15.1 View Article Online View Article Online LE SUEUR AND WI'I'HELiS : HENEICOSOIC ACID. 736 Making this assumption, the values of 15 may be expressed propor-
tionally in round numbers as follows: Making this assumption, the values of 15 may be expressed propor-
tionally in round numbers as follows: Published on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52. 15. Downloaded by University of Chicago on 29/10/2014 17:47:52. hed on 01 January 1915. Downloaded by University of Chicago on 29/10/2014 17:47:52 Group. 0. m. p. Ring. CH, ............... 15
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formanilide is the unit,. Published on 01 January 1915. Downloaded by University of Chicago on The similar results obtained from the halogens point to the
conclusion that the extra weight of the bromine atom is counter-
balanced by the, chemical activity of the chlorine, the two atoms
exercising an equal hindering effect. Published on 01 January 1915. Downloaded by University I f steric influence is proportional to the weight of the substituent
group, the factor H might be divided by the weight of t,he group
in question ; the quotient would be a proportional representation
of what might be termed "chemical hindrance." The above method of treatment is purely tentative, and is sug-
gested as possibly allowing a direct comparison t o be drawn
between the effects produced by substituting different groups in a
reacting system, and a discrimination to be made between certain
of the reaction factors. The' expenses in connexion with the work have been partly met
by a grant from the Royal Society, for which the authors express
their thanks, CHEMICAL DEPARTMEN
BRISTOL
UNIVERSITY [Received, February 20th, 19 15.1
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https://openalex.org/W4317852656
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https://figshare.com/articles/preprint/3D_Quantum-inspired_Self-supervised_Tensor_Network_for_Volumetric_Segmentation_of_Brain_MR_Images/12909860/4/files/38883345.pdf
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English
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3D Quantum-inspired Self-supervised Tensor Network for Volumetric Segmentation of Medical Images
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3D Quantum-inspired Self-supervised Tensor Network for
Volumetric Segmentation of Medical Images This paper was downloaded from TechRxiv (https://www.techrxiv.org). LICENSE SUBMISSION DATE / POSTED DATE
02-09-2020 / 24-01-2023 SUBMISSION DATE / POSTED DATE
02-09-2020 / 24-01-2023 g
Debanjan Konar, SMIEEE, Siddhartha Bhattacharyya, SMIEEE, Tapan K. Gandhi, SMIEEE,
Bijaya K. Panigrahi, SMIEEE, and Richard Jiang, SMIEEE Debanjan Konar, SMIEEE, Siddhartha Bhattacharyya, SMIEEE, Tapan K. Gandhi, SM
Bijaya K. Panigrahi, SMIEEE, and Richard Jiang, SMIEEE for accurate and fast segmentation of volumetric medical
images with different modalities.i Abstract—This paper introduces a novel shallow 3D self-
supervised tensor neural network for volumetric segmentation of
medical images with merits of obviating training and supervision. The proposed network is referred to as the 3D Quantum-inspired
Self-supervised Tensor Neural Network (3D-QNet). The underly-
ing architecture of 3D-QNet is composed of a trinity of volumetric
layers viz. input, intermediate, and output layers inter-connected
using an S-connected third-order neighborhood-based topology
for voxel-wise processing of 3D medical image data, suitable for
semantic segmentation. Each of the volumetric layers contains
quantum neurons designated by qubits or quantum bits. The
incorporation of tensor decomposition in quantum formalism
leads to faster convergence of the network operations to preclude
the inherent slow convergence problems faced by the classical
supervised and self-supervised networks. The segmented volumes
are obtained once the network converges. The suggested 3D-QNet
is tailored and tested on the BRATS 2019 Brain MR image data
set and Liver Tumor Segmentation Challenge (LiTS17) data set
extensively in our experiments. 3D-QNet has achieved promising
dice similarity as compared to the time intensive supervised
convolutional neural network-based models like 3D-UNet, Vox-
ResNet, DRINet, and 3D-ESPNet, thereby showing a potential
advantage of our self-supervised shallow network on facilitating
semantic segmentation. The unified concept of quantum-inspired neural networks
(QINN) enhances the approximation and generalization capa-
bilities of classical neural networks and has emerged to process
information faster in the field of computer science [8], [9]. Of
late, quantum-inspired neural networks are becoming popular
in solving problems in the domain of pattern recognition and
classification [10], [11], [14], [15], employing the inherent
characteristics of quantum computation. However, the complex
and time-intensive quantum back-propagation algorithm in the
aforementioned QINN models suffers from slow convergence
problems. In addition, the fixed activation schemes adopted in
the QINN models restrict their application to gray-scale image
segmentation. Given 3D medical image data, the primary aim of our proposed
3D-QNet architecture is to perform volumetric organ and
lesions segmentation with expert-level accuracy for tumor
identification alleviating supervision or training. Our pro-
posed 3D-QNet architecture centres on the self-supervised bi-
directional counter propagation of the quantum states obviating
the time-intensive quantum back-propagation algorithm for
faster convergence. D. Konar is with the CASUS - Center for Advanced Systems Understand-
ing, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Görlitz, Germany,
Email: d.konar@hzdr.de g
Debanjan Konar, SMIEEE, Siddhartha Bhattacharyya, SMIEEE, Tapan K. Gandhi, SMIEEE,
Bijaya K. Panigrahi, SMIEEE, and Richard Jiang, SMIEEE The network hyper-parameters associated
with the gray-level thresholding process are adaptive, and
voxel-wise context-sensitive information is exhibited in quan-
tum formalism, as reported in this article. Index Terms—Quantum Computing, Volumetric Medical Im-
age Segmentation, QIS-Net, Tensor Network CITATION Konar, Debanjan; Bhattacharyya, Siddhartha; Gandhi, Tapan Kumar; Panigrahi, Bijaya Ketan; Jiang, Richard
(2020): 3D Quantum-inspired Self-supervised Tensor Network for Volumetric Segmentation of Medical
Images. TechRxiv. Preprint. https://doi.org/10.36227/techrxiv.12909860.v4 10.36227/techrxiv.12909860.v4 10.36227/techrxiv.12909860.v4 1 T. K. Gandhi, and B. K. Panigrahi are with the Department of Electrical
Engineering, Indian Institute of Technology Delhi, New Delhi, India, Email:
tgandhi@ee.iitd.ac.in and bkpanigrahi@ee.iitd.ac.in S. Bhattacharyya is with Rajnagar Mahavidyalaya, Birbhum, West Bengal,
India, Email: dr.siddhartha.bhattacharyya@gmail.com D. Konar is with the CASUS - Center for Advanced Systems Understand-
ing, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Görlitz, Germany,
Email: d.konar@hzdr.de
T. K. Gandhi, and B. K. Panigrahi are with the Department of Electrical
Engineering, Indian Institute of Technology Delhi, New Delhi, India, Email:
tgandhi@ee.iitd.ac.in and bkpanigrahi@ee.iitd.ac.in
S. Bhattacharyya is with Rajnagar Mahavidyalaya, Birbhum, West Bengal,
India, Email: dr.siddhartha.bhattacharyya@gmail.com
R. Jiang is with the School of Computing and Communications, Lancaster
University, Lancaster, UK, Email: r.jiang2@lancaster.ac.uk R. Jiang is with the School of Computing and Communications, Lancaster
University, Lancaster, UK, Email: r.jiang2@lancaster.ac.uk I. INTRODUCTION Despite popu-
larity among the medical and computer vision researchers,
U-Net architectures [24] fall short in scalability and cannot
distinguish the distinctive features (shape, size, intensity, lo-
cation etc.) learned at the convolutional layers. Moreover, they
suffer from the vanishing gradient problem when the number
of feature layers is increased to better represent the features. Various deeper network architectures obviating the vanishing
gradient problem have been proposed concurrently for voxel-
wise medical image segmentation including VoxResNet [26],
DRINet [27], and 3D-ESPNet [28]. The current voxel-wise segmentation work has significant
contributions over 2D medical image segmentation [1]–[3],
[14] as given below. 1) We propose a novel quantum-inspired self-supervised
shallow voxel-wise neural network referred to as 3D-
QNet, which has relevance on volumetric medical image
segmentation. 2) In this work, an S-connected quantum fuzzy context-
sensitive voxel information is processed to integrate
the appearance of low-level and high-level local image
features with wide intensity variations and implicit shape
of the VOIs, thereby enabling accurate volumetric seg-
mentation of 3D medical images. 3) A novel generalized quantum-inspired self-supervised
learning is proposed using a tensor representation of the
weight vectors for high dimensional data employed in
our suggested 3D-QNet for 3D medical image segmen-
tation. The non-tensorized implementation of 3D-QNet,
referred to as 3D-QNet-NonTensor, is also demonstrated
in our experiments. 4) The convergence analysis of the proposed 3D-QNet is
also demonstrated with super-linearity. The primary aim
of incorporating quantum computing in our proposed 3D
network architecture is to exploit the features of quantum
correlation and accelerate the speed of convergence of
the network operation, thereby simultaneously improv-
ing the discrimination ability to yield fast and accurate
segmentation. However, these deeply supervised network architectures suffer
in computational complexity and slow-convergence with an
increase in the number of feature layers in the network ar-
chitecture. Currently, self-supervised/semi-supervised/weakly
supervised networks have gained significant attention among
the computer vision and medical imaging research community
due to lack of annotated images for deep supervision [29]–
[32]. Nevertheless, these self-supervised networks [29]–[32]
for volumetric medical image segmentation rely on pre-trained
3D CNN models, and hence these are not fully self-supervised
networks. Moreover, these networks are characterized by
significant memory footprints which often poses a serious
obstacle in employing them in various medical imaging ap-
plication settings. It inspires us to develop 3D self-supervised
neural network architectures for volumetric medical image
segmentation. I. INTRODUCTION A A
utomatic volumetric medical image segmentation as-
sisted by contextual information yields Volumes of In-
terest (VOIs), which are critical to cancer patients. Deeply su-
pervised Convolutional Neural Networks (CNN) have achieved
respectable accuracy in 2D medical image segmentation [1]–
[4]. However, in automatic 3D medical image data segmen-
tation, deeply supervised 3D-CNNs suffer from manually
affected challenges viz. acquiring sufficient 3D annotated data
for suitable training, high heterogeneity and dimensionality of
3D medical images, complex anatomical environments, and
the need for optimizing the 3D neural networks [5]–[7]. Hence,
3D medical imaging research calls for self-supervised learning Translational medicine (TM) is an emerging concept and
practice that facilitates the rapid transfer of medical break-
throughs from scientists to clinicians. Recently, there have
been a number of instructive instances in which the trans-
lation of research has resulted in undesirable effects need-
ing prompt intervention [12], [13]. A greater emphasis on
three-dimensional (3D) simulation, biomarkers, and artificial
intelligence may enable orthopaedic surgeons to forecast the
optimal surgical techniques prior to surgery. Utilizing the most
advanced imaging methods may enhance the precision and
accuracy of tumor resections. This article is aimed at young
surgeon scientists, specifically orthopaedic residents, to help
them better understand how 3D quantum-inspired models can
be used to process high mega pixel volumetric medical images
in a faster, self-supervised manner, with merits of obviating
training or very limited training. Heuristically, the suggested
3D-QNet has the ability to investigate the inherent features
of quantum parallelism in order to concurrently compute
high-resolution image voxels. This expedites the transfer of 2 information from the laboratory to the patient’s bedside. The current voxel-wise segmentation work has significant
contributions over 2D medical image segmentation [1]–[3],
[14] as given below. volumetric medical images [22]–[24]. Kamnitas et al. [22]
suggested a dual path 3D CNN incorporating local and larger
contextual feature information to obviate the computationally
complex 3D medical image processing and exhibit dense
inference on medical image segmentation. A flexible network,
3D-UNet architecture [24] achieved remarkable success on
brain MR image semantic segmentation. Of late, to exploit
the 3D contextual information, Brebisson et al. [25] employed
2D CNNs on three orthogonal 2D patches and formed 3D
patches in combination to reduce the memory requirements. However, 3D CNN networks suffer from slow convergence
problems owing to computationally exhaustive 3D convolution
operations and extensive training procedures. I. INTRODUCTION The
organization
of
the
remaining
sections
of
the
manuscript is as follows: a comprehensive literature review
about various deep learning-based volumetric segmentation of
medical images and the challenges are presented in Section II. Section III illustrates the fundamental concepts of quantum
computing. The novel self-supervised 3D-QNet architecture
with a quantum-inspired tensor network model is introduced
in Section IV. Section V elucidates voxel-wise segmentation of
3D medical images using the proposed 3D-QNet architecture. The experimental datasets, experimental setup, and outcome
are provided in Section VI. The advantages and limitations
of the proposed work have been discussed in Section VII. Section VIII states the concluding remarks of the proposed
work and sheds light on the future directions of research. The main problem with the classical self-supervised neural
network models lies in the fact that they do not converge
fast and hence the segmented outcome is distorted due to
the slower convergence problems [33], [34]. Numerous quan-
tum neural networks have been evolved in the last few
decades replicating classical neural networks and offering
faster processing while compared with the classical counter-
parts [9], [35]–[38]. The quantum versions of the classical
self-supervised neural network architectures [10], [11], [39],
[40] offer a potential candidate for faster and efficient image
segmentation and surpasses the classical counterparts. Konar et
al. recently developed quantum-inspired neural network mod-
els referred to as QIS-Net [14], QFS-Net [15], and QIBDS-
Net [41] suitable for brain MR image segmentation. These
networks have been found to attain a promising outcome in
complete brain tumor segmentation and serve as the motivation
behind the assimilation of quantum-inspired computing in the
current 3D-QNet architecture. A. Quantum Bits and Tensor Products The basic element equivalent to classical bits in quantum
computing is known as quantum bit or qubit and is represented
using Dirac notations |0⟩and |1⟩. However, unlike classical
computing, quantum bits are expressed as a linear combination
of probability amplitudes often known as superposition as
follows [9]. (7) III. FUNDAMENTALS OF QUANTUM COMPUTING To enhance and extract the contextual information from high
dimensional data, Tucker tensor decomposition is suitable for
neural network layer decomposition [43]. The inner product
of the two equal sized tensors V, Ψ ∈Rm×n×p is defined as
follows. The basic concept of quantum computing deals with the
principles of quantum mechanics and offers to demonstrate
the quantum computing algorithms which rely on quantum
bits having quantum operations on qubits [42]. |ξ⟩= ⟨V|Ψ⟩=
X
l
⟨V(l), Ψ(l)⟩=
M
X
i=1
θi|i⟩·
N
X
j=1
ϕj|j⟩
=
θ1
θ2
... θM
·
ϕ1
ϕ2
... ϕN
, θi ∈V, ϕj ∈Ψ
(7) IV. 3D QUANTUM-INSPIRED SELF-SUPERVISED TENSOR
NEURAL NETWORK (3D-QNET) ARCHITECTURE In quantum formalism, the
tensor products of the subspace form the full Hilbert space,
H as (2) H = ⊗n
t=1Ht
(2) H = ⊗n
t=1Ht
(2) A set of n basis states (designated as |ϕj⟩) comprising 0 −1
can form a qubit system |ψ⟩, of size log n in the Hilbert space,
H as follows. n A set of n basis states (designated as |ϕj⟩) comprising 0 −1
can form a qubit system |ψ⟩, of size log n in the Hilbert space,
H as follows. n |ψ⟩=
n
X
j
pj|ϕj⟩
(3) (3) where, pj is the probability amplitude and |ϕj⟩= |ϕ1⟩⊗
|ϕ2⟩⊗. . . |ϕn⟩. For example, using two qubits, four distinct
tensor sub-spaces can be created as basis |0⟩⊗|0⟩, |0⟩⊗|1⟩,
|1⟩⊗|0⟩and |1⟩⊗|1⟩often represented as |00⟩, |01⟩, |10⟩,
and |11⟩, respectively. II. RELATED WORKS Recent years have witnessed a surge in the application of
deep learning networks in various tumor segmentation [16]–
[20] tasks with respectable accuracy in 2D medical image
segmentation [2], [21]. However, in contrast to automated
volumetric segmentation of medical images, 2D convolutional
neural network architectures (CNNs) [16], [18], [19] process
the medical images in a 2D independent slice-wise fashion
which leads to non-optimal use of the 3D contextual feature
information of volumetric medical image data (3D Computed
Tomography (CT) and Magnetic Resonance Imaging (MRI)). In turn, 3D CNN based architectures extract rich spatial and
contextual features and perform voxel-wise segmentation of This manuscript presents a novel fully 3D self-supervised 3 functions defined over α ∈[0, 1]N. Considering the dimen-
sions of the input feature vector restricted to N = 2, ϕ(α) is
defined as quantum-inspired shallow neural network architecture for vol-
umetric medical image segmentation to obviate the compre-
hensive challenges faced by deep supervision of complex 3D
CNNs. ϕ(0) = [0, 1], ϕ(1) = [1, 0]
(6) (6) IV. 3D QUANTUM-INSPIRED SELF-SUPERVISED TENSOR
NEURAL NETWORK (3D-QNET) ARCHITECTURE |ϕ⟩= cos α
2 |0⟩+ ei θ
2 sin α
2 |1⟩
(1) (1) In this article, a 3D Quantum-inspired Self-supervised
Tensor Network (3D-QNet) architecture with self-supervised
tensor learning is proposed for automatic voxel-wise segmen-
tation of medical images. The 3D-QNet architecture comprises
a trinity of volumetric layers of quantum neurons arranged as
input, intermediate and output layers. A schematic outline of
the proposed 3D-QNet architecture is shown in Figure 1. The
input volume (M ×N ×P) is normalized and propagated from
the 3D input layer to the successive 3D hidden and output
layers of the 3D-QNet architecture for processing through S-
connected voxels. Each of the three volumetric layers of the
3D-QNet architecture is fully intra-linked with qubits using
a 3D-matrix representation. Each 3D layer of the proposed
architecture is intra-connected through quantum neurons with
intra-connection strengths set to
π
2 (quantum 1 logic). The
basic processing unit of each volumetric layer of the 3D-QNet
architecture is the S-connected neighborhood-based voxel-
wise orientation of each candidate neuron as illustrated in
Figure 2. The inter-layer connection between the 3D input to
3D intermediate and the 3D intermediate to 3D output layer
is formed using the S-connected voxel-wise neighborhood
orientation. The contribution of the S number of neighborhood
quantum neurons (pixels) of a candidate neuron at one 3D
layer is propagated in the forward direction and accumulated
at the corresponding candidate neuron of the subsequent 3D
layer. Consequently, the voxel-wise information from the 3D
output layer to the 3D intermediate layer is counter-propagated
for further processing. The voxel-wise processing of each 3D
layer is performed along with the depth of the 3D layer for
semantic segmentation. The inter-linked connections between
two successive 3D layers are represented using 3D weight
matrices of qubits and each inter-connection weight is updated
using rotation gate for faster processing. The relevant details
about the principle of operation of the proposed 3D-QNet
architecture for volumetric segmentation are provided in the
following subsections using a self-supervised tensor learning
model in quantum formalism. where, 0 ≤α ≤π and 0 ≤θ ≤2π. where, 0 ≤α ≤π and 0 ≤θ ≤2π. Hence, qubits reside in the Hilbert space parametrized by
the continuous variables θ and α. B. Input Data Encoding and Tensor Decomposition A tensor product basis relies on the local input feature map
{Φdj(αj)} in the Hilbert space of functions over αj ∈[0, 1]
as [38] |Φd1,d2,...dN (α)⟩= |ϕd1(α1)⟩⊗|ϕd2(α2)⟩⊗. . . ⊗|ϕdN (αN)⟩
(4) where, dj varies from 1 . . . N (N-dimensional vector). A
function, f l(α) can be realized using the inner product of
the input local feature map ϕdj(αj) and the network weight
decomposition Ψ, as follows. f l(α) = ⟨Ψl|Φ(α)⟩
= ⟨Ψd1d2...dN |ϕ(αd1
1 )⟩⊗|ϕ(αd2
2 )⟩. . . ⊗|ϕ(αdN
N )⟩
(5) (5) Hence, the local feature map ϕdj(αj) forms a basis for a
Hilbert space of functions defined over α ∈[0, 1] and the
tensor product basis Φd1,d2,...dN (α) forms a Hilbert space of Fig. 1: 3D Quantum-inspired Self-supervised Tensor Neural Network (3D-QNet) architecture. For every candidate neuron
as marked in red color, it forms a S-connected neighborhood oriented Inter-layer connections and here only three Inter-
layer connection is illustrated for better visibility. A classical information, α is mapped to quantum bits or qubits as, ϕ(α) =
cos( π
2 α) sin( π
2 α)
. The inter-connection weights between the input and hidden layers of the 3D-QNet architecture are denoted
by |φl,d
k,j⟩, for the hidden layers to the output layers are indicated by |φl,d
j,i⟩and for the output to the hidden layers are |φl,d
i,j⟩
at a layer l with depth d. The classical interconnection weight [0, 1] is transformed in quantum formalism as |φ(ω)⟩=
cos( π
2 ω) sin( π
2 ω)
, where the angle of rotation (ω) is measured using the relative difference of fuzzy intensities of the
candidate pixel and the neighborhood pixels in S-connected (here, S = 26) neighborhood oriented quantum neurons in a
3 × 3 × 3 voxel (v) as ωi,j = 1 −(αi −αi,j); j ∈{1, 2, 3, . . . S}. 3D-volumes of dimensions 3 × 3 × 3 are processed along
the depth of the 3D-layers as shown in the Figure. Fig. 1: 3D Quantum-inspired Self-supervised Tensor Neural Network (3D-QNet) architecture. For every candidate neuron
as marked in red color, it forms a S-connected neighborhood oriented Inter-layer connections and here only three Inter-
layer connection is illustrated for better visibility. A classical information, α is mapped to quantum bits or qubits as, ϕ(α) =
cos( π
2 α) sin( π
2 α)
. B. Input Data Encoding and Tensor Decomposition The inter-connection weights between the input and hidden layers of the 3D-QNet architecture are denoted
by |φl,d
k,j⟩, for the hidden layers to the output layers are indicated by |φl,d
j,i⟩and for the output to the hidden layers are |φl,d
i,j⟩
at a layer l with depth d. The classical interconnection weight [0, 1] is transformed in quantum formalism as |φ(ω)⟩=
cos( π
2 ω) sin( π
2 ω)
, where the angle of rotation (ω) is measured using the relative difference of fuzzy intensities of the
candidate pixel and the neighborhood pixels in S-connected (here, S = 26) neighborhood oriented quantum neurons in a
3 × 3 × 3 voxel (v) as ωi,j = 1 −(αi −αi,j); j ∈{1, 2, 3, . . . S}. 3D-volumes of dimensions 3 × 3 × 3 are processed along
the depth of the 3D-layers as shown in the Figure. Fig. 2: S-connected neighborhood oriented quantum neurons
form a voxel (The red pixel is the candidate neuron and
the black pixels represent the corresponding neighborhood
neurons). function ϕ(αi) as follows. ϕ(αi) =
h
cos(π
2 αi) sin(π
2 αi)
i
∀i = 1, . . . M, j = 1, . . . N
(8) (8) The classical interconnection weight [0, 1] is transformed into
quantum formalism as |φ(ωi,j)⟩=
h
cos(π
2 ωi,j) sin(π
2 ωi,j)
i
(9) (9) Fig. 2: S-connected neighborhood oriented quantum neurons
form a voxel (The red pixel is the candidate neuron and
the black pixels represent the corresponding neighborhood
neurons). Hence, the strength of inter-connection between neuron j
(neighborhood of the candidate neuron i) of a layer to the cor-
responding candidate neuron of the adjacent layer is mapped
using φ. Also, ωi,j is designated as the angle of rotation
and measured as the relative intensity difference between the
candidate pixel (αi) and one of its neighborhood pixels αi,j
as follows. Hence, the strength of inter-connection between neuron j
(neighborhood of the candidate neuron i) of a layer to the cor-
responding candidate neuron of the adjacent layer is mapped
using φ. Also, ωi,j is designated as the angle of rotation
and measured as the relative intensity difference between the
candidate pixel (αi) and one of its neighborhood pixels αi,j
as follows. A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model V, Ψ are third-order
tensors (1 ≤m ≤M, 1 ≤n ≤N, 1 ≤p ≤P)). According to
Tucker Tensor decomposition [43] X = V ×1 Ψ1 ×2 Ψ2 ×3 Ψ3
(11) (11) where, X
∈Rm×n×p is the tensor outcome, Ψn is the
weight matrix in terms of n factor matrix and ×n is the
mod −n product of a tensor with a matrix. Each layer of
the proposed 3D-QNet architecture is transformed to lower-
dimensional tensors. Such types of M×N×P tensors in voxel
form each layer in the underlying network architecture. Each
volumetric layer of the 3D-QNet architecture forms M×N×P
volumetric patches (voxels) of size S corresponding to the
candidate pixels as where, X
∈Rm×n×p is the tensor outcome, Ψn is the
weight matrix in terms of n factor matrix and ×n is the
mod −n product of a tensor with a matrix. Each layer of
the proposed 3D-QNet architecture is transformed to lower-
dimensional tensors. Such types of M×N×P tensors in voxel
form each layer in the underlying network architecture. Each
volumetric layer of the 3D-QNet architecture forms M×N×P
volumetric patches (voxels) of size S corresponding to the
candidate pixels as Quantum fuzzy context-sensitive thresholding determines the
bi-directional propagation of quantum information between
the layers of the 3D-QNet architecture by means of self-
organization of the inter-linked weight matrices. Reduction of
feature dimensions using tensor decomposition followed by
voxel-wise information processing of the proposed 3D-QNet
architecture is inspired by the basic quantum neural network
input-output model [14] as follows. v = vox(V)
(12) (12) Here, V comprises all the 3D-patches (voxels), v ∈Rm×n×p
for a network layer in the proposed 3D-QNet architecture. The spatial features in terms of the neighborhood pixels
of every seed pixel at the network layer, are extracted and
propagated to the next subsequent layers as inputs guided by
a Quantum-inspired voxel-wise multi-level Sigmoidal (Vox-
QSig) activation function, σ3D−QNet as follows. Here, V comprises all the 3D-patches (voxels), v ∈Rm×n×p
for a network layer in the proposed 3D-QNet architecture. The spatial features in terms of the neighborhood pixels
of every seed pixel at the network layer, are extracted and
propagated to the next subsequent layers as inputs guided by
a Quantum-inspired voxel-wise multi-level Sigmoidal (Vox-
QSig) activation function, σ3D−QNet as follows. A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model In the suggested 3D-QNet architecture, the high dimensional
weight vector Ψ is represented using a tensor to optimize the
network operations and to facilitate the extraction of significant
semantic feature information in the quantum-inspired self-
supervised model. The internal kernels associated with the
network operate in parallel, thereby accelerating convergence
of the 3D-QNet architecture. The input quantum neurons
containing the pixel intensity are expressed as qubits and
the inter-connection weights are represented using quantum
rotation gates. The classical intensity of any ith normalized
gray-scale image pixel of MR or CT volume (denoted as
αi ∈[0, 1]) is transformed into quantum state using a mapping ωi,j = 1 −(αi −αi,j); j ∈{1, 2, 3, . . . S}
(10) (10) The angle of rotation is measured using the relative difference
of fuzzy intensities of the candidate pixel and the neighbor-
hood pixels in quantum formalism. This relative measure en-
coding scheme [14], [15], [39] helps to segment the foreground
and background regions of an image. However, there are other
encoding schemes like variational and amplitude encoding,
which are often used for variational quantum circuits. The angle of rotation is measured using the relative difference
of fuzzy intensities of the candidate pixel and the neighbor-
hood pixels in quantum formalism. This relative measure en-
coding scheme [14], [15], [39] helps to segment the foreground
and background regions of an image. However, there are other
encoding schemes like variational and amplitude encoding,
which are often used for variational quantum circuits. In this proposed tensor network model, the 3D-QNet layer
is decomposed as voxel (core tensor) using Tucker Tensor
decomposition [43] to reduce the input dimensions and the 5 TABLE I: Variational parameters used in the counter-
propagation algorithm of the proposed 3D-QNet
Symbol
Description
αl,d
i
The intermediate output at pixel i at layer l and depth
d
ωl,d
The rotation angle for inter-connection weight at layer
l with depth d
χS
It corresponds the contribution of S-connected third-
order neighborhood pixels
ϑl
i
The fuzzy context sensitive activation in quantum for-
malism at layer l
δl,d
i
The phase transformation parameters at layer l−1 with
depth d interconnection wights as factor matrices. Let us consider
tensor V, Ψ ∈Rm×n×p, where V is the voxel-wise input
of 3D medical images and the corresponding inter-connection
3D weight matrix is evaluated in Eq. 9, respectively (m, n, p
denote the row, column, and slice number. A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model |ϕl(αd
i )⟩= σ3D−QNet(
m×n×p
X
j
f l−1(αd
i )⟨φl
j|χl,d
i ⟩)
(18) (18) yl = σ3D−QNet(vl−1 · Ψl(ω))
(13) (13) where, vl−1 ∈RM×N×P , Ψl(ω) ∈RM×N×P ×S at the
network layer l = 2, 3, yl ∈RS, and “·” is the inner product
operator. The Vox-QSig activation function, σ3D−QNet with
slope λ and activation ϑ, is defined as where, |ϕl(αd
i )⟩denotes the intermediate output of the ith
seed quantum neuron at the 3D network layer in the lth
sample with depth (slice#) d = 1, 2, . . . P. σ3D−QNet is the
Quantum-inspired voxel-wise multi-level Sigmoidal activation
(Vox-QSig) function with activation as |χl,d
i ⟩. The output
|ϕl(αd
i )⟩can be written as σ3D−QNet(x) =
1
βτ + e−λ(x−ϑ) , 0 ≤βτ ≤π
2
(14) (14) |ϕl(αd
i )⟩=
f(π
2 δl,d
i
−arg{
m×n×p
X
j
f l(ωd
j,i)f l−1(αd
i ) −f l(χd
i )})
= σ3D−QNet(
m×n×p
X
j
f l−1(αd
i )(cos(ωl,d
j,i −ϑl
i)+
γ sin(ωl,d
j,i −ϑl
i)))
(19) where, βτ describes the multi-level class responses exhibited
by the S-connected third-order neighborhood pixels expressed βτ =
χS
ρτ −ρτ−1
(15) (15) (19) where, ρτ and ρτ are the τ th and (τ −1)th class outcome,
respectively and the contribution of the S-connected neighbor-
hood gray-level pixels is χS. The fuzzy context-sensitive activation (designated as χi) for
semantic segmentation in quantum formalism is defined as
follows.
The fuzzy context-sensitive activation (designated as χi) for
semantic segmentation in quantum formalism is defined as
follows.
Here, the designated rotation angles associated with the inter-
connection weights between input neuron j to output neuron i
are represented by ωl,d
j,i and δl,d
i
is the phase transfer parameter. Here, the designated rotation angles associated with the inter-
connection weights between input neuron j to output neuron i
are represented by ωl,d
j,i and δl,d
i
is the phase transfer parameter. The variational parameters used in the counter-propagation
algorithm are provided in Table I. The true classical output
state (|1⟩) from the ith quantum neuron is obtained by consid-
ering the imaginary part (sin) of the above expression where,
γ is an imaginary unit. V. VOLUMETRIC MEDICAL IMAGE SEGMENTATION USING
3D-QNET where, L corresponds to the number of class levels. The multi-
class responses for various hyper-parameters employed in the
Vox-QSig activation functions are provided in Figure 3. where, L corresponds to the number of class levels. The multi-
class responses for various hyper-parameters employed in the
Vox-QSig activation functions are provided in Figure 3. Minimum pre-processing is performed before the medical
image slices are fed as inputs to 3D-QNet. The input medical
image volume is normalized as a fuzziness measure ([0, 1])
before transforming it to qubits as follows. A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model (a) L = 3 (b) L = 5 |ϕl(αd
i )⟩= σ3D−QNet(
m×n×p
X
j
f(π
2 yl,d
j )⟨φl,d
ji |ϑl,d
j ⟩)
= σ3D−QNet(
m×n×p
X
j
f(π
2 × σ3D−QNet(
m×n×p
X
k
f(π
2 yl,d
j )
⟨φl,d
kj |ϑl,d
k ⟩)⟨φl,d
ji |ϑl,d
j ⟩)
(20) |ϕl(αd
i )⟩= σ3D−QNet(
m×n×p
X
j
f(π
2 yl,d
j )⟨φl,d
ji |ϑl,d
j ⟩) = σ3D−QNet(
m×n×p
X
j
f(π
2 × σ3D−QNet(
m×n×p
X
k
f(π
2 yl,d
j )
⟨φl,d
kj |ϑl,d
k ⟩)⟨φl,d
ji |ϑl,d
j ⟩)
(20) (a) L = 3 (b) L = 5 (c) L = 6 (d) L = 8 i.e., |ϕl(αd
i )⟩= σ3D−QNet(
m×n×p
X
j
Net(
m×n×p
X
k
f(π
2 yl,d
j ) cos(ωl,d
k,j −ϑl,d
j )
γ sin(ωl,d
k,j −ϑl,d
j ) sin(ωl,d
j,i −ϑl,d
i ))))
(21) |ϕl(αd
i )⟩= σ3D−QNet(
m×n×p
X
j
f(π
2 × σ3D−QNet(
m×n×p
X
k
f(π
2 yl,d
j ) cos(ωl,d
k,j −ϑl,d
j )
cos(ωl,d
j,i −ϑl,d
i ) + γ sin(ωl,d
k,j −ϑl,d
j ) sin(ωl,d
j,i −ϑl,d
i ))))
(21) (d) L = 8 (c) L = 6 f(π
2 × σ3D−QNet(
m×n×p
X
k
f(π
2 yl,d
j ) cos(ωl,d
k,j −ϑl,d
j )
cos(ωl,d
j,i −ϑl,d
i ) + γ sin(ωl,d
k,j −ϑl,d
j ) sin(ωl,d
j,i −ϑl,d
i ))))
(21) Fig. 3: Multi-level class response of Vox-QSig activation
function for λ = 15, 20, 25. ))
(21) and and where, γ is an imaginary number. where, γ is an imaginary number. ϑι+1,d = ϑι,d + △ϑι,d
(26) (26) Equations 25 and 26 refer to updating the angles of rota-
tion and activation, respectively. The error or loss function,
ζ(ωι,d, ϑι,d) in the suggested 3D-QNet architecture is eval-
uated in terms of Root Mean Square Error (RMSE) of the
3D-weight matrices at depth d (or the slice #d) in the lth
epoch and is defined on the phase angles ωι,d, ϑι,d as Equations 25 and 26 refer to updating the angles of rota-
tion and activation, respectively. The error or loss function,
ζ(ωι,d, ϑι,d) in the suggested 3D-QNet architecture is eval-
uated in terms of Root Mean Square Error (RMSE) of the
3D-weight matrices at depth d (or the slice #d) in the lth
epoch and is defined on the phase angles ωι,d, ϑι,d as B. A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model Quantum-inspired Voxel-wise multi-level Sigmoidal (Vox-
QSig) activation function Equations 25 and 26 refer to updating the angles of rota-
tion and activation, respectively. The error or loss function, ζ(ωι,d, ϑι,d) in the suggested 3D-QNet architecture is eval-
uated in terms of Root Mean Square Error (RMSE) of the
3D-weight matrices at depth d (or the slice #d) in the lth
epoch and is defined on the phase angles ωι,d, ϑι,d as In this 3D-QNet architecture, a Quantum-inspired Voxel-
wise multi-level Sigmoidal (Vox-QSig) activation function is
proposed for voxel-wise processing of S-connected spatially
oriented neighborhood-based pixels. The generalized form of
the Vox-QSig activation function is obtained by leveraging the
activation function hyper-parameters employed in Equation 14
as ζ(ωι,d, ϑι,d) =
1
N
N
X
i=1
S
X
j=1
h
φij(ωι+1,d
ij
, ϑι+1,d
i
) −φij(ωι,d
ij , ϑι,d
i )
i2
(27) ζ(ωι,d, ϑι,d) = (27) σV ox−QSig(x; βτ, ρτ) =
L
X
τ=1
1
βτ + e−λ(x−(τ−1)ρτ−1−ϑ)
(22) A. 3D-Quantum-Inspired
Self-supervised
Tensor
Network
Model Assume that the inter-connection
weights between the input and hidden layers of the 3D-QNet
architecture are denoted by |φl,d
k,j⟩and for the hidden layer to
the output layer, are indicated by |φl,d
j,i⟩in the lth sample sets. The activation at the hidden and output layers are designated
using |χl,d
j ⟩and |χl,d
i ⟩, respectively. Considering any quantum |χi⟩=
cos ϑi
sin ϑi
(16) (16) where, the angle of rotation, ϑi is evaluated using the sum-
mation of the intensities of the third-order S-connected neigh-
borhood pixels (denoted as αi,j, j = 1, 2, . . . S) of a candidate
pixel i (neuron) in quantum formalism using the following
equation. ϑi = 2π × (
S
X
j=1
αi,j)
(17) (17) 6 seed neuron k from the sample of input neurons at the input
layer, the corresponding seed neuron at the hidden layer be j
and the output seed neuron be i, the response at the ith neuron
with depth d in the lth sample sets is expressed as (a) L = 3
(b) L = 5
(c) L = 6
(d) L = 8
Fig. 3: Multi-level class response of Vox-QSig activation
function for λ = 15, 20, 25. C. Experimental Results Experiments have been carried out in the current setup, and
the results have been reported together with numerical and
statistical analysis using the proposed 3D-QNet, 3D-QNet-
NonTensor, 3D-UNet [24], VoxResNet [26], DRINet [27], and
3D-ESPNet [28] on BRATS 2019 data set [47] and LiTS17
data set [48]. Table II presents the complete tumor (WT)
segmentation on BraTS19-CBICA-AAG-1-flair-slice#69. It
is evident from the experimental data reported in Table II
that the proposed 3D-QNet performs optimally for com-
plete brain tumor segmentation of four different modalities
of MR volumes (viz. T1, T1 −CE, FLAIR, and T2)
using the activation guided by 26-connected heterogeneous
voxel intensities (υξ) with L = 8 in comparison with other
thresholding schemes under the four evaluation parameters
(AC, DS, PV, SS) [18]. The 3D-QNet segmented brain MR
slices collected from two different volumes BRATS19-CBICA-
AAG and BRATS19-CBICA-AAB using class level L = 8
with activation scheme υξ, are shown in Figures 4 and A. Data Set The proposed 3D-QNet and its non-tensorized implementa-
tion (3D-QNet-NonTensor) are validated extensively using the
BRATS 2019 data set [47] and the Liver Tumor Segmentation
Benchmark (LiTS17) data set [48]. The BRATS 2019 data
set is composed of 315 (239 HGG and 76 LGG) 3D MRI
volumes. Each MRI volume comprises 155 slices of resolution
240×240 with the ground truth segmented labels and includes
four different modalities of 3D MR images viz. T1, T1
with Contrast-Enhanced (T1 −CE), T2, and FLAIR. The
segmented labels are annotated with three distinct tumor sub-
regions, viz. tumor core (TC), tumor enhancing (TE), necrosis
and non-enhancing core region. These three annotations form
a complete tumor (WT). The BRATS 2019 data set is divided
into 8 : 2 ratio for training (252) and testing (63) due to
GPU limitations. The Liver Tumor Segmentation Benchmark
(LiTS17) data set [48] consists of 131 CT scans with various
types of tumors. The LiTS17 data set is also divided into 8 : 2
ratio for training and testing in the study. Each CT volume
consists of variable number of slices of resolution 512 × 512. C. Adjustment of Inter-connection Weights of 3D-QNet and
Loss Function We have trained 3D-UNet [24]
and VoxResNet [26] rigorously with the Stochastic Gradient
Descent (SGD) algorithm on Caffe library1 using an Nvidia
Tesla V 100 −SXM2 GPU Cluster with 32 GB of memory
and 640 Tensor cores with 8 cores of Intel(R) Xeon(R) CPU
E5-2683 v4@2.1GHz. The 3D-ESPNet is implemented using
Pytorch from the code available in Github2 with 100 epochs
using adam optimizer with an initial learning rate of 0.01. The DRINet is also implemented using adam optimizer with
an initial learning rate of 0.01 and kernel size of 3 × 3. In
order to detect a complete tumor, the segmented output images
are resized to match the binary mask’s dimensions, with the
result 1 representing the tumor region and 0 representing the
backdrop. The dice similarity (DS) [18], which is regarded as
a standard assessment technique in automatic medical image
segmentation, can be evaluated by pixel to pixel comparison
with manually segmented regions of interest or lesion mask. The ground truth for the evaluation is a manually segmented
lesion mask, and each 2D pixel is predicted as True Positive
(TRP ) or True Negative (TRN) or False Positive (TRN)
or False Negative (FLN). The empirical goodness measures
[Positive Predictive Value (PV ), Sensitivity (SS), Accuracy
(AC) and Dice Similarity (DS) [18]] are assessed to evaluate
the results. ξ
(4) Activation guided by S-connected fuzzy voxel cardinality
estimates (υκ). In addition, to investigate a number of optimal thresh-
olds
{T1, T2, · · · , TCl−1}
in
multi-class
settings,
Otsu’s
method [45] is explored. The optimal thresholds maximize
the class variance as follows [45]. O = hn{T1, T2, · · · , TCl−1}θi(µi −ω)
(29) (29) where, Cl represents the number of defined classes in C
={C1, C2, . . . , CCl} and θi =
X
i∈Cl
pi , µi =
X
i∈Cl
ipi/θl
(30) (30) where, the ith pixel is defined as pi. The probability of class
Ci is represented as µi and its mean value is given by µi. ω
is known to be the mean of class C.i To refine the segmentation accuracy and dice score for false-
positive reduction in brain tumor detection, 3D-QNet seg-
mented highly representative volumetric intensity features are
post processed using the k-Means algorithm [46] for initial
label segmentation and segmented into pre-defined number of
clusters. C. Adjustment of Inter-connection Weights of 3D-QNet and
Loss Function Moreover, the Vox-QSig is guided
by four distinct activation schemes (υβ, υξ, υζ, υκ) [14], [34],
[44]. Experiments have also been performed using the 3D-
UNet [24] architecture, Deep Voxel-wise Residual Network
(VoxResNet) [26], Dense-Res-Inception Net (DRINet) [27],
and 3D-ESPNet [28] on the BRATS 2019 data set [47] and
on the LiTS17 data set [48]. We have trained 3D-UNet [24]
and VoxResNet [26] rigorously with the Stochastic Gradient
Descent (SGD) algorithm on Caffe library1 using an Nvidia
Tesla V 100 −SXM2 GPU Cluster with 32 GB of memory
and 640 Tensor cores with 8 cores of Intel(R) Xeon(R) CPU
E5-2683 v4@2.1GHz. The 3D-ESPNet is implemented using
Pytorch from the code available in Github2 with 100 epochs
using adam optimizer with an initial learning rate of 0.01. The DRINet is also implemented using adam optimizer with
an initial learning rate of 0.01 and kernel size of 3 × 3. In
order to detect a complete tumor, the segmented output images
are resized to match the binary mask’s dimensions, with the
result 1 representing the tumor region and 0 representing the
backdrop. The dice similarity (DS) [18], which is regarded as
a standard assessment technique in automatic medical image
segmentation, can be evaluated by pixel to pixel comparison
with manually segmented regions of interest or lesion mask. The ground truth for the evaluation is a manually segmented
lesion mask, and each 2D pixel is predicted as True Positive
(TRP ) or True Negative (TRN) or False Positive (TRN)
or False Negative (FLN). The empirical goodness measures
[Positive Predictive Value (PV ), Sensitivity (SS), Accuracy
(AC) and Dice Similarity (DS) [18]] are assessed to evaluate
the results. been observed that in majority of cases, λ = 0.232 and S = 26
(3 × 3 × 3 volume) yield optimal performance. For the other
two modalities T1 and T1-CE, λ = 0.238 and S = 26 (3×3×3
volume) yield optimal performance. On contrary, the LiTS17
CT volume data has performed optimally for λ = 0.239 and
S = 26 (3×3×3 volume). Moreover, the Vox-QSig is guided
by four distinct activation schemes (υβ, υξ, υζ, υκ) [14], [34],
[44]. Experiments have also been performed using the 3D-
UNet [24] architecture, Deep Voxel-wise Residual Network
(VoxResNet) [26], Dense-Res-Inception Net (DRINet) [27],
and 3D-ESPNet [28] on the BRATS 2019 data set [47] and
on the LiTS17 data set [48]. C. Adjustment of Inter-connection Weights of 3D-QNet and
Loss Function αd
i,j =
αd
i,j −min(αd
i,j)
max(αd
i,j) −min(αd
i,j)
(28) (28) Each inter-connection link for each candidate pixel of the
S-connected medical image volume and its corresponding
activation are updated using quantum rotation gates, thereby
enabling faster convergence of the proposed 3D-QNet archi-
tecture. The inter-connection weight, φl,d and its activation,
χl,d are updated as follows. Medical image volumes exhibit heterogeneous responses over
the local intensities in the S-connected neighborhood regions, Medical image volumes exhibit heterogeneous responses over
the local intensities in the S-connected neighborhood regions,
owing to the wide variations of gray-levels. Inspired by
the authors’ previous works [14], [34], [44], the proposed
Vox-QSig activation function employs four different adaptive
thresholding schemes suitable for efficient gray-scale segmen-
tation in the 3D-QNet architecture. Medical image volumes exhibit heterogeneous responses over
the local intensities in the S-connected neighborhood regions,
owing to the wide variations of gray-levels. Inspired by
the authors’ previous works [14], [34], [44], the proposed
Vox-QSig activation function employs four different adaptive
thresholding schemes suitable for efficient gray-scale segmen-
tation in the 3D-QNet architecture. |φι+1,d⟩=
cos △ωι+1,d
−sin △ωι+1,d
sin △ωι+1,d
cos △ωι+1,d
|φι,d⟩
(23)
|χι+1,d⟩=
cos △ϑι+1,d
−sin △ϑι+1,d
sin △ϑι+1,d
cos△ϑι+1,d
|χι,d⟩
(24)
where,
ωι+1,d
ωι,d + △ωι,d
(25) |φι+1,d⟩=
cos △ωι+1,d
−sin △ωι+1,d
sin △ωι+1,d
cos △ωι+1,d
|φι,d⟩
(23)
|χι+1,d⟩=
cos △ϑι+1,d
−sin △ϑι+1,d
sin △ϑι+1,d
cos△ϑι+1,d
|χι,d⟩
(24)
where,
ωι+1,d = ωι,d + △ωι,d
(25) |φι+1,d⟩=
cos △ωι+1,d
−sin △ωι+1,d
sin △ωι+1,d
cos △ωι+1,d
|φι,d⟩
(23)
|χι+1,d⟩=
cos △ϑι+1,d
−sin △ϑι+1,d
sin △ϑι+1,d
cos△ϑι+1,d
|χι,d⟩
(24) (1) Activation guided by β-distribution of the intensity of S-
connected neighborhood voxels (υβ). (2) Activation guided by S-connected voxels based on Skew-
ness (υχ). (2) Activation guided by S-connected voxels based on Skew-
ness (υχ). ωι+1,d = ωι,d + △ωι,d
(25) ωι+1,d = ωι,d + △ωι,d (25) (3) Activation guided by S-connected heterogeneous voxel (3) Activation guided by S-connected heterogeneous voxel 7 been observed that in majority of cases, λ = 0.232 and S = 26
(3 × 3 × 3 volume) yield optimal performance. For the other
two modalities T1 and T1-CE, λ = 0.238 and S = 26 (3×3×3
volume) yield optimal performance. On contrary, the LiTS17
CT volume data has performed optimally for λ = 0.239 and
S = 26 (3×3×3 volume). 1https://doi.org/10.1145/2647868.2654889
2https://github.com/sacmehta/3D-ESPNet B. Experimental Setup Table IV presents the
results reported using the proposed 3D-QNet, 3D-UNet [24],
VoxResNet [26], 3D-QNet-NonTensor, DRINet [27], and 3D-
ESPNet [28] on LiTS17 data set [48] in detecting complete
Liver tumor regions. A sample of segmented Liver tumor using
3D-QNet with manually segmented tumors is shown in Fig-
ure 7. It is observed from Table IV that 3D-QNet has reported
with an average Dice Score (DS) of 0.958. Furthermore, a
one-sided two-sample Kolmogorov-Smirnov (KS) [49] test is
performed with a significance level of α = 0.05, and the
experimental data given in Table III and Table IV show that
3D-QNet is capable of segmenting 3D medical image data. Despite being characterized by fully self-supervised quantum-
inspired learning, the 3D-QNet has demonstrated compara- 5, respectively. The human expert annotated ground truth
slices for all the four different modalities are illustrated in
Figure 6. 3D-UNet [24], VoxResNet [26], DRINet [27], 3D-
ESPNet [28], and 3D-QNet-NonTensor segmented brain MR
volumes from BRATS19-CBICA-AAG are also demonstrated in
the Supplementary Materials. It has been observed from the
segmented MR slices that the proposed 3D-QNet is suitable in
segmenting the correct position and size of the complete tumor
while compared with the ground truth segmentation. However,
it is not efficient in mapping the sharp contour of the core and
enhanced tumor sub-regions outlined in the annotated slices. The convergence analysis of the proposed 3D-QNet architec-
ture is discussed in the Appendix and experimentally demon-
strated with the non-tensorized implementation of the network
(3D-QNet-NonTensor). The convergence analysis of the pro-
posed 3D-QNet and 3D-QNet-NonTensor architectures for all
types of brain tumor images (T1, T1 −CE, Flair, and T2)
from the BRATS 2019 datasets [47] and for LiTS17 liver
volumes [48] are provided in the Supplementary Materials. Table V reports the average number of iterations required
to converge the proposed 3D-QNet architecture and its non-
tensorized implementation, 3D-QNet-NonTensor. It is evident
from Table V that an optimal convergence of the proposed
3D-QNet architecture is observed for Flair with υξ. Hence,
the non-tensorized implementation (3D-QNet without tensor
decomposition) underperforms in terms of average iterations
in comparison to 3D-QNet. It serves as the inspiration behind
the incorporation of tensor-decomposition in 3D-QNet imple-
mentation. it is not efficient in mapping the sharp contour of the core and
enhanced tumor sub-regions outlined in the annotated slices. B. Experimental Setup Table III presents the quantitative results reported using
the proposed 3D-QNet, 3D-QNet-NonTensor, 3D-UNet [24],
VoxResNet [26], DRINet [27], and 3D-ESPNet [28] on evalu-
ating the average accuracy (AC), dice similarity score (DS),
positive prediction value (PV ), and sensitivity (SS) [18]. It has been observed from the 3D-QNet segmented brain
MR slices and the results reported in Table III, that optimal
segmentation is achieved for FLAIR reported with an average
of 0.821 dice score (DS). The proposed 3D-QNet marginally
outperforms the convolutional architectures (3D-UNet [24],
VoxResNet [26], DRINet [27], 3D-ESPNet [28]), and 3D-
QNet-NonTensor in predicting complete brain tumor detection. However, it may be noted that the proposed 3D-QNet does not
intend to predict the core, enhanced tumor and necrosis sub
regions owing to lack of optimization of the parameters in
the suggested 3D-QNet architecture. The box plots are also
demonstrated in the Supplementary Materials citing the out-
come reported in Table III. Moreover, to show the effectiveness
of the proposed 3D-QNet architecture over 3D-UNet [24],
VoxResNet [26], DRINet [27], and 3D-ESPNet [28], we have
also conducted experiments on the Liver Tumor Segmentation
Benchmark (LiTS17) data set [48]. Table IV presents the
results reported using the proposed 3D-QNet, 3D-UNet [24],
VoxResNet [26], 3D-QNet-NonTensor, DRINet [27], and 3D-
ESPNet [28] on LiTS17 data set [48] in detecting complete
Liver tumor regions. A sample of segmented Liver tumor using
3D-QNet with manually segmented tumors is shown in Fig-
ure 7. It is observed from Table IV that 3D-QNet has reported
with an average Dice Score (DS) of 0.958. Furthermore, a
one-sided two-sample Kolmogorov-Smirnov (KS) [49] test is
performed with a significance level of α = 0.05, and the
experimental data given in Table III and Table IV show that
3D-QNet is capable of segmenting 3D medical image data. Despite being characterized by fully self-supervised quantum-
inspired learning, the 3D-QNet has demonstrated compara-
ble accuracy (AC) and dice similarity (DS) in comparison
to 3D-UNet [24], VoxResNet [26], DRINet [27], and 3D-
ESPNet [28]. Hence, the performance of the 3D-QNet model
on the BRATS 2019 and LiTS17 data sets are statistically
significant and offers a promising alternative to self-supervised
deep learning for 3D-medical image segmentation. B. Experimental Setup Further-
more, in case of brain MR image segmentation, the num-
ber of parameters required in the 3D-UNet [24] architecture
is 19, 069, 955, whereas the proposed 3D-QNet architecture
employs maximum 2, 995, 200 parameters (considering each
voxel as a candidate in a 240 × 240 dimensional slice, there
are total 240 × 240 × 26 × 2 connections) in bi-directional VII. DISCUSSIONS The proposed 3D-QNet is computed and tested on a clas-
sical system. Hence, the proposed model architecture is not
quantum in the real sense. Instead, it is quantum-inspired. The pixel intensities and interconnection weight matrices
are expressed in quantum formalism in classical simulations
using real vectors. Here, each pixel information and weight
term are presented as a vector with better expressibility and
non-linearity than classical neurons. The 3D-QNet network
architecture and the use of a unitary matrix representation in
network weights ensure the authenticity of quantum analogies
without sacrificing efficacy or efficiency, as shown by a full
comparison with state-of-the-art models on two benchmark
datasets. The incorporation of quantum-inspired computing
and tensor-based learning in the suggested network model aims
to provide faster convergence of the 3D-QNet architecture over
its non-tensorized implementation, thereby enabling accurate
segmentation results. g
However, the proposed 3D-QNet architecture may be im-
plemented utilizing a quantum-classical hybrid paradigm for
NISQ devices on the real quantum processor. In this hybrid
quantum classical framework, the N-connected third-order
neighborhood-based interconnection needed N (here N = 26)
input qubits, Hadamard gates, and Rotation gates, as well as
N(N −1) numbers of CNOT gates [15], which resulted in
quantum superposition and entanglement of the input states. This quantum-classical model lays the way for the imple-
mentation of quantum machine learning on near-term NISQ
devices using variational quantum circuits (VQCs) — a kind
of Quantum circuits with improved gate settings. It is worth noting that 3D-QNet has the potential to be
significantly more computationally efficient than the models
presented in the experiments, especially in multi-level seg-
mentation of BRATS 2019 MR images. The performance
of many quantum-inspired algorithms derives directly from
quantum parallelism, which is a fundamental characteristic of
many quantum systems. Heuristically, the proposed 3D-QNet However, the proposed 3D-QNet architecture may be im-
plemented utilizing a quantum-classical hybrid paradigm for
NISQ devices on the real quantum processor. In this hybrid
quantum classical framework, the N-connected third-order
neighborhood-based interconnection needed N (here N = 26)
input qubits, Hadamard gates, and Rotation gates, as well as
N(N −1) numbers of CNOT gates [15], which resulted in
quantum superposition and entanglement of the input states. This quantum-classical model lays the way for the imple-
mentation of quantum machine learning on near-term NISQ
devices using variational quantum circuits (VQCs) — a kind
of Quantum circuits with improved gate settings. B. Experimental Setup Experiments have been carried out using 3D-QNet and 3D-
QNet-NonTensor on 3D brain MR volumes collected from the
BRATS 2019 dataset of size 240×240 and on the LiTS17 data
set of size 512×512 with MATLAB 2020a. The proposed 3D-
QNet and 3D-QNet-NonTensor architectures are implemented
with the multi-level gray-scale images using distinct multi-
class levels L = 4, 6, and 8 characterized by the Vox-QSig
activation function. The steepness λ is varied in the range
0.23 to 0.24 with step size of 0.001. For FLAIR and T2, it has 8 5, respectively. The human expert annotated ground truth
slices for all the four different modalities are illustrated in
Figure 6. 3D-UNet [24], VoxResNet [26], DRINet [27], 3D-
ESPNet [28], and 3D-QNet-NonTensor segmented brain MR
volumes from BRATS19-CBICA-AAG are also demonstrated in
the Supplementary Materials. It has been observed from the
segmented MR slices that the proposed 3D-QNet is suitable in
segmenting the correct position and size of the complete tumor
while compared with the ground truth segmentation. However,
it is not efficient in mapping the sharp contour of the core and
enhanced tumor sub-regions outlined in the annotated slices. Table III presents the quantitative results reported using
the proposed 3D-QNet, 3D-QNet-NonTensor, 3D-UNet [24],
VoxResNet [26], DRINet [27], and 3D-ESPNet [28] on evalu-
ating the average accuracy (AC), dice similarity score (DS),
positive prediction value (PV ), and sensitivity (SS) [18]. It has been observed from the 3D-QNet segmented brain
MR slices and the results reported in Table III, that optimal
segmentation is achieved for FLAIR reported with an average
of 0.821 dice score (DS). The proposed 3D-QNet marginally
outperforms the convolutional architectures (3D-UNet [24],
VoxResNet [26], DRINet [27], 3D-ESPNet [28]), and 3D-
QNet-NonTensor in predicting complete brain tumor detection. However, it may be noted that the proposed 3D-QNet does not
intend to predict the core, enhanced tumor and necrosis sub
regions owing to lack of optimization of the parameters in
the suggested 3D-QNet architecture. The box plots are also
demonstrated in the Supplementary Materials citing the out-
come reported in Table III. Moreover, to show the effectiveness
of the proposed 3D-QNet architecture over 3D-UNet [24],
VoxResNet [26], DRINet [27], and 3D-ESPNet [28], we have
also conducted experiments on the Liver Tumor Segmentation
Benchmark (LiTS17) data set [48]. VII. DISCUSSIONS Heuristically, the proposed 3D-QNet 9 TABLE II: Results obtained using proposed 3D-QNet for complete tumor (WT) segmentation on BraTS19-CBICA-AAG-1-
flair-slice#69 TABLE II: Results obtained using proposed 3D-QNet for complete tumor (WT) segmentation on BraTS19-CBICA-AAG-1-
flair-slice#69 flair-slice#69
Level
Modality
AC =
TRP +TRN
TRP +FLP +TRN +FLN
DS =
2TRP
2TRP +FLP +FLN
PV =
TRP
TRP +FLP
SS =
TRP
TRP +FLN
υβ
υχ
υξ
υκ
υβ
υχ
υξ
υκ
υβ
υχ
υξ
υκ
υβ
υχ
υξ
υκ
L = 4
T 1
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.65
0.65
0.99
0.99
0.99
0.99
T 1 −CE
0.99
0.99
0.99
0.99
0.79
0.80
0.79
0.80
0.66
0.66
0.66
0.66
0.99
0.99
0.99
0.99
F LAIR
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.66
0.65
0.99
0.99
0.99
0.99
T 2
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.66
0.65
0.99
0.99
0.99
0.99
L = 6
T 1
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.66
0.65
0.99
0.99
0.99
0.99
T 1 −CE
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.66
0.65
0.99
0.99
0.99
0.99
F LAIR
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.66
0.65
0.99
0.99
0.99
0.99
T 2
0.99
0.99
0.99
0.99
0.62
0.80
0.80
0.79
0.44
0.66
0.66
0.65
0.99
0.99
0.99
0.99
L = 8
T 1
0.99
0.99
0.99
0.99
0.82
0.82
0.82
0.81
0.70
0.69
0.69
0.68
0.99
0.99
0.99
0.99
T 1 −CE
0.99
0.99
0.99
0.99
0.81
0.81
0.81
0.81
0.68
0.68
0.68
0.68
0.99
0.99
0.99
0.99
F LAIR
0.99
0.99
0.99
0.99
0.84
0.84
0.84
0.84
0.73
0.73
0.73
0.72
0.99
0.99
0.98
0.98
T 2
0.99
0.99
0.99
0.99
0.82
0.82
0.82
0.82
0.69
0.69
0.70
0.70
0.99
0.99
0.99
0.99 TABLE III: Comparative analysis of proposed 3D-QNet
with 3D-QNet-NonTensor, 3D-UNet [24], VoxResNet [26],
DRINet [27], and 3D-ESPNet [28] [The bold numbers rep-
resent evaluation metrics completed with a one-sided two-
sample KS test with a significance threshold of α = 0.05 [49]] TABLE III: Comparative analysis of proposed 3D-QNet
with 3D-QNet-NonTensor, 3D-UNet [24], VoxResNet [26],
DRINet [27], and 3D-ESPNet [28] [The bold numbers rep-
resent evaluation metrics completed with a one-sided two-
sample KS test with a significance threshold of α = 0.05 [49]] TABLE IV: Comparative Results on Liver Segmentation using
proposed 3D-QNet, 3D-UNet [24], VoxResNet [26], 3D-QNet-
NonTensor, DRINet [27], and 3D-ESPNet [28] [The bold
numbers represent evaluation metrics completed with a one-
sided two-sample KS test with a significance threshold of
α = 0.05 [49]] Methods
Modality
AC
DS
PV
SS
3D-UNet [24]
T 1
0.990
0.811
0.736
0.941
T 1 −CE
0.990
0.807
0.732
0.938
F LAIR
0.992
0.823
0.737
0.943
T 2
0.989
0.812
0.735
0.944
VoxResNet [26]
T 1
0.990
0.810
0.737
0.937
T 1 −CE
0.989
0.813
0.732
0.943
F LAIR
0.991
0.822
0.751
0.942
T 2
0.990
0.807
0.729
0.944
DRINet [27]
T 1
0.989
0.793
0.701
0.958
T 1 −CE
0.988
0.800
0.711
0.959
F LAIR
0.989
0.805
0.708
0.969
T 2
0.987
0.789
0.700
0.958
3D-ESPNet [28]
T 1
0.989
0.801
0.709
0.961
T 1 −CE
0.989
0.813
0.721
0.966
F LAIR
0.989
0.800
0.715
0.959
T 2
0.988
0.802
0.714
0.957
3D-QNet
T 1
0.989
0.801
0.736
0.965
T 1 −CE
0.989
0.811
0.740
0.957
F LAIR
0.991
0.821
0.751
0.957
T 2
0.990
0.814
0.736
0.960
3D-QNet-NonTensor
T 1
0.987
0.776
0.678
0.959
T 1 −CE
0.987
0.772
0.678
0.958
F LAIR
0.989
0.786
0.697
0.956
T 2
0.988
0.788
0.696
0.957 Methods
AC
DS
PV
SS
VoxResNet [26]
0.991
0.961
0.798
0.973
3D-QNet
0.989
0.958
0.801
0.965
3D-UNet [24]
0.991
0.959
0.830
0.964
3D-QNet-NonTensor
0.982
0.953
0.799
0.879
DRINet [27]
0.980
0.951
0.802
0.988
3D-ESPNet [28]
0.987
0.943
0.798
0.961 TABLE V: Average number of iterations required for conver-
gence of the proposed 3D-QNet and 3D-QNet-NonTensor TABLE V: Average number of iterations required for conver-
gence of the proposed 3D-QNet and 3D-QNet-NonTensor
Methods
Modality
Number of iterations
υβ
υχ
υξ
υκ
3D-QNet
T 1
30.89
30.64
30.38
30.91
T 1−CE
31.60
31.46
31.43
31.68
F lair
30.78
30.23
30.18
30.57
T 2
32.25
32.66
32.14
32.73
Liver
27.11
27.89
26.99
27.08
3D-QNet-NonTensor
T 1
31.46
31.59
31.43
31.69
T 1−CE
32.98
32.33
32.39
32.56
F lair
31.69
31.12
31.19
31.26
T 2
32.88
32.34
32.51
32.79
Liver
28.52
28.16
28.12
28.17 architecture has the potential to explore the intrinsic properties
of quantum parallelism to simultaneously compute the image
pixels. VII. DISCUSSIONS However, 3D-QNet is computed and experimented on
a classical system, and hence the quantum parallelism has
not been fully explored in the proposed quantum-inspired
framework. Despite the fact that quantum simulation requires
a great deal of resources in general, the proposed quantum-
inspired model, 3D-QNet, requires a lesser number of param-
eters compared to the 3D classical CNN models. 3D-QNet architecture to promote automatic semantic segmen-
tation of 3D medical images in real-time with minimum human
intervention, still being considered as an uphill task in the field
of volumetric medical image segmentation. Despite being a 3D self-supervised network model, 3D-QNet
achieved similar dice similarity score on complete tumor
detection as deeply supervised 3D-UNet, Vox-ResNet, DRINet
and ESPNet, thus promoting self-supervised network learning
for volumetric segmentation of medical images. In principle,
the proposed 3D-QNet is a general self-supervised network
architecture which can be extended to many other 3D medical
image segmentation avenues, where the segmented annotations
are limited. Furthermore, the proposed 3D self-supervised
model can be used immediately in any application (e.g.,
medical IoT devices) where 3D deep learning models face VII. DISCUSSIONS It is worth noting that 3D-QNet has the potential to be
significantly more computationally efficient than the models
presented in the experiments, especially in multi-level seg-
mentation of BRATS 2019 MR images. The performance
of many quantum-inspired algorithms derives directly from
quantum parallelism, which is a fundamental characteristic of
many quantum systems. VIII. CONCLUSION (a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69 (d) Slice#69 (e) Slice#44
(f) Slice#59
(g) Slice#64
(h) Slice#69 (f) Slice#59 (g) Slice#71 (h) Slice#95 (h) Slice#95 Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA-
AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the
BRATS 2019 data set [47] (Complete tumor (WT) region
comprises a union of brown, light green and green yellow,
core tumor (TC) is the union of light green and green yellow,
and green yellow corresponds to the tumor enhancing (TE)). Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA-
AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the
BRATS 2019 data set [47] (Complete tumor (WT) region
comprises a union of brown, light green and green yellow,
core tumor (TC) is the union of light green and green yellow,
and green yellow corresponds to the tumor enhancing (TE)). (m) Slice#44 (a) Slice#172 (b) Slice#181 (c) Slice#198 (d) Slice#206
(e) Slice#172 (f) Slice#181 (g) Slice#198 (h) Slice#206
Fig. 7: (a −d) 3D-QNet segmented volumetric Liver image
slices (scan#64), (e −h) manually segmented image slices
(scan#64) from data set [48] (Union of overlapped brown
and red corresponds to a complete tumor (WT) region). Fig. 4: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG-
1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19-
CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). Fig. 4: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG-
1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19-
CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). ( ) Sli
#172 (f) Sli
#181 ( ) Sli
#198 (h) Sli
#206 plete tumor (WT) region). (a) Slice#52
(b) Slice#60
(c) Slice#71
(d) Slice#95
(e) Slice#52
(f) Slice#60
(g) Slice#71
(h) Slice#95
(i) Slice#52
(j) Slice#60
(k) Slice#71
(l) Slice#95
(m) Slice#52
(n) Slice#60
(o) Slice#71
(p) Slice#95
Fig. 5: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAB-1-flair, (e−h) BraTS19-CBICA-AAB-
1-t2, (i −l) BraTS19-CBICA-AAB-1-t1ce, (m −p) BraTS19-
CBICA-AAB-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). p
(
)
g
) (e) Slice#172 (f) Slice#181 (e) Slice#172 (f) Slice#181 (g) Slice#198 (h) Slice#206 (g) Slice#198 (h) Slice#206 Fig. VIII. CONCLUSION 7: (a −d) 3D-QNet segmented volumetric Liver image
slices (scan#64), (e −h) manually segmented image slices
(scan#64) from data set [48] (Union of overlapped brown
and red corresponds to a complete tumor (WT) region). (a) Slice#52
(b) Slice#60
(c) Slice#71
(d) Slice#95 (b) Slice#60 significant challenges. However, the 3D-QNet fails to yield
optimal outcome for multi-level segmentation on the BRATS
2019 data set. The authors are currently engaged in extend-
ing the 3D-QNet architecture by up-scaling the intermediate
volumetric features in the network and optimizing its hyper-
parameters to yield optimal segmentation outcome. (e) Slice#52
(f) Slice#60
(g) Slice#71
(h) Slice#95 VIII. CONCLUSION A 3D Quantum-inspired Self-supervised Tensor Network
(3D-QNet) architecture characterized by S-connected voxel-
wise processing for fully automated semantic segmentation of
Brain MR volumes and 3D Liver CT scans, is presented in this
work. Intensive validation using the BRATS 2019 and LiTS17
data sets shows the efficacy of the proposed self-supervised 10 10
(a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69
(e) Slice#44
(f) Slice#59
(g) Slice#64
(h) Slice#69
(i) Slice#44
(j) Slice#59
(k) Slice#64
(l) Slice#69
(m) Slice#44
(n) Slice#59
(o) Slice#64
(p) Slice#69
Fig. 4: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG-
1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19-
CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). (a) Slice#52
(b) Slice#60
(c) Slice#71
(d) Slice#95
(e) Slice#52
(f) Slice#60
(g) Slice#71
(h) Slice#95
(i) Slice#52
(j) Slice#60
(k) Slice#71
(l) Slice#95
(m) Slice#52
(n) Slice#60
(o) Slice#71
(p) Slice#95
Fig. 5: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAB-1-flair, (e−h) BraTS19-CBICA-AAB-
1-t2, (i −l) BraTS19-CBICA-AAB-1-t1ce, (m −p) BraTS19-
CBICA-AAB-1-t1 from the BRATS 2019 data set [47] (Union
(a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69
(e) Slice#52
(f) Slice#60
(g) Slice#71
(h) Slice#95
Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA-
AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the
BRATS 2019 data set [47] (Complete tumor (WT) region
comprises a union of brown, light green and green yellow,
core tumor (TC) is the union of light green and green yellow,
and green yellow corresponds to the tumor enhancing (TE)). (a) Slice#172 (b) Slice#181 (c) Slice#198 (d) Slice#206
(e) Slice#172 (f) Slice#181 (g) Slice#198 (h) Slice#206
Fig. 7: (a −d) 3D-QNet segmented volumetric Liver image
slices (scan#64), (e −h) manually segmented image slices
(scan#64) from data set [48] (Union of overlapped brown
and red corresponds to a complete tumor (WT) region). significant challenges. However, the 3D-QNet fails to yield
optimal outcome for multi-level segmentation on the BRATS
2019 data set. The authors are currently engaged in extend-
ing the 3D-QNet architecture by up-scaling the intermediate
volumetric features in the network and optimizing its hyper-
parameters to yield optimal segmentation outcome. APPENDIX
A. Convergence Analysis of 3D-QNet
Le us assume the optimal phase angles at depth d for the weighted matrix and the
activation are denoted as ωd and ϑd, respectively. VIII. CONCLUSION Now, consider
Wι,d = ωι,d −ωd
(31)
Vι,d = ϑι,d −ϑd
(32)
and
Dι,d = ωι+1,d −ωι,d = Wι+1,d −Wι,d
(33)
Pι,d = ϑι+1,d −ϑι,d = Vι+1,d −Vι,d
(34)
The loss function ζ(ωι,d, ϑι,d) is differentiable with respect to ωι,d and ϑι,d as
∂ζ(ωι,d, ϑι,d)
∂ωι,d
ij
= 2
N
N
X
i=1
S
X
j=1
△φι,d
ij (ωι,d
ij , ϑι,d
j
)
"
∂φι+1,d
ij
(ωι+1,d
ij
, ϑι+1,d
j
)
−
∂φι,d
ij (ωι,d
ij , ϑι,d
j
)
#
(35) (a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69
(e) Slice#44
(f) Slice#59
(g) Slice#64
(h) Slice#69
(i) Slice#44
(j) Slice#59
(k) Slice#64
(l) Slice#69
(m) Slice#44
(n) Slice#59
(o) Slice#64
(p) Slice#69
Fig. 4: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG-
1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19-
CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). (a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69
(e) Slice#52
(f) Slice#60
(g) Slice#71
(h) Slice#95
Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA-
AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the
BRATS 2019 data set [47] (Complete tumor (WT) region
comprises a union of brown, light green and green yellow,
core tumor (TC) is the union of light green and green yellow,
and green yellow corresponds to the tumor enhancing (TE)). (a) Slice#172 (b) Slice#181 (c) Slice#198 (d) Slice#206 (a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69
(e) Slice#52
(f) Slice#60
(g) Slice#71
(h) Slice#95
Fig. 6: Annotated Brain MR volume (a−d) BraTS19-CBICA-
AAG-1-seg, (e −h) BraTS19-CBICA-AAB-1-seg from the
BRATS 2019 data set [47] (Complete tumor (WT) region
comprises a union of brown, light green and green yellow,
core tumor (TC) is the union of light green and green yellow,
and green yellow corresponds to the tumor enhancing (TE)). (a) Slice#44
(b) Slice#59
(c) Slice#64
(d) Slice#69
(e) Slice#44
(f) Slice#59
(g) Slice#64
(h) Slice#69
(i) Slice#44
(j) Slice#59
(k) Slice#64
(l) Slice#69
(m) Slice#44
(n) Slice#59
(o) Slice#64
(p) Slice#69
Fig. 4: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAG-1-flair, (e−h) BraTS19-CBICA-AAG-
1-t2, (i −l) BraTS19-CBICA-AAG-1-t1ce, (m −p) BraTS19-
CBICA-AAG-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). A. Convergence Analysis of 3D-QNet Le us assume the optimal phase angles at depth d for the weighted matrix and the
activation are denoted as ωd and ϑd, respectively. Now, consider (j) Slice#60 (k) Slice#71 (k) Slice#71
(l) Sli (k) Slice#71 Wι,d = ωι,d −ωd
(31)
Vι,d = ϑι,d −ϑd
(32) and Dι,d = ωι+1,d −ωι,d = Wι+1,d −Wι,d
(33)
Pι,d = ϑι+1,d −ϑι,d = Vι+1,d −Vι,d
(34) (o) Slice#71 (m) Slice#52 (m) Slice#52 (p) Slice#95 (n) Slice#60 The loss function ζ(ωι,d, ϑι,d) is differentiable with respect to ωι,d and ϑι,d as n ζ(ωι,d, ϑι,d) is differentiable with respect to ωι,d and ϑι,d as Fig. 5: 3D-QNet segmented Brain MR volume (a −d)
BraTS19-CBICA-AAB-1-flair, (e−h) BraTS19-CBICA-AAB-
1-t2, (i −l) BraTS19-CBICA-AAB-1-t1ce, (m −p) BraTS19-
CBICA-AAB-1-t1 from the BRATS 2019 data set [47] (Union
of overlapped brown/yellow and green corresponds to a com-
plete tumor (WT) region). REFERENCES ζ(ωι+1,d) ≤ζ(ωι,d) + ⟨∇ωζ(ωι,d), ωι+1,d −ωι,d⟩+ ι
2 ||ωι+1,d −ωι,d||2
= ζ(ωι,d) + ⟨∇ωζ(ωι,d) −ρ∇ωζ(ωι,d)⟩+ L
2 || −ρ∇ωζ(ωι,d)||2
= ζ(ωι,d) −ρ||∇ωζ(ωι,d)||2 + ρ2 L
2 ||∇ωζ(ωι,d)||2
= ζ(ωι,d) −ρ(1 −ρ L
2 )||∇ωζ(ωι,d)||2
≤ζ(ωι,d) −ρ
2 ||∇ωζ(ωι,d)||2 (Assuming, ρ ∈(0, 1
L ])
≤ζ(ωd) + ⟨∇ωζ(ωι,d), ωι,d −ωd⟩−ρ
2 ||∇ωζ(ωι,d)||2, (ζ is convex)
= ζ(ωd) + ⟨∇ωζ(ωι,d), ωι,d −ωd⟩−ρ
2 ||∇ωζ(ωι,d)||2+
1
2ρ (||ωι,d −ωd||2 −||ωι,d −ωd||2)
= ζ(ωd) + 1
2ρ (||ωι,d −ωd||2 −(||ωι,d||2 −2⟨ωι,d, ωd⟩+
||ωd||2 −2ρ⟨∇ωζ(ωι,d), ωι,d −ωd⟩+ ρ2||∇ωζ(ωι,d)||2))
= ζ(ωd) + 1
2ρ (||ωι,d|| −ωd||2 −(||ωι,d −ρ∇ωζ(ωι,d)||2−
2⟨ωι,d −∇ωζ(ωι,d), ωd⟩+ ||ωd||2))
= ζ(ωd) + 1
2ρ (||ωι,d −ωd||2 −||ωι+1,d −ωd||2)
∴, ζ(ωι+1,d) −ζ(ωd) ≤1
2ρ (||ωι,d −ωd||2 −||ωι+1,d −ωd||2) [7] Q. Doua, L. Yua, H. Chena, Y.Jina, X. Yanga, J. Q. Pheng, and A. Heng,
“3D deeply supervised network for automated segmentation of volumetric
medical images," Medical Image Analysis, vol. 41, pp. 40–54, 2017, doi:
https://doi.org/10.1016/j.media.2017.05.001. [8] V. Gandhi, G. Prasad, D. Coyle, L. Behera, and T. M. McGinnity,
“Quantum neural network-based EEG filtering for a brain-computer
interface,” IEEE Transaction on Neural Network and Learning Systems,
vol. 25, no. 2, pp. 278-–288, 2014, doi: 10.1109/TNNLS.2013.2274436. [9] P. Li, H. Xiao, F. Shang, X. Tong, X. Li, and M. Cao, “A hybrid quantum-
inspired neural networks with sequence inputs,” Neurocomputing, vol. 117, pp. 81-–90, 2013, doi: https://doi.org/10.1016/j.neucom.2013.01.029. [10] N. Masuyama, C. K. Loo, M. Seera, and N. Kubota, “Quantum-
Inspired Multidirectional Associative Memory With a Self-Convergent
Iterative Learning,” IEEE Transaction on Neural Network and Learning
Systems, vol. 29, no. 4, pp. 1058—1068, 2018, doi: 10.1109/TNNLS. 2017.2653114. [11] S. Bhattacharyya, P. Pal and S. Bhowmick, “Binary Image Denoising
Using a Quantum Multilayer Self Organizing Neural Network," Applied
Soft Computing, vol. 24, pp. 717–729, 2014, doi: https://doi.org/10.1016/
j.asoc.2014.08.027. [12] M. Mediouni, D. R. Schlatterer, H. Madry, M. Cucchiarini and B. Rai,
“A review of translational medicine. The future paradigm: how can we
connect the orthopedic dots better?," Current Medical Research and
Opinion, vol. 34, no. 7, pp. 1217–1229, 2018, doi: 10.1080/03007995. 2017.1385450. Similarly, it can also be shown that Similarly, it can also be shown that ζ(ϑι+1,d) −ζ(ϑι,d) ≤1
2ρ (||ϑι+1,d −ϑd||2 −||ϑι+1,d −ϑd||2)
(45)
Now, according to Thaler formula ζ(ϑι+1,d) −ζ(ϑι,d) ≤1
2ρ (||ϑι+1,d −ϑd||2 −||ϑι+1,d −ϑd||2)
(45)
[ ζ(ϑι+1,d) −ζ(ϑι,d) ≤1
2ρ (||ϑι+1,d −ϑd||2 −||ϑι+1,d −ϑd||2)
(45)
Now, according to Thaler formula [13] M. Mediouni, R. Madiouni, M. Gardner, N. A. Convergence Analysis of 3D-QNet ∂ζ(ωι,d, ϑι,d)
∂ωι,d
ij
= 2
N
N
X
i=1
S
X
j=1
△φι,d
ij (ωι,d
ij , ϑι,d
j
)
"
∂φι+1,d
ij
(ωι+1,d
ij
, ϑι+1,d
j
)
∂ωι+1,d
ij
−
∂φι,d
ij (ωι,d
ij , ϑι,d
j
)
∂ωι,d
ij
#
(35) (35) 11 ∂ζ(ωι,d, ϑι,d)
∂ϑι,d
j
= 2
N
N
X
i=1
S
X
j=1
△φι,d
ij (ωι,d
ij , ϑι,d
j
)
"
∂φι+1,d
ij
(ωι+1,d
ij
, ϑι+1,d
j
)
∂ϑι+1,d
j
−
∂φι,d
ij (ωι,d
ij , ϑι,d
j
)
∂ϑι,d
j
#
(36)
≈
"
{−ρι,d
ij
∂ζ(ωι,d, ϑι,d)
∂ωι,d
ij
}2 + {−κι,d
j
∂ζ(ωι,d, ϑι,d)
∂ϑl,d
j
}2
#
{ζ(ωι,d, ϑι,d)}
1
ι
(47)
It is obvious that (ζ(ωι+1,d, αι+1,d) −ζ(ωι,d, ϑι,d)) ≤0 and the sequences of
{ωι,d} and {ϑι,d} are monotonically decreasing as ≈
"
{−ρι,d
ij
∂ζ(ωι,d, ϑι,d)
∂ωι,d
ij
}2 + {−κι,d
j
∂ζ(ωι,d, ϑι,d)
∂ϑl,d
j
}2
#
{ζ(ωι,d, ϑι,d)}
1
ι
(47) (47) (47) It is obvious that (ζ(ωι+1,d, αι+1,d) −ζ(ωι,d, ϑι,d)) ≤0 and the sequences of
{ωι,d} and {ϑι,d} are monotonically decreasing as where, where, lim
ι→∞ζ(ωι,d, ϑι,d) = (ωd, ϑ
d)
(48) lim
ι→∞ζ(ωι,d, ϑι,d) = (ωd, ϑ
d) (48) △φι,d
ij (ωι,d
ij ϑj) = |φι+1,d
ij
(ωι+1,d
ij
, ϑι+1,d
j
) −φι,d
ij (ωι,d
ij , ϑι,d
j
)|
(37) (37) and The following equations evaluate the change in phase or angles (△ω and △α) of the
rotation gates as lim
ι→∞
||ζ(ωι+1,d, ϑι+1,d) −(ωd, ϑ
d)||
||ζ(ωι,d, ϑι,d) −(ωd, ϑ
d)||
≤1
(49) (49) △ωι,d
ij
= −ρι,d
ij { ∂φ(ωι,d, ϑι,d)
∂ωι,d
ij
φ(ωι,d, ϑι,d)}
1
τ
(38)
△ϑι,d
j
= −κι,d
j
{ ∂φ(ωι,d, ϑι,d)
∂ϑι,d
i
φ(ωι,d, ϑι,d))}
1
τ
(39) (38) B. Code Availability 3D-QNet implementation is made available in GitHub: https://github.com/konar1987/
3D-QNet for brain volume image segmentation with few samples, tailored and tested for
FLAIR and T2 from the BRATS 2019 data set [47]. where, ρij and κj refer to the learning rates for the adjustments of weights and
activation, respectively and are evaluated as REFERENCES ρι,d
ij
= X ι,d
i
−X ι,d
ij ∀j = 1, 2 . . . 8
κι,d
j
= (
X
j
X ι,d
i,j )∀j = 1, 2 . . . 8
(40) [1] G. Litjens et al., “A survey on deep learning in medical image analysis,”
Medical Image Analysis, vol. 42, pp. 60—88, 2017. doi: https://doi.org/
10.1016/j.media.2017.07.005. (40) j
[2] M. Huang, W. Yang, Y. Wu, J. Jiang, W. Chen, and Q. Feng, “Brain Tumor
Segmentation Based on Local Independent Projection-Based Classifica-
tion," IEEE Transactions on Biomedical Engineering, vol. 61, no. 10, pp. 2633–2645, 2014, doi: 10.1109/TBME.2014.2325410. The sequences of {ωι,d} and {ϑι,d} converge super-linearly subject to the following
conditions [15]. ||
ι+1 d
d|| lim
ι→∞
||ωι+1,d −ωd||
||ωι,d −ωd||
≤1 lim
ι→∞
||ωι+1,d −ωd||
||ωι,d −ωd||
≤1
(41) lim
ι→∞
||ωι+1,d −ωd||
||ωι,d −ωd||
≤1
(41)
and
||Wι+1,d|| = O||Dι,d||
(42)
Also,
lim
ι→∞
||ϑι+1,d −ϑ
d||
||ϑι −ϑ
d||
≤1
(43)
and
||Vι+1,d|| = O||Pι,d||
(44) (41) [3] Z. Wu, K. D. Paulsen, and J. M. Sullivan, “Adaptive model initialization
and deformation for automatic segmentation of T1-weighted brain MRI
data", IEEE Transactions on Biomedical Engineering, vol. 52, no. 6, pp. 1128–1131, 2005, doi: 10.1109/TBME.2005.846709. and ||Wι+1,d|| = O||Dι,d|| (42) Also, lim
ι→∞
||ϑι+1,d −ϑ
d||
||ϑι −ϑ
d||
≤1 [4] M. Chung, J. Lee, M. Lee, J. Lee, Y-G. Shin, “Deeply self-supervised
contour embedded neural network applied to liver segmentation," Com-
puter Methods and Programs in Biomedicine, vol. 192, pp. 105447, 2020,
https://doi.org/10.1016/j.cmpb.2020.105447. (43) and ||Vι+1,d|| = O||Pι,d|| (44) [5] F. Milletari, N. Navab, and S. A. Ahmadi, “V-Net: Fully Convolutional
Neural Networks for Volumetric Medical Image Segmentation" 2016
Fourth International Conference on 3D Vision (3DV), pp. 565-–571, 2016
doi:10.1109/3DV.2016.79. The convergence of the sequence {ωι,d} according to L-Lipschitz continuity is
illustrated as [50] [6] O. Ronneberger, P. Fischer, T. Brox, “U-Net: convolutional networks
for biomedical image segmentation. International Conference on Med-
ical Image Computing and Computer-Assisted Intervention (MICCAI
2015), vol. 9351, pp. 234-–241. 2016. doi: https://doi.org/10.1007/
978-3-319-46723-8_4. REFERENCES Maulik, “Multilevel image segmen-
tation with adaptive image context based thresholding,” Applied Soft
Computing, vol. 11, no.1, pp. 946–962, 2011, doi: https://doi.org/10.1016/
j.asoc.2010.01.015. [25] A. Brebisson, and G. Montana, “Deep Neural Networks for Anatomical
Brain Segmentation,” In: Proceedings of the IEEE Conference on Com-
puter Vision and Pattern Recognition Workshops, pp. 20-–28, 2015, doi:
10.1109/CVPRW.2015.7301312. [45] N. Otsu, “A threshold selection method from gray level histograms,"
IEEE Transactions on Systems, Man, and Cybernetics, vol. 9, no. 1, pp. 62–66, 1979, doi: 10.1109/TSMC.1979.4310076. [26] H. Chen, Q. Dou, L. Yu, J. Qin, and P. A. Heng, 2017. “VoxResNet:
Deep voxelwise residual networks for brain segmentation from 3D MR
images,” NeuroImage, vol. 170, pp. 446-455, 2017, doi: https://doi.org/
10.1016/j.neuroimage.2017.04.041. [46] H. P. Ng, S. H. Ong, K. W. C. Foong, P. S. Goh, and W. L. Nowinski,
“Medical Image Segmentation Using K-Means Clustering and Improved
Watershed Algorithm,"
2006 IEEE Southwest Symposium on Image
Analysis and Interpretation, 2006, doi: 10.1109/SSIAI.2006.1633722. [27] L.Chen , P. Bentley, K. Mori, K. Misawa, M. Fujiwara, and D. Rueckert,
“DRINet for Medical Image Segmentation,” IEEE Transaction on Medical
Imaging, vol. 37, no. 11, 2018, doi: 10.1109/TMI.2018.2835303.i [47] B. H. Menze et al., “The multimodal brain tumor image segmentation
benchmark (BRATS)," IEEE Transactions on Medical Imaging, vol. 34,
no. 10, pp. 1993-–2024, 2015, doi: 10.1109/TMI.2014.2377694. [28] N. Nuechterlein and S. Mehta, “3D-ESPNet with Pyramidal Refinement
for Volumetric Brain Tumor Image Segmentation,” International MICCAI
Brainlesion Workshop (BrainLes 2018), pp. 245–253, 2018, doi: https:
//doi.org/10.1007/978-3-030-11726-9_22. [48] P. Bilic et al., “The Liver Tumor Segmentation Benchmark (LiTS),"
arXiv, 2019, arXiv:1901.04056. [29] G. Wang et al., “Interactive Medical Image Segmentation Using Deep
Learning With Image-Specific Fine Tuning,” IEEE Transactions on Med-
ical Imaging, vol. 37, no. 7, 2018, doi: 10.1109/TMI.2018.2791721. [49] M. H. Gail and S. B. Green, “Critical values for the one-sided two-
sample Kolmogorov–Smirnov statistic," J. Am. Stat. Assoc., vol. 71, pp. 757–760, 1976. [30] N. Tajbakhsh et al., “Convolutional Neural Networks for Medical Image
Analysis: Full Training or Fine Tuning?,” IEEE Transactions on Medical
Imaging, vol. 35, no. 5, pp. 1299–1312, 2016, doi: 10.1109/TMI.2016. 2535302. [50] H. Kim, J. Kang, W. Park, S. Ko, Y. Cho, D. Yu, Y. Song, and J. Choi,
“Convergence Analysis of Optimization Algorithms," 2017, arXiv:1707. 01647. [31] X. Zhuang, Y. Li, Y. Hu, K. Ma, Y. Yang, and Y. REFERENCES Vries,
M. J. N. L. Benders and I. Išgum, “Automatic Segmentation of MR
Brain Images With a Convolutional Neural Network,”IEEE Transactions
on Medical Imaging, vol.35, no.5, 2016, doi: 10.1109/TMI.2016.2548501. [39] D. Konar, S. Bhattacharya, B. K. Panigrahi, K. Nakamatsu “A quantum
bi-directional self-organizing neural network (QBDSONN) architecture
for binary object extraction from a noisy perspective,” Applied Soft
Computing, vol.46, pp. 731–752, 2016, doi: https://doi.org/10.1016/j.asoc. 2015.12.040. [20] G. Wang et al., “DeepIGeoS: A Deep Interactive Geodesic Framework
for Medical Image Segmentation," IEEE Transactions on Pattern Analysis
and Machine Intelligence, vol. 41, no. 7, pp. 1559–1572, 2019, doi: 10. 1109/TPAMI.2018.2840695. [40] D. Konar, S. Bhattacharya, U. Chakraborty, T. K.Gandhi, B. K. Pani-
grahi, “A quantum parallel bi-directional self-organizing neural network
(QPBDSONN) architecture for extraction of pure color objects from
noisy background,” Proc. IEEE International Conference on Advances in
Computing, Communications and Informatics (ICACCI), 2016, pp. 1912–
1918, 2016, doi: 10.1109/ICACCI.2016.7732330. [21] S. Banerjee, L. Magee, D. Wang et al., “Semantic segmentation of
microscopic neuroanatomical data by combining topological priors with
encoder–decoder deep networks," Nature Machine Intelligence, vol. 2,
pp. 585—594, 2020, doi: https://doi.org/10.1038/s42256-020-0227-9.i [22] K. Kamnitsas, “Efficient multi-scale 3D CNN with fully connected CRF
for accurate brain lesion segmentation,” Medical Image Analysis, vol. 36,
pp. 61–78, 2017, doi: https://doi.org/10.1016/j.media.2016.10.004. [41] D. Konar, S. Bhattacharyya and B. K. Panigrahi, “QIBDS Net: A
Quantum-Inspired Bi-Directional Self-supervised Neural Network Archi-
tecture for Automatic Brain MR Image Segmentation,” Proc. 8th Inter-
national Conference on Pattern Recognition and Machine Intelligence
(PReMI 2019), vol. 11942, pp. 87–95, 2019, doi:https://doi.org/10.1007/
978-3-030-34872-4_64. [23] Q. Dou, H. Chen, L. Yu, L. Zhao, J. Qin, D. Wang, V. C. Mok, L. Shi,
and P. A. Heng. “Automatic Detection of Cerebral Microbleeds From
MR Images via 3D Convolutional Neural Networks, IEEE Transactions
on Medical Imaging, vol. 35, no. 5, pp. 1182-–1195, 2016, doi: 10.1109/
TMI.2016.2528129. [42] M. A. Nielson and I. L. Chung, Quantum computation and quantum
information, Cambridge University press, 2002. [24] Ö. Çiçek, A. Abdulkadir, S. S. Lienkamp, T. Brox, and O. Ronneberger,
“3D U-Net: Learning Dense Volumetric Segmentation from Sparse An-
notation,” International Conference on Medical Image Computing and
Computer-Assisted Intervention (MICCAI 2016), pp. 424-–432, 2016, doi:
https://doi.org/10.1007/978-3-319-46723-8_49. [43] T. G. Kolda, B. W. Bader, “Tensor Decompositions and Applications,”
SIAM Review, vol. 51, no. 3, pp. 455-–500, 2009, doi: https://doi.org/10. 1137/07070111X. [44] S. Bhattacharyya, P. Dutta and U. REFERENCES Vaughan, Neil, “Trans-
lational medicine: Challenges and new orthopaedic vision (Mediouni-
Model)," Current Orthopaedic Practice, vol. 31. no. 2, pp. 196-200, 2020,
doi: 10.1097/BCO.0000000000000846. Now, according to Thaler formula Now, according to Thaler formula ζ(ωι+1,d, ϑι+1,d) −ζ(ωι,d, ϑι,d) =
(46)
d
d ζ(ωι+1,d, ϑι+1,d) −ζ(ωι,d, ϑι,d) =
(46) [14] D. Konar, S. Bhattacharyya, T. K. Gandhi and B. K. Panigrahi, “A
quantum-inspired self-supervised Network model for automatic segmen-
tation of brain MR images," Applied Soft Computing, vol. 93, 2020, doi:
https://doi.org/10.1016/j.asoc.2020.106348. ζ(
,
)
ζ(
,
)
(
h
△ωι,d
ij
△ϑι,d
j
i
∂ζ(ωι,d,ϑι,d)
∂ωι,d
ij
∂ζ(ωι,d,ϑι,d
∂ϑι,d
j
+ O
h
||△ωι,d
ij
△ϑι,d
i
||
i 12 [15] D. Konar, S. Bhattacharyya, B. K. Panigrahi and E. C. Behrman, “Qutrit-
Inspired Fully Self-Supervised Shallow Quantum Learning Network for
Brain Tumor Segmentation," IEEE Transactions on Neural Networks and
Learning Systems, vol. 33, no. 11, pp. 6331–6345, Nov. 2022, doi: 10. 1109/TNNLS.2021.3077188. [34] S. Bhattacharyya, P. Dutta and U. Maulik, “A parallel bi-directional
self-organizing neural network (PBDSONN) architecture for color image
extraction and segmentation," Neurocomputing, vol. 86, pp. 1–23, 2012,
doi: https://doi.org/10.1016/j.neucom.2011.11.025. [35] G. Purushothaman, N. B. Karayiannis, “Quantum neural networks
(QNNs): inherently fuzzy feedforward neural networks,” IEEE Transac-
tions on Neural Networks, vol. 8 , no. 3, 1997, doi: 10.1109/72.572106. [16] M. Havaei et al., “Brain tumor segmentation with deep neural networks,”
Medical Image Analysis, vol. 35, pp. 18–31, 2017, doi: https://doi.org/10. 1016/j.media.2016. [36] T. C. Lu, G. R. Yu, and J. C. Juang, “Quantum-based algorithm
for optimizing artificial neural networks,” IEEE Transaction on Neural
Network and Learning Systems, vol. 24, no. 8, pp. 1266—1278, 2013,
doi: 10.1109/TNNLS.2013.2249089. j
[17] E. Shelhamer, J. Long, and T. Darrell, “Fully convolutional networks
for semantic segmentation,” IEEE Transactions on Pattern Analysis and
Machine Intelligence, vol. 39, no. 4, pp. 640–651, 2017, doi: 10.1109/
TPAMI.2016.2572683. [37] Nam-H. Nguyen ; E. C. Behrman, A. Moustafa, J. E. Steck, “Bench-
marking Neural Networks For Quantum Computations,” IEEE Trans-
actions on Neural Networks and Learning Systems, pp. 1-10, 2019,
doi:10.1109/TNNLS.2019.2933394. [18] S. Pereira, A. Pinto, V. Alves, and C. A. Silva, “Brain Tumor Seg-
mentation Using Convolutional Neural Networks in MRI Images,” IEEE
Transactions on Medical Imaging, vol.35, no. 5, 2016, doi: 10.1109/TMI. 2016.2538465. [38] E. M. Stoudenmire,
and
D. J. Schwab,
“Supervised
Learning
with Quantum-Inspired Tensor Networks,” https://www.arxiv-vanity.com/
papers/1605.05775/, 2020. [19] P. Moeskops,
M. A. Viergever,
A. M. Mendrik,
L. S. REFERENCES Zheng, “Self-supervised
Feature Learning for 3D Medical Images by Playing a Rubik’s Cube,”
International Conference on Medical Image Computing and Computer-
Assisted Intervention (MICCAI 2019), pp. 420–428, https://doi.org/10. 1007/978-3-030-32251-9_46. [32] M. Blendowski, H. Nickisch, M. P. Heinrich, “How to Learn from Unla-
beled Volume Data: Self-supervised 3D Context Feature Learning," Proc. Medical Image Computing and Computer Assisted Intervention – MIC-
CAI 2019, vol 11769, 2019, https://doi.org/10.1007/978-3-030-32226-7_
72. [33] A. Ghosh, N. R. Pal, and S. K. Pal, “Self organization for object
extraction using a multilayer neural network and fuzziness measures,"
IEEE Transactions on Fuzzy Systems, vol. 1, no.1, pp. 54–68, 1993, doi:
10.1109/TFUZZ.1993.390285.
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https://openalex.org/W3011224673
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https://periodicos.ufrn.br/bibliocanto/article/download/18864/12498
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Portuguese
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Um Olhar ao estudante com surdez da Universidade Federal do Rio Grande do Norte:
|
BiblioCanto
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cc-by
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2 Doutora em Educação - UFRGS; Mestra em Educação - UFRGS; Especialização em Museologia
Patrimônio
Cultural
-
UFRGS;
Graduação
em
Artes
Plásticas
-
UFRGS.
Lattes:
http://lattes.cnpq.br/1990500214696545 1 Especialização em andamento em LIBRAS - UFRN; Especialista em Gestão Documental - UFRN;
Graduada em Biblioteconomia - UFRN. Lattes: http://lattes.cnpq.br/0834125840536553 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 1 Especialização em andamento em LIBRAS - UFRN; Especialista em Gestão Documental UM OLHAR AO ESTUDANTE COM SURDEZ DA UNIVERSIDADE FEDERAL DO
RIO GRANDE DO NORTE: ANÁLISE SOBRE A ACESSIBILIDADE À
INFORMAÇÃO NA BIBLIOTECA CENTRAL ZILA MAMEDE
A LOOK AT THE STUDENT WITH AMAZING FEDERAL UNIVERSITY OF RIO
GRANDE DO NORTE: ANALYSIS ON INFORMATION ACCESSIBILITY IN THE
ZILA MAMEDE CENTRAL LIBRARY Michele Rodrigues Dias
1
michele-1011@hotmail.com
Gabriela Bon
2
gabibon@gmail.com Resumo: Este estudo trata do atendimento ao aluno com surdez em bibliotecas
universitárias, em especial, na Biblioteca Central Zila Mamede, integrante do
Sistema de Bibliotecas da Universidade Federal do Rio Grande Norte. Aborda
também a legislação vigente acerca do Acesso à informação e da Acessibilidade
Universal nestes ambientes, bem como, apresenta a Política de Inclusão e
Acessibilidade para os estudantes com deficiência na Universidade Federal do Rio
Grande Norte. Discorre sobre as políticas e diretrizes criadas e disponibilizadas para
que as bibliotecas se adequem em prol da inclusão informacional dos usuários com
surdez nos ambientes biblioteconômicos. Utiliza como metodologia, pesquisa de
caráter exploratório. Como instrumento de coleta de informações foi aplicada técnica
de
observação
e
entrevistas
com
docentes
do
curso
de
Letras/LIBRAS e
bibliotecários da Biblioteca Central Zila Mamede. Em decorrência, é explicitado como
é realizado o atendimento para este público no Laboratório de Acessibilidade da
Biblioteca Central Zila Mamede. Sugere recomendações, se adotadas, podem
oferecer ao aluno com surdez acesso aos serviços das bibliotecas de forma BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 83 igualitária, derrubando as barreiras informacionais e comunicacionais existentes, e
por fim, prioriza a criação de coleções acessíveis para este público. Palavras- chave: Acessibilidade. Surdez. Desenvolvimento de coleções. Acervo
acessível. Biblioteca Universitária. 3 Documento não paginado. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 1 INTRODUÇÃO Ao longo da história, as pessoas com deficiência sempre foram rotuladas em
consequência
de
conceitos
errôneos
e
representações
construídas
por sua
condição, carregando com isso um estigma da deficiência e da incapacidade. De
acordo com dados do Censo Demográfico do Instituto Brasileiro de Geografia e
Estatística - IBGE (2010) , 23,9% da população brasileira, em torno de 46 milhões de
3
pessoas possui pelo menos uma deficiência. Deste total, uma grande parcela é
excluída do acesso à informação, apesar dos avanços legislativos em nosso país
nas últimas décadas. Dentre as deficiências existentes, enfatiza-se neste artigo a deficiência
auditiva, apresentando a necessidade da aplicabilidade das políticas públicas a partir
da evolução da legislação brasileira. Cabe ressaltar, que estas leis foram criadas a
fim de garantir o acesso da pessoa surda à informação em todos os níveis sociais,
dentre os quais, se incluem os acervos biblioteconômicos. Além disso, este acesso
só pode ser assegurado através de um processo de gestão da informação nas
bibliotecas e da formação e desenvolvimento de coleções adequadas a este público
específico dado a complexidade de sua cultura própria. Some-se a isso, a relevância
das bibliotecas como elemento essencial para o desenvolvimento da cidadania de
forma incontestável. Apesar de o termo ‘Acessibilidade’ ser um tema abordado em diversas áreas
do conhecimento e em fontes teóricas, nota-se que no âmbito das bibliotecas, o
trabalho com a pessoa com deficiência auditiva ainda é bastante insipiente. Somado BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 84 84 4
https://digitaispuccampinas.wordpress.com/2012/10/06/numero-de-alunos-com-deficiencia-no-ensino-
superior-aumentou-9336-aponta-mec/. Acesso em: 30 nov. 2019.
BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Além disso, foi identificada uma pequena quantidade de publicações na área
da ciência da informação, sobre acessibilidade à informação nas bibliotecas para
este público específico. 2 METODOLOGIA E DISCUSSÃO DOS RESULTADOS Os dados abordados no trabalho foram obtidos a partir de pesquisa de caráter
exploratório que, segundo Gil, tem “como principal finalidade desenvolver, esclarecer
e modificar conceitos e ideias, tendo em vista a formulação de problemas mais
precisos ou hipótese pesquisáveis para estudos posteriores” (GIL, 2008, p. 27). E,
para a obtenção de uma análise mais profunda do assunto a ser estudado foi
utilizado o método quanti-qualitativo, utilizando como técnica de pesquisa a coleta de
dados e, como procedimentos para essa coleta, foram aplicadas 02 entrevistas com
docentes do curso de Letras/LIBRAS e com 02 bibliotecários do LA, bem como,
técnica de observação no atendimento fornecido pela BCZM. Depois de obtidos os dados, o passo seguinte foi a análise e discussão das
questões referentes à pessoa surda e seus avanços legais, bem como a Inclusão de
alunos com Necessidades Educacionais Especiais na UFRN e a necessidade de
uma Gestão de Coleção Acessível ao Estudante com Surdez no SISBI da UFRN. a isso, percebe-se a crescente demanda de pessoas com surdez no ensino superior. Apontou o Ministério da Educação (MEC) que entre 2000 e 2010, a quantidade de
4
matrículas em ensino superior aumentou 933,6%. Por estes motivos, o enfoque
deste artigo será o atendimento da pessoa com surdez em bibliotecas universitárias,
em especial na Biblioteca Central Zila Mamede (BCZM), integrante do Sistema de
Bibliotecas (SISBI) da Universidade Federal do Rio Grande do Norte (UFRN). Além disso, esta pesquisa pretende verificar em que medida a Política de
Acessibilidade do SISBI atende às necessidades informacionais dos estudantes
surdos, identificando também, quais parâmetros são adotados pela BCZM na
acessibilidade à informação para este público, bem como, identificar quais as
barreiras que impedem o acesso destes estudantes ao Laboratório de Acessibilidade
da Biblioteca Central Zila Mamede (LA). O papel das bibliotecas em nossa sociedade é bastante claro: garantir o
acesso
à
informação
para
toda
a
sua
comunidade,
consequentemente,
o
bibliotecário assume a postura de intermediário entre a informação e o usuário e,
para isso, precisa estar habilitado a comunicar-se com ele. Da mesma maneira,
quando o bibliotecário atende um usuário com surdez, deve estar minimamente
habilitado a comunicar-se através da Língua Brasileira de Sinais (LIBRAS), que é a
língua usada pela comunidade surda. Em consequência disso, a pesquisa se justifica também, quanto ao currículo
do curso de graduação em Biblioteconomia da UFRN, que não oferece disciplina
obrigatória que desperte nos futuros profissionais a sensibilização para a prestação
de serviço às pessoas com deficiência. Existe a disciplina LIBRAS como optativa,
com a carga horária de 60h, tendo sido inserida na matriz curricular do curso no
período letivo 2011.1. Sendo que, nesta categoria não é possível adquirir todo
conhecimento
necessário
para
este
atendimento
específico,
tendo
que
ser
complementada com cursos básicos e/ou especializações na área. 85 85 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 5 Documento não paginado. 3
ACESSIBILIDADE UNIVERSAL E ACESSO À INFORMAÇÃO PARA AS
PESSOAS COM DEFICIÊNCIA Quando uma instituição possui o intuito de promover um local onde todas as
pessoas fazem parte, no qual é permitido o acesso de toda e qualquer pessoa, tenha
ela alguma necessidade específica ou não, devemos colocar em prática os conceitos
de Acessibilidade Universal, que segundo GelpI, KaliL e Becker: Pode ser entendida como o direito de ir e vir de todos os cidadãos, inclusive
daquelas pessoas com deficiências permanentes ou ocasionais. Isto incluía BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 86 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. todos, em todos os momentos e períodos de sua vida, quer sejam usuários
de cadeiras de rodas, pessoas com deficiências visuais ou auditivas,
gestantes, obesos, idosos ou ainda, as crianças. Os espaços devem permitir
o trânsito e acesso a todos os espaços da cidade, prédios públicos,
institucionais, usar transporte, equipamentos públicos e mobiliários urbanos,
como telefones, sanitários, rede bancária, cabinas, assentos, bebedouros,
etc. O importante e prioritário, está na abordagem da arquitetura, do
urbanismo e da mobilidade urbana através do desenho universal, acessível,
criando uma cidade ao alcance de todos os cidadãos com mobilidade
reduzida ou não, democratizando todos os espaços, entendendo o desenho
universal como a capacidade de comunicar e integrar a todos. (GELPI;
KALIL; BECKER, 2015, p. 5). todos, em todos os momentos e períodos de sua vida, quer sejam usuários
de cadeiras de rodas, pessoas com deficiências visuais ou auditivas,
gestantes, obesos, idosos ou ainda, as crianças. Os espaços devem permitir
o trânsito e acesso a todos os espaços da cidade, prédios públicos,
institucionais, usar transporte, equipamentos públicos e mobiliários urbanos,
como telefones, sanitários, rede bancária, cabinas, assentos, bebedouros,
etc. O importante e prioritário, está na abordagem da arquitetura, do
urbanismo e da mobilidade urbana através do desenho universal, acessível,
criando uma cidade ao alcance de todos os cidadãos com mobilidade
reduzida ou não, democratizando todos os espaços, entendendo o desenho
universal como a capacidade de comunicar e integrar a todos. (GELPI;
KALIL; BECKER, 2015, p. 5). Com isso, promovesse também, a valorização da dignidade humana, que
segundo Moraes é “Um valor espiritual e moral inerente da pessoa, que se manifesta
singularmente na autodeterminação consciente e responsável da própria vida e que
traz consigo a pretensão ao respeito por parte das demais pessoas” (MORAES,
2005, p. 41). Em 1988, surge a primeira lei que trata sobre o assunto na Constituição
Federal Brasileira, a qual estabelece um título próprio aos Princípios Fundamentais
da Dignidade Humana. p g
7 Documento não paginado. 6 Documento não paginado. Em seu artigo 1º, inciso I ao V, ela nos traz como
fundamentos: A soberania, a cidadania, a dignidade da pessoa humana, os valores sociais
do trabalho e da livre iniciativa e o pluralismo político [...] E, como objetivos
fundamentais, se constitui em “construir uma sociedade livre, justa e
solidária; garantir o desenvolvimento nacional; erradicar a pobreza e a
marginalização e reduzir as desigualdades sociais e regionais; promover o
bem de todos, sem preconceitos de origem, raça, sexo, cor, idade e
quaisquer outras formas de discriminação. (BRASIL, 1988) . 5 A soberania, a cidadania, a dignidade da pessoa humana, os valores sociais
do trabalho e da livre iniciativa e o pluralismo político [...] E, como objetivos
fundamentais, se constitui em “construir uma sociedade livre, justa e
solidária; garantir o desenvolvimento nacional; erradicar a pobreza e a
marginalização e reduzir as desigualdades sociais e regionais; promover o
bem de todos, sem preconceitos de origem, raça, sexo, cor, idade e
quaisquer outras formas de discriminação. (BRASIL, 1988) . 5 Com isso, a lei deixa clara a importância de se respeitar, acima de qualquer
coisa o outro, independente de suas manifestações culturais e sociais, sendo
primordial a não exclusão. Sabe-se que não é uma tarefa fácil, mas este marco legal
está avançando, ajudando na criação de políticas públicas mais ajustadas com os
Direitos Humanos e com a Acessibilidade. Nesta mesma perspectiva e com o intuito de promover a Acessibilidade e de
reduzir
as
desigualdades
sociais
(um
dos
princípios
defendidos
por
nossa BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 87 87 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. [...] possibilidade e condição de alcance para utilização, com segurança e
autonomia, de espaços, mobiliários, equipamentos urbanos, edificações,
transportes,
informação
e
comunicação,
inclusive
seus
sistemas
e
tecnologias, bem como de outros serviços e instalações abertos ao público,
de uso público ou privados de uso coletivo, tanto na zona urbana como na
rural, por pessoa com deficiência ou com mobilidade reduzida. (BRASIL,
2015, grifo nosso) .
6 autoridades nacionais devem encontrar meio para evitar efeitos possíveis de
exclusão nas áreas de acesso à informação e ao conhecimento, de difusão de
Novas Tecnologias de Informação e Comunicação (NTIC) e de desenvolvimento do
plurilinguismo na internet. Serão
abordadas
em
seguida,
peculiaridades
sobre
uma
deficiência
específica, a surdez, onde serão apresentados alguns conceitos de estudiosos da
área, bem como, avanços na legislação acerca desse público. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Artigo 2º - garante, por parte do poder público em geral e empresas concessionárias de serviços
públicos, formas institucionalizadas de apoiar o uso e difusão da Língua Constituição), a gestão da informação é vista como uma ferramenta essencial no
desenvolvimento das organizações, do ponto de vista de Miranda e Streit (2007, p. 4) “As organizações processam e gerem informações para diminuir a ambiguidade e
a incerteza provindas do ambiente em que atuam, e/ou para elaborar produtos e
serviços de informação”. Ou seja, a gestão da informação vem dar suporte ao
trabalho de organizar a informação, de maneira que os serviços e produtos possam
ser úteis aos seus clientes, sem distinção. Desde 1988, após amplos debates nacionais e internacionais sobre o tema, a
legislação brasileira foi sendo aprimorada e complementada com novas leis. Dentre
estes avanços, podemos citar a introdução do conceito de acessibilidade à
informação e à comunicação presente na Lei 13.146/15, de 6 de julho de 2015, Lei
Brasileira de Inclusão da Pessoa com Deficiência (Estatuto da Pessoa com
Deficiência), que conceitua acessibilidade como sendo: [...] possibilidade e condição de alcance para utilização, com segurança e
autonomia, de espaços, mobiliários, equipamentos urbanos, edificações,
transportes,
informação
e
comunicação,
inclusive
seus
sistemas
e
tecnologias, bem como de outros serviços e instalações abertos ao público,
de uso público ou privados de uso coletivo, tanto na zona urbana como na
rural, por pessoa com deficiência ou com mobilidade reduzida. (BRASIL,
2015, grifo nosso) . 6 Vale salientar que em outros artigos específicos, esta lei reafirma um conjunto
de direitos das pessoas com deficiência, mas também define, de forma mais clara,
sanções relativas ao seu não cumprimento. Com isso, esta lei desde a sua
promulgação em 2015, vem influenciando e alavancando a criação de outras
políticas, programas e projetos voltados para a temática do acesso à informação. É importante ressaltar que a informação deve estar acessível a toda e
qualquer pessoa, sem distinção. Podemos evidenciar tal afirmação, de acordo com o
manifesto da Declaração Universal dos Direitos Humanos da Organização das
Nações Unidas para a Educação, a Ciência e a Cultura (UNESCO) (ASSEMBLÉIA
GERAL DAS NAÇÕES UNIDAS, 1998)
onde as instituições internacionais e as
7 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 88 8 Documento não paginado. 3.1 A Pessoa Surda e seus Avanços Legais Para as pessoas surdas, o aspecto cultural é que define sua necessidade
especial, até porque, possuem sua própria língua e forma de se comunicar, a Língua
Brasileira de Sinais (LIBRAS), a qual envolve muito mais do que a simples tradução
da Língua Portuguesa sob forma de sinais, o que para a sociedade é difícil de
entender, uma vez que alguns ainda veem a surdez exclusivamente como um
fenômeno físico, conforme afirma Gesser (2008, p. 230) que “infelizmente, os surdos
têm sido narrados e definidos exclusivamente a partir da realidade física da falta de
audição e, portanto, aos olhos da sociedade majoritária ouvinte tem sido vistos
exclusivamente a partir desse fato”. É importante abordar o marco principal de avanço da comunidade surda, que
de fato oficializou a Língua Brasileira de Sinais como meio legítimo de comunicação
da pessoa com surdez, que foi a criação da Lei nº 10.436, de 24 de abril de 2002, a
qual ficou conhecida na comunidade surda como a “Lei de LIBRAS”. Os artigos 1º e
2º desta Lei, respectivamente, tratam: Artigo 1º - é reconhecido como meio legal de comunicação e expressão a Língua Brasileira de Sinais
- LIBRAS e outros recursos de expressão a ela associados. Artigo 2º - garante, por parte do poder público em geral e empresas concessionárias de serviços
públicos, formas institucionalizadas de apoiar o uso e difusão da Língua BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 89 Brasileira de Sinais - LIBRAS como meio de comunicação objetiva e de
utilização corrente das comunidades surdas do Brasil (BRASIL, 2002) . 8 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. unicamente porque não ouve, mas porque desenvolve potencialidades psicoculturais
diferentes das dos ouvintes” (BEHARES, 2000, p. 2). Desta forma, pode-se dizer que a pessoa com surdez tem sua própria cultura,
possui sua própria identidade, com seus valores, regras, comportamentos e
tradições inerentes a elas. Além disso, por várias questões sociais e políticas,
costumam criar comunidades onde vivem e compartilham juntos anseios e metas,
além de se ajudarem nas suas lutas e na diminuição dos preconceitos que os
cercam. Dentre os avanços das pessoas com deficiência, a seguir, serão relatadas
as políticas de inclusão dos estudantes com necessidades especiais no âmbito da
UFRN. 9 Documento não paginado Brasileira de Sinais - LIBRAS como meio de comunicação objetiva e de
utilização corrente das comunidades surdas do Brasil (BRASIL, 2002) .
8 Portanto, somente a partir desta data foi possível realizar, em âmbito nacional,
discussões relacionadas às necessidades das pessoas surdas, deixando claro para
as pessoas que a LIBRAS é uma língua formada por sinais, com significados
específicos, possuidora de uma composição gramatical própria, podendo expressar
conceitos dentro da realidade em que o surdo está inserido. Strobel, afirma que: Ela [LIBRAS] é uma das principais marcas da identidade de um povo surdo
por ser uma das peculiaridades da cultura surda. É uma forma de
comunicação que capta as experiências visuais dos sujeitos surdos, sendo
que é esta língua que vai levar o surdo a transmitir e proporcionar-lhe a
aquisição de conhecimento universal (STROBEL, 2008, p. 42-43). É notória a importância da LIBRAS na vida das pessoas surdas, sendo usada
como um meio de garantia deste grupo menorizado na socialização e interação na
sociedade. Abordasse o conceito de surdez, uma vez que já foi discorrido sobre a
legislação que configura a LIBRAS como língua reconhecida no Brasil. Sá, ao definir
o termo surdo, diz que: [...] é o termo com o qual as pessoas que não ouvem referem-se a si
mesmos e a seus pares. Podemos definir uma pessoa surda como àquela
que vivencia um déficit de audição que o impede de adquirir, de maneira
natural, a língua oral/auditiva usada pela comunidade majoritária e que
constrói
sua
identidade
calcada
principalmente
nesta
diferença,
utilizando-se de estratégias cognitivas e de manifestações comportamentais
e culturais diferentes da maioria das pessoas que ouvem. (SÁ, 2006, p. 2,
grifo nosso). Para este grupo, o termo deficiente não é bem-aceito e o uso desta
terminologia reforça a segregação e a exclusão que já perdura desde seus
antepassados. Além disso, o termo pode ser associado ao fato de que muitas
pessoas com deficiência sofriam torturas, abandono e discriminação, por parte,
principalmente, de sua própria família. Behares (2000) nos mostra que nos estudos
sobre os surdos, é enfatizado a diferença, e não a deficiência, por que: “Cremos que
é nela que se baseia a essência psicossocial da surdez: ele (o surdo) não é diferente BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 90 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. especializados; V – oferta de capacitação que possa contribuir para o
aperfeiçoamento do processo de ensino-aprendizagem (Ibidem). Este documento formal tem a missão de instituir a Política de Inclusão e
Acessibilidade para as Pessoas com Deficiência na Universidade Federal do Rio
Grande do Norte, para fim de sua aplicação. No entanto, é a Comissão de Apoio a
Estudantes com Necessidades Educacionais Especiais (CAENE), a responsável
pela institucionalização da Política de Inclusão dos alunos com Necessidades
Educacionais Especiais (NEE), e afiança o direito da pessoa com deficiência à
educação superior na UFRN. Esta Comissão atua sob demanda de solicitação
espontânea do aluno com NEE, em seguida, a solicitação é enviada à coordenação
do curso do aluno para identificação e formalização de acompanhamento e triagem
com o intuito de investigar mais sobre sua realidade, bem como suas necessidades
efetivas. A seguir, será discorrido sobre a acessibilidade à informação da BCZM para o
atendimento ao NEE, focando no público surdo. 4 INCLUSÃO DE ALUNOS COM NECESSIDADES EDUCACIONAIS ESPECIAIS
NA UFRN De
acordo
com
a
Resolução
193/2010
da
Política
de
Inclusão
e
Acessibilidade para as Pessoas com Deficiência na UFRN em seu artigo 1º,
entende-se por estudante com necessidade educacional especial aquele com: “I –
deficiência nas áreas: auditiva, visual, física, intelectual ou múltipla; II – transtornos
globais do desenvolvimento; III - Altas habilidades/superdotação; IV – transtornos
específicos” (UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE, 2010) . 9 E, em seu artigo 2º, a mesma resolução determina: Que os dirigentes das unidades acadêmicas deverão prover iniciativas que
contemplem o princípio da inclusão social nas propostas curriculares de
seus cursos presenciais e à distância, garantindo ações voltadas para o
atendimento às demandas dos estudantes com necessidades educacionais
especiais. § 2º A inclusão mencionada no caput deste artigo refere-se às
responsabilidades
concernentes
dos
estudantes
com
necessidades
educacionais
especiais,
como:
I
–
recursos
didático-pedagógicos
adequados; II – acesso às dependências das unidades acadêmicas; III –
pessoal
docente
e
técnico
especializado;
IV
–
serviços
de
apoio BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 91 especializados; V – oferta de capacitação que possa contribuir para o
aperfeiçoamento do processo de ensino-aprendizagem (Ibidem). BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Em
relação
aos
usuários
com
deficiência,
as
suas
necessidades
informacionais em linhas gerais não diferem das necessidades dos demais
usuários, o que diferencia é o suporte físico da informação e o acesso a
esta. Nesse contexto, a problemática está no acesso à informação e tipos
de suporte que obedeçam ao conceito de desenho universal. (MELO, 2015,
p. 33). 4.1 Acessibilidade à informação ao estudante com surdez da ufrn e a gestão de
coleções acessíveis da BCZM De acordo com dados fornecidos pela CAENE, existem, atualmente na UFRN,
395 alunos com deficiência, e desse total, 15 correspondem a alunos com surdez. Para complementar este quantitativo de pessoas com surdez na UFRN, em visita ao
departamento de Letras, foi informado que atualmente existem 05 professores com
surdez no curso de graduação Letras/LIBRAS – Departamento de Letras do Centro
de Ciências Humanas, Letras e Artes (CCHLA) e 01 professor surdo no Curso de
Letras - Departamento de Letras (DLC) do Centro de Ensino Superior do Seridó
(CERES) no Campus de Currais Novos. Diante do quantitativo apresentado e da presença do curso de Letras/ LIBRAS,
que visa a formar professores para atuarem no ensino de Língua Brasileira de Sinais
e de Língua Portuguesa para surdos, é de suma importância que o SISBI e a BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 92 92 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. CAENE
disponibilizem
acervo
informacional
acessível
que
atenda
às
reais
necessidades informacionais dos alunos e professores com surdez da UFRN,
auxiliando-os no desenvolvimento social, científico e acadêmico. Nesse sentido, Miranda enfatiza o papel das bibliotecas no contexto da
formação do ser humano, “[...] uma vez que oferece aos seus usuários o acesso à
informação que irá contribuir para a formação de um cidadão mais consciente de
seus direitos e deveres” (MIRANDA, 2015, p. 23). No entanto, as bibliotecas devem
garantir um ambiente democrático e acessível a todos, contribuindo para o
estabelecimento de produtos e serviços adequados as necessidades informacionais
das pessoas com surdez, que também necessitam de informação para seu
conhecimento. Diante desse contexto das bibliotecas, será enfatizada sua abordagem no
âmbito
das universidades. Leitão nos traz uma definição da biblioteca nas
Universidades, uma vez que, tem o papel de “[...] estimular, apoiar, fomentar e
desenvolver o saber em seus múltiplos aspectos por meio de seus acervos e das
relações que nela se estabelecem” (LEITÃO, 2005, p. 25). Ou seja, a biblioteca além
de garantir material bibliográfico para os estudantes, deve também promover a
Acessibilidade Universal, que visa facilitar a aquisição à informação por todas as
pessoas. Apesar de o termo acessibilidade ser tema de diversas áreas do conhecimento,
nota-se que no âmbito das bibliotecas ainda existem limitações e necessitam de
contribuições mais pontuais. Baseando-se nisso, Melo nos traz uma abordagem
mais específica quanto às necessidades informacionais dos estudantes com surdez,
uma vez que: Em
relação
aos
usuários
com
deficiência,
as
suas
necessidades
informacionais em linhas gerais não diferem das necessidades dos demais
usuários, o que diferencia é o suporte físico da informação e o acesso a
esta. Nesse contexto, a problemática está no acesso à informação e tipos
de suporte que obedeçam ao conceito de desenho universal. (MELO, 2015,
p. 33). Vale salientar que, uma das atividades mais importantes de gestão nas
bibliotecas é a formação e desenvolvimento de coleções, que nada mais é que a BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 93 93 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. seleção do material que irá compor o acervo da mesma. Miranda nos relata a
relevância desta atividade: seleção do material que irá compor o acervo da mesma. Miranda nos relata a
relevância desta atividade: A gestão da coleção é fator fundamental à plena consecução dos objetivos
das bibliotecas. A formação, desenvolvimento e organização do acervo
devem ser encarados como um processo permanente no qual as atividades
de seleção, aquisição e avaliação de materiais devem permanecer em
contínua sintonia com as necessidades de informação da comunidade de
usuários. (MIRANDA, 2007, p. 87). Fica claro que a formação e desenvolvimento de coleções é um processo
fundamental e de grande importância para as bibliotecas, principalmente, devido à
explosão bibliográfica. Tal informação pode ser corroborada com as diretrizes da
Federação Internacional de Associações e Instituições Bibliotecárias (IFLA) (2010, p. 59) onde relata que “uma grande coleção não significa uma boa coleção,
especialmente no novo mundo digital. A relevância da coleção para as necessidades
da comunidade local é mais importante do que o seu tamanho”. É evidente que uma
biblioteca
com
um
grande
acervo
é
fundamental,
mas
deve-se
garantir,
principalmente, que estes materiais sejam utilizados, que serão encontrados por
seus usuários, e vice-versa. Baseando-se nisso, o Manual Orientador de Fortalecimento de Bibliotecas
Inclusivas e Acessíveis (2016) sugere que é imprescindível que se tenha acervos em
diferentes formatos, em especial, nos formatos acessíveis. Este manual ainda define
como seriam estes formatos de acervos: “[...] são aqueles que incluem livros e
outros materiais com recursos de acessibilidade (livros em braille, em tinta e braille,
audiolivros, livros digitais bilíngües Português/LIBRAS etc. Que possibilitam o
acesso ao livro e à leitura para pessoas com deficiência” (BRASIL, 2016, p. 25). O acervo acessível é a adaptação da informação registrada nos diferentes
materiais da biblioteca para os formatos que sejam acessíveis aos usuários com
NEE. Melo (2015) relata que a formação do acervo acessível só ocorre após o
levantamento das necessidades informacionais dos usuários, o qual se pode iniciar o
processo de (re)formulação do acervo para torná-lo acessível aos usuários com
deficiência. Com isso, faz-se necessário que os bibliotecários tenham conhecimento BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 94 94 10 Documento não paginado. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. De acordo com Melo, Costa e Soares, Tecnologias Assistivas são: [...]
recursos e serviços que visam facilitar o desenvolvimento de atividades da vida
diária por pessoas com deficiências. Procuram aumentar capacidades funcionais e
assim promover a autonomia e a independência de quem as utiliza. (MELO; COSTA;
SOARES, 2008, apud ARAÚJO, 2017, p. 4). sobre possíveis formatos que possam ser utilizados para que ocorra a acessibilidade
à informação para os usuários com surdez. Visto isso, pode-se contar também com
a parceria das editoras, que no ato da compra podem disponibilizar o arquivo digital
das obras, onde é possível converter para o formato acessível desenvolvido para
cada deficiência. Inclusive, de acordo com o disposto na Lei n. 13.146/2015, que em
seu Art. 68, afirma que: O poder público deve adotar mecanismos de incentivo à produção, à edição,
à
difusão, à distribuição e à comercialização de livros em formatos
acessíveis,
inclusive
em
publicações
da
administração
pública
ou
financiadas com recursos públicos, com vistas a garantir à pessoa com
deficiência o direito de acesso à leitura, à informação e à comunicação. § 1o Nos editais de compras de livros, inclusive para o abastecimento ou a
atualização de acervos de bibliotecas em todos os níveis e modalidades de
educação e de bibliotecas públicas, o poder público deverá adotar cláusulas
de impedimento à participação de editoras que não ofertem sua produção
também em formatos acessíveis. § 2o Consideram-se formatos acessíveis os arquivos digitais que possam
ser reconhecidos e acessados por softwares leitores de telas ou outras
tecnologias assistivas que vierem a substituí-los, permitindo leitura com voz
sintetizada, ampliação de caracteres, diferentes contrastes e impressão em
Braille. (BRASIL, 2015) . 10 Diante do exposto, a BCZM tem a possibilidade de adequar sua Política de
Formação e Desenvolvimento de Coleções de acordo com o disposto das exigências
legais, para que assim, possa oferecer um atendimento adequado para esta
demanda de usuários existentes. Para que isso ocorra, conta-se também com a CAENE, que segundo Ferreira,
uma das suas missões é “propor ações através da consolidação de redes de apoio e
serviço
institucional
que
incidam
na
eliminação
de
barreiras
arquitetônicas,
atitudinais, pedagógicas e de comunicação na UFRN” (FERREIRA, 2016, p. 73). Em
conformidade a isto, foi criado em 2011, o Laboratório de Acessibilidade (LA)
localizado na BCZM. O LA tem o intuito de promover a inclusão informacional das
pessoas com NEE com limitações e/ou dificuldades na leitura impressa, aliado aos BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 95 95 equipamentos e recursos da Tecnologia Assistiva. Dentre suas atividades estão:
digitalização de textos; produção de materiais em formatos acessíveis; empréstimo
de tecnologias Assistivas; revisão Braille e Repositório de Informação Acessível
(RIA). 96 Ter materiais referentes à surdez e a deficiência auditiva é importante não
apenas para os próprios usuários com essa deficiência, mas para que todos
tenham acesso a informações que transmitam mais conhecimentos sobre
essa temática contribuindo para mudanças no comportamento, nas ações
das pessoas em geral em relação aos surdos e pessoas com deficiência
auditiva (MIRANDA, 2015, p. 127). BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. equipamentos e recursos da Tecnologia Assistiva. Dentre suas atividades estão:
digitalização de textos; produção de materiais em formatos acessíveis; empréstimo
de tecnologias Assistivas; revisão Braille e Repositório de Informação Acessível
(RIA). De acordo com Melo, Costa e Soares, Tecnologias Assistivas são: [...]
recursos e serviços que visam facilitar o desenvolvimento de atividades da vida
diária por pessoas com deficiências. Procuram aumentar capacidades funcionais e
assim promover a autonomia e a independência de quem as utiliza. (MELO; COSTA;
SOARES, 2008, apud ARAÚJO, 2017, p. 4). Como uma das missões da BCZM é tornar a informação acessível ao usuário,
foi criado em 2012, o Repositório de Informações Acessíveis (RIA) que tem o
objetivo de reunir, integrar e disponibilizar os textos adaptados pelo LA. Em consulta
ao site da BCZM, o RIA atualmente só tem acessibilidade ao estudante com
deficiência visual. Pode-se constatar tal informação abaixo: Seu principal objetivo é armazenar, preservar, divulgar e permitir acesso ao
estudante
com
deficiência
visual
dos
cursos
de
Graduação
e
Pós-Graduação ao material de estudo e pesquisa necessários à sua
formação acadêmica. O material disponibilizado no RIA é destinado à
pessoa com deficiência visual e representa uma tentativa de promoção à
igualdade
de
condições
no
acesso
ao
conhecimento
(BIBLIOTECA
CENTRAL ZILA MAMEDE, [201-]). Em resultante, é possível notar a inexistência de suporte informacional na
biblioteca ao usuário surdo da UFRN. Uma vez que vai de encontro ao compromisso
formal da Instituição em gerar acessibilidade à informação para todos os estudantes
com NEE. Pode-se notar também, a não inclusão das pessoas com surdez nos
objetivos do Laboratório de Acessibilidade, que é amparado pelo Decreto 5.296, de
02 de dezembro de 2004, que determina a garantia da acessibilidade e utilização de
serviços e atendimentos das pessoas portadoras de deficiência, ou com mobilidade
reduzida. Quanto ao termo “portador”, recomenda-se a não utilização do termo,
sendo aconselhado utilizar “Pessoa com Deficiência (PCD)”, sendo empregado para
demonstrar a existência de alguma deficiência, sem indicar qual (BRASIL, 2004). Retomando ao suporte informacional ao usuário surdo, concordasse com
Miranda ao dizer que: BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. Ter materiais referentes à surdez e a deficiência auditiva é importante não
apenas para os próprios usuários com essa deficiência, mas para que todos
tenham acesso a informações que transmitam mais conhecimentos sobre
essa temática contribuindo para mudanças no comportamento, nas ações
das pessoas em geral em relação aos surdos e pessoas com deficiência
auditiva (MIRANDA, 2015, p. 127). As bibliotecas e os bibliotecários não podem mais se comportar como se as
pessoas surdas não existissem, como se fossem invisíveis, principalmente, porque
seus usuários estão cada vez mais conscientes de seus direitos a produtos e
serviços com qualidade. Miranda (2015, p. 61-63) relata que a IFLA preocupada com este público
pouco assistido nas bibliotecas, elaborou um documento chamado “Diretrizes para
Serviços de Bibliotecas para Surdos”. Enfatiza ainda, que estas diretrizes foram
idealizadas em 1988, durante um simpósio sobre serviços de bibliotecas para surdos
que aconteceu na Austrália. As diretrizes servem para nortear as bibliotecas nas
adaptações necessárias para que possam oferecer um atendimento adequado para
as pessoas com surdez. Essas recomendações são divididas em 5 categorias:
pessoal, comunicação, acervo, serviços e divulgação dos programas das bibliotecas. Destaca ainda, que é de responsabilidades das bibliotecas “garantir que suas
coleções e serviços sejam acessíveis aos surdos e que os surdos estejam cientes
dos serviços que as bibliotecas podem lhes prover” (IFLA, 2000, p. 6, apud
MIRANDA, 2015, p. 20). Vale ressaltar que a biblioteca e seus profissionais não
precisam esperar essa demanda vir até a biblioteca para que se planejem, para que
se inicie um projeto de acessibilidade, visto que, podem se antecipar iniciando uma
gestão informacional para esse público, criando um fluxo que atenda às suas
especificidades, fazendo uso das recomendações sugeridas pelas diretrizes da IFLA. Atualmente, o SISBI realiza o atendimento por demanda direcionada pela CAENE. À frente, além de expor os resultados das observações realizadas sobre o
atendimento ao estudante com surdez da UFRN na BCZM, serão apresentadas
algumas recomendações como sugestão. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 97 97 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 5 CONSIDERAÇÕES FINAIS 5 CONSIDERAÇÕES FINAIS Diante das investigações que embasaram esta pesquisa e considerando a
eficácia da Política de Acessibilidade do SISBI em atender as necessidades
informacionais dos estudantes surdos, os resultados demonstraram que o Sistema
de Bibliotecas da UFRN: (a) atende, somente parcialmente, as necessidades desse
grupo, e (b) não dispõe de pessoal habilitado em LIBRAS para facilitar a
comunicação com os usuários surdos. Além disso, os resultados também apontaram
a existência de um desejo por parte desses alunos de frequentar as bibliotecas do
SISBI. Esta
disposição,
porém,
só
não
se
realiza
em
razão
de barreiras
comunicacionais e humanas que acabam por desmotivar o público surdo. Como já mencionado, a BCZM possui um Laboratório de Acessibilidade
destinado, a princípio, a atender todas as pessoas com deficiência. No entanto, este
Laboratório se restringe atualmente a atender apenas o público com deficiência
visual. Apesar de o Laboratório estar aberto às demandas das pessoas surdas,
identifica-se também que os bibliotecários que lá atuam não têm habilidade em
LIBRAS, mas que existe o anseio por aprender. Além disso, verificamos que o local não dispõe de tecnologias específicas,
como, por exemplo: o software Vlibras, que possui uma série de ferramentas para
tradução de conteúdos de sites, áudio e textos para a Língua Brasileira de Sinais –
LIBRAS. Outra ferramenta é a WIKILIBRAS, que consiste em um sistema de
correção e inclusão de novos sinais. Outro bastante utilizado é o Hand Talk, esse
aplicativo transforma as imagens e textos em linguagens de sinais. Existe também o
Prodeaf Móvel, onde seu principal objetivo é a comunicação entre as pessoas com
necessidades
especiais
(OLIVEIRA,
2016). Estas
ferramentas
favorecem
a
autonomia das pessoas com deficiência auditiva na busca de informações. A partir destas corroborações, conclui-se que a BCZM não atende aos
anseios da comunidade surda da UFRN. E, como forma de aprimorar os serviços
oferecidos pelo Laboratório de Acessibilidade e o Sistema de Bibliotecas da UFRN
como um todo, foram produzidas algumas recomendações, baseadas no Manual BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 98 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. orientador para fortalecimento de bibliotecas acessíveis e inclusivas, como também,
recomendações da IFLA, e de autores referenciados no trabalho. Acredita-se que
assim, possam ser minimizadas algumas das barreiras informacionais que impedem
o acesso à informação aos usuários com surdez na BCZM. São elas: 1. Realizar coletas de materiais informacionais digitais, como trabalhos de
conclusão de cursos de outras universidades, artigos, publicações em revistas,
nacionais e internacionais, que estejam relacionados especificamente à surdez, com
a finalidade de criar um repositório sobre a temática; 2. Verificar se existe banco de dados com materiais acessíveis às pessoas com
surdez, almejando possíveis parcerias; 3. Explicitar
de
forma
mais
objetiva
e
transparente
a
Política
de
Desenvolvimento de Coleções voltadas para as pessoas com NEE atendidas pelo
LA; 4. Solicitar às editoras, na etapa de compra, arquivo digital compatível para que
seja convertido pelas tecnologias assistivas e adaptado no formato acessível para o
usuário com surdez; 5. Implantar e disponibilizar tecnologia assistiva para o usuário surdo, tais como
tradutores automáticos da Língua Portuguesa para a LIBRAS; 6. Organizar visualmente as informações nas estantes e demais espaços da
biblioteca, utilizando a datilologia ou imagens dos sinais em LIBRAS, de maneira que
a escrita em Língua Portuguesa não seja a única forma de sinalização; 7. Disponibilizar cursos de capacitação em LIBRAS para bibliotecários, técnicos
administrativos
e
demais
profissionais
do
SISBI,
visando
uma
melhoria da
comunicação no atendimento ao usuário surdo da BCZM. 7. Disponibilizar cursos de capacitação em LIBRAS para bibliotecários, técnicos
administrativos
e
demais
profissionais
do
SISBI,
visando
uma
melhoria da
comunicação no atendimento ao usuário surdo da BCZM. 8. Divulgar os serviços oferecidos pela BCZM de forma que atraia os estudantes
e docentes surdos para este ambiente informacional. Todas estas recomendações e estratégias se coadunam com Aranha (2000)
que nos afirma que os profissionais da informação não podem ser meros
organizadores e controladores do acesso às estantes, mas sim, se adequarem às BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 99 99 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. and communication barriers, and ultimately prioritizing the creation of collections
accessible to this audience. Keywords: Acessibility. Deafness. Collection development. Accessible colllection. University library Keywords: Acessibility. Deafness. Collection development. Accessible colllection. Keywords: Acessibility. Deafness. Collection development. Accessible colllection. University library. University library. necessidades informacionais de interesse dos seus clientes, cumprindo seu papel de
intermediador entre o leitor e a informação. O bibliotecário tem que estar atento às
necessidades de seus usuários, buscando se qualificar e ter uma postura em prol da
inclusão das pessoas com deficiência, independente de qual seja, passando da
simples tarefa de organizar a informação que se encontra dentro da biblioteca e se
adaptar à comunidade usuária da instituição que nela está inserida. Deve também
acompanhar os avanços das tecnologias, estando atento sobre novas soluções que
estejam disponíveis que facilite o acesso à informação para as pessoas com surdez. Desta forma, para que as bibliotecas universitárias em geral se tornem
espaços
realmente
profícuos
para
as
pessoas
com
deficiência
auditiva,
é
imprescindível que sejam tomadas medidas que viabilizem seu acesso. Ademais,
para que o aluno surdo possa usufruir igualitariamente de todos os serviços do
SISBI, as adaptações sugeridas neste artigo, dentre outras, promovem a autonomia
para a pessoa com surdez inserida na UFRN e que anseiam por fazer uso efetivo de
suas bibliotecas. Abstract: This study deals with the care of students with deafness in university
libraries, especially in the Central Library Zila Mamede, member of the Library
System of the Federal University of Rio Grande Norte. It also discusses the current
legislation
on
Access
to
Information
and
Universal
Accessibility
in
these
environments, as well as presents the Inclusion and Accessibility Policy for students
with disabilities at the Federal University of Rio Grande Norte. It discusses the
policies and guidelines created and made available for libraries to adapt to the
informational inclusion of deaf users in library environments. It uses as methodology,
exploratory research. As an instrument for collecting information, observation
technique and interviews with teachers of the course of Letters / LIBRAS and
librarians of the Central Library Zila Mamede were applied. As a result, it is explained
how the attendance for this public is performed in the Accessibility Laboratory of the
Central Library Zila Mamede. It suggests recommendations, if adopted, may offer the
deaf student equal access to library services, breaking down existing informational BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 100 REFERÊNCIAS ARANHA, Francisco. E- Service em Bibliotecas: geração de valor para
pesquisadores por meio de cooperação indireta. Revista de Administração de
Empresas, São Paulo, n. 4, v. 49, out/dez. 2000. p. 84-93. Disponível em:
http://www.scielo.br/pdf/rae/v40n4/v40n4a08.pdf. Acesso em: 08 ago. 2019. ARANHA, Francisco. E- Service em Bibliotecas: geração de valor para
pesquisadores por meio de cooperação indireta. Revista de Administração de
Empresas, São Paulo, n. 4, v. 49, out/dez. 2000. p. 84-93. Disponível em:
http://www.scielo.br/pdf/rae/v40n4/v40n4a08.pdf. Acesso em: 08 ago. 2019. ARAÚJO, A. K. DA S. O livro acessível: um auxiliar no acesso à informação para
deficientes visuais. Revista Informação na Sociedade Contemporânea, v. 1, n. 2,
p. 1-25, 5 abr. 2017. Disponível em:
https://periodicos.ufrn.br/informacao/article/view/11353/7996. Acesso em: 08 ago. 2019. ARAÚJO, A. K. DA S. O livro acessível: um auxiliar no acesso à informação para
deficientes visuais. Revista Informação na Sociedade Contemporânea, v. 1, n. 2,
p. 1-25, 5 abr. 2017. Disponível em:
https://periodicos.ufrn.br/informacao/article/view/11353/7996. Acesso em: 08 ago. 2019. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. BRASIL. Ministério da Cultura. Fortalecimento de bibliotecas acessíveis e inclusivas:
manual orientador. São Paulo: Diferenças, 2016. FERREIRA, Érika Luzia Lopes da Silva. A Política de Inclusão de estudantes
cegos na educação superior na UFRN: do acesso a permanência. 2016. Dissertação (Mestrado em Serviço social) - Centro de Ciências Sociais Aplicadas,
Programa de Pós- Graduação em Serviço Social, Universidade Federal do Estado
do Rio Grande do Norte, Natal, 2016. FERREIRA, Érika Luzia Lopes da Silva. A Política de Inclusão de estudantes
cegos na educação superior na UFRN: do acesso a permanência. 2016. Dissertação (Mestrado em Serviço social) - Centro de Ciências Sociais Aplicadas,
Programa de Pós- Graduação em Serviço Social, Universidade Federal do Estado
do Rio Grande do Norte, Natal, 2016. FERREIRA, Érika Luzia Lopes da Silva. A Política de Inclusão de estudante
cegos na educação superior na UFRN: do acesso a permanência. 2016. GELPI, Adriana; KALIL, Rosa Maria Locatelli; BECKER, Alcione. Acessibilidade
universal em instituições de ensino. In: SEMINÁRIO NACIONAL DE
CONSTRUÇÕES SUSTENTÁVEIS, 4.; FÓRUM DESEMPENHO DAS
EDIFICAÇÕES, 1., 2015, Rio Grande do Sul. Anais [...]. 2015. Disponível em:
file:///C:/Users/01176410490/Desktop/Acessibilidade%20Universal%20em%20Institui
ções%20de%20Ensino.pdf. Acesso em: 01 dez. 2019. GELPI, Adriana; KALIL, Rosa Maria Locatelli; BECKER, Alcione. Acessibilidade
universal em instituições de ensino. In: SEMINÁRIO NACIONAL DE
CONSTRUÇÕES SUSTENTÁVEIS, 4.; FÓRUM DESEMPENHO DAS
EDIFICAÇÕES, 1., 2015, Rio Grande do Sul. Anais [...]. 2015. Disponível em:
file:///C:/Users/01176410490/Desktop/Acessibilidade%20Universal%20em%20Institui
ções%20de%20Ensino.pdf. Acesso em: 01 dez. 2019. GESSER, Audrei. Do patológico ao cultural na surdez: para além de um e de outro
ou para uma reflexão crítica dos paradigmas. Trabalhos em lingüística aplicada,
Campinas, v. 47, n. 1, p. 223 – 239, 2008. Disponível em:
http://www.scielo.br/pdf/tla/v47n1/v47n1a12.pdf. Acesso em: 30 nov. 2019. GESSER, Audrei. Do patológico ao cultural na surdez: para além de um e de outro
ou para uma reflexão crítica dos paradigmas. Trabalhos em lingüística aplicada,
Campinas, v. 47, n. 1, p. 223 – 239, 2008. Disponível em:
http://www.scielo.br/pdf/tla/v47n1/v47n1a12.pdf. Acesso em: 30 nov. 2019. GIL, Antonio Carlos. Como elaborar projetos de pesquisa. 6. ed. São Paulo: Atlas,
2008. ASSEMBLÉIA GERAL DAS NAÇÕES UNIDAS. Declaração Universal dos Direitos
Humanos. 1998. Disponível em: ASSEMBLÉIA GERAL DAS NAÇÕES UNIDAS. Declaração Universal dos Direitos
Humanos. 1998. Disponível em: p
https://unesdoc.unesco.org/ark:/48223/pf0000139423. Acesso em: 22 jul. 2019. BEHARES, Luis Ernesto. Novas correntes na educação do surdo: dos enfoques
clínicos aos culturais. Santa Maria: UFSM, 2000. BIBLIOTECA CENTRAL ZILA MAMEDE. Repositório de informação acessível da
UFRN – RIA. [201-]. Disponível em:
http://www.sisbi.ufrn.br/bczm/pagina.php?a=ria#.Xe-I8pNKgdU. Acesso em: 05 ago. 2019. BIBLIOTECA CENTRAL ZILA MAMEDE. Repositório de informação acessível da
UFRN – RIA. [201-]. Disponível em:
http://www.sisbi.ufrn.br/bczm/pagina.php?a=ria#.Xe-I8pNKgdU. Acesso em: 05 ago. UFRN – RIA. [201-]. Disponível em:
http://www.sisbi.ufrn.br/bczm/pagina.php?a=ria#.Xe-I8pNKgdU. Acesso em: 05 ago. 2019. BRASIL. Constituição (1988). Constituição da República Federativa do Brasil. Diário
Oficial da União, Brasília, DF. Disponível em:
http://www.planalto.gov.br/ccivil_03/constituicao/constituicao.htm. Acesso em: 30 jul. 2019. BRASIL. Lei nº 10.436 de 24 de abril de 2002. Dispõe sobre a Língua Brasileira de
Sinais - Libras e dá outras providências. Brasília, DF. 2002. Disponível em:
http://www.planalto.gov.br/ccivil_03/LEIS/2002/L10436.htm. Acesso em: 30 jul. 2019. BRASIL. Lei nº 10.436 de 24 de abril de 2002. Dispõe sobre a Língua Brasileira de
Sinais - Libras e dá outras providências. Brasília, DF. 2002. Disponível em:
http://www planalto gov br/ccivil 03/LEIS/2002/L10436 htm Acesso em: 30 jul 2019 BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 101 BRASIL. Decreto 5.296, de 02 de dezembro de 2004. Regulamenta as Leis
nos 10.048, de 8 de novembro de 2000, que dá prioridade de atendimento às
pessoas que especifica. 2004. Disponível em:
http://www.planalto.gov.br/ccivil_03/_ato2004-2006/2004/decreto/d5296.htm. Acesso
em: 30 jul. 2019. BRASIL. Lei nº 13.146 de 06 de julho de 2015. Institui a Lei Brasileira de Inclusão
da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência). Brasília, DF. Disponível em: BRASIL. Lei nº 13.146 de 06 de julho de 2015. Institui a Lei Brasileira de Inclusão
da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência). Brasília, DF. Disponível em:
http://www.planalto.gov.br/ccivil_03/_ato2015-2018/2015/lei/l13146.htm. Acesso em:
30 jul. 2019. BRASIL. Lei nº 13.146 de 06 de julho de 2015. Institui a Lei Brasileira de Inclusão
da Pessoa com Deficiência (Estatuto da Pessoa com Deficiência). Brasília, DF. Disponível em:
http://www.planalto.gov.br/ccivil_03/_ato2015-2018/2015/lei/l13146.htm. Acesso em:
30 jul. 2019. Disponível em:
http://www.planalto.gov.br/ccivil_03/_ato2015-2018/2015/lei/l13146.htm. Acesso em:
30 jul. 2019. BRASIL. Ministério da Cultura. Fortalecimento de bibliotecas acessíveis e inclusivas:
manual orientador. São Paulo: Diferenças, 2016. BRASIL. Ministério da Cultura. Fortalecimento de bibliotecas acessíveis e inclusivas:
manual orientador. São Paulo: Diferenças, 2016. https://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf. Acesso em: 14 set.
2019. LEITÃO, Bárbara Júlia Menezello. Avaliação qualitativa e quantitativa numa
biblioteca universitária: grupos de foco. Rio de Janeiro: Intertexto; Interciência,
2005. MELO, Érika Simony Fernandes de. Formação de acervos acessíveis em
bibliotecas universitárias: o caso da Biblioteca Central Zila Mamede. BiblioCanto,
Natal, v. 1, n. 1, p. 31-44, 2015. MIRANDA, Ana Claudia Carvalho de. Desenvolvimento de coleções em bibliotecas
universitárias. Revista Digital de Biblioteconomia e Ciência da Informação,
Campinas, v. 4, n. 2, p. 01-19, 2007. Disponível em:
http://www.brapci.inf.br/_repositorio/2010/05/pdf_8d3e5e1ab3_0010629.pdf. Acesso
em: 09 ago. 2019. MIRANDA, Silvânia Vieira de; STREIT, Rosalvo Ermes. O processo de gestão da
informação em organizações públicas. In: ENCONTRO DE ADMINISTRAÇÃO DA
INFORMAÇÃo, 1., 2007, Florianopolis. Anais [...]. Florianopolis, 2007. Disponível
em: file:///C:/Users/01176410490/Documents/BIBLIOCANTO/enadi309.pdf. Acesso
em: 01 dez. 2019. MIRANDA, Sulamita Nicolau de. Acessibilidade ao usuário surdo e com
deficiência auditiva em bibliotecas universitárias: o caso da UNIRIO. 2015. Dissertação (Mestrado Profissional em Biblioteconomia) - Programa de Pós-
Graduação em Biblioteconomia, Universidade Federal do Estado do Rio de Janeiro,
Rio de Janeiro, 2015. Disponível em: http://www.unirio.br/ppgb/arquivo/Dissertacao%20Sulamita%20Miranda.pdf. Acesso
em: 12 jun. 2019. INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. Censo Demográfico.
2010. Disponível em: https://www.ibge.gov.br/estatisticas/sociais/populacao/9662-censo-demografico-2010
.html?edicao=9748&t=destaques. Acesso em: 04 ago. 2019. https://www.ibge.gov.br/estatisticas/sociais/populacao/9662-censo-demografico-2010
.html?edicao=9748&t=destaques. Acesso em: 04 ago. 2019. INTERNATIONAL FEDERATION OF LIBRARY ASSOCIATION (IFLA). UNITED
NATIONS EDUCATIONAL, SCIENTIFIC AND CULTURAL ORGANIZATION
(UNESCO). Diretrizes da IFLA para biblioteca escolar. 2010. Disponível em: BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 102 https://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf. Acesso em: 14 set.
2019 ttps://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf. Acesso em: 14 https://www.ifla.org/files/assets/hq/publications/series/147-pt.pdf. Acesso em: 14 set. 2019 MORAES, Alexandre de. Constituição do Brasil Interpretada. 5. ed. São Paulo:
Atlas, 2005. Disponível em:
//
/
/
/
/
/
O CO S
C O MORAES, Alexandre de. Constituição do Brasil Interpretada. 5. ed. São Paulo:
Atlas, 2005. Disponível em:
https://jornalistaslivres org/wp-ontent/uploads/2017/02/DIREITO CONSTITUCIONAL ,
p
https://jornalistaslivres.org/wp-ontent/uploads/2017/02/DIREITO_CONSTITUCIONAL
-1.pdf. Acesso em: 20 jun. 2019. OLIVEIRA, Nazareth Ana Lúcia de. As Tecnologias e a Educação de Alunos
Surdos. 2016. Disponívem em:
https://monografias.brasilescola.uol.com.br/imprimir/15432. Acesso em: 01 dez. 2019. OLIVEIRA, Nazareth Ana Lúcia de. As Tecnologias e a Educação de Alunos
Surdos. 2016. Disponívem em:
https://monografias brasilescola uol com br/imprimir/15432 Acesso em: 01 dez Surdos. 2016. Disponívem em:
https://monografias.brasilescola.uol.com.br/imprimir/15432. Acesso em: 01 dez. 2019. SÁ, Nídia Limeira de. Cultura, poder e educação de surdos. São Paulo: Paulinas,
2006. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. SÁ, Nídia Limeira de. Cultura, poder e educação de surdos. São Paulo: Paulinas,
2006. STROBEL, K. As imagens do outro sobre a cultura surda. Florianópolis: Editora
da UFSC, 2008. BiblioCanto, Natal, v. 5, n.1, p. 83 – 104, 2019. 103 UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. Resolução nº
193/2010, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a
estudantes com necessidades educacionais especiais na Universidade Federal do
Rio Grande do Norte. 2010. Disponível em:
file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10
ago. 2019. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. Resolução nº
193/2010, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a
estudantes com necessidades educacionais especiais na Universidade Federal do
Rio Grande do Norte. 2010. Disponível em:
file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10
ago. 2019. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. Resolução nº
193/2010, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a
estudantes com necessidades educacionais especiais na Universidade Federal do
Rio Grande do Norte. 2010. Disponível em:
file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10
ago. 2019. UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE. Resolução nº
193/2010, de 21 de setembro de 2010. Dispõe sobre o atendimento educacional a
estudantes com necessidades educacionais especiais na Universidade Federal do
Rio Grande do Norte. 2010. Disponível em:
file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10
ago. 2019. p
file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10
ago. 2019. p
file:///C:/Users/Michele/Downloads/RESOLUO_N_193%20(2).pdf. Acesso em: 10
ago. 2019. 104
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|
English
| null |
First Observation of Top Quark Production in the Forward Region
|
Physical review letters
| 2,015
|
cc-by
| 8,525
|
DOI: 10.1103/PhysRevLett.115.112001 to integrated luminosities of 1.0 and 2.0 fb−1 collected at
center-of-mass energies of
ffiffiffis
p ¼ 7 and 8 TeV in pp
collisions with the LHCb detector. The W bosons are
reconstructed using the W →μν decay with muons having
a transverse momentum, pT, larger than 25 GeV (c ¼ 1
throughout this Letter) in the pseudorapidity range,
2.0 < η < 4.5. The analysis is performed using jets clus-
tered with the anti-kT algorithm [5] using a distance
parameter
R ¼ 0.5. The
jets
are
required
to
have
50 < pT < 100 GeV and 2.2 < η < 4.2. The muon and
jet (j) must be separated by ΔRðμ; jÞ > 0.5, with ΔR ≡
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Δη2 þ Δϕ2
p
. Here ΔηðΔϕÞ is the difference in pseudor-
apidity (azimuthal angle) between the muon and jet
momenta. The transverse component of the sum of
the muon and jet momenta must satisfy pTðμ þ jÞ ≡
½~pðμÞ þ ~pðjÞT > 20 GeV. The production of top quarks (t) from proton-proton
(pp) collisions in the forward region is of considerable
experimental and theoretical interest. In the standard model
(SM), four processes make significant contributions to top
quark production: t¯t pair production, single-top production
via processes mediated by a W boson in the t channel
(qb →q0t) or in the s channel (q¯q0 →t¯b), and single top
produced in association with a W boson (gb →tW). The
initial-state b quarks arise from gluon splitting to b¯b pairs
or from the intrinsic b quark content in the proton. Top
quarks decay almost entirely via t →Wb. The SM predicts
that about 75% of t →Wb decays in the forward region are
due to t¯t pair production. The remaining 25% are mostly
due to t-channel single-top production, with s-channel and
associated single-top production making percent-level
contributions. The enhancement at forward rapidities of t¯t production
via q¯q and qg scattering, relative to gg fusion, can result in
larger charge asymmetries, which may be sensitive to
physics beyond the SM [1,2]. Forward t¯t events can be
used to constrain the gluon parton distribution function
(PDF) at a large momentum fraction, resulting in reduced
theoretical uncertainty for many SM predictions [3]. Furthermore, both single-top and t¯t cross-section measure-
ments in the forward region will provide important exper-
imental tests of differential next-to-next-to-leading order
theoretical calculations as they become available [4]. *Full author list given at the end of the article.
Published by the American Physical Society under the terms of
the Creative Commons Attribution 3.0 License. Further distri-
bution of this work must maintain attribution to the author(s) and
the published article’s title, journal citation, and DOI. First Observation of Top Quark Production in the Forward Region R. Aaij et al.*
(LHCb Collaboration) j
(LHCb Collaboration) (Received 3 June 2015; revised manuscript received 8 July 2015; published 8 September 2 Top quark production in the forward region in proton-proton collisions is observed for the first time. The
W þ b final state with W →μν is reconstructed using muons with a transverse momentum, pT, larger than
25 GeV in the pseudorapidity range 2.0 < η < 4.5. The b jets are required to have 50 < pT < 100 GeV
and 2.2 < η < 4.2, while the transverse component of the sum of the muon and b-jet momenta must satisfy
pT > 20 GeV. The results are based on data corresponding to integrated luminosities of 1.0 and 2.0 fb−1
collected at center-of-mass energies of 7 and 8 TeV by LHCb. The inclusive top quark production
cross sections in the fiducial region are σðtopÞ½7 TeV ¼ 239 53ðstatÞ 33ðsystÞ 24ðtheoryÞ fb;
σðtopÞ½8 TeV ¼ 289 43ðstatÞ 40ðsystÞ 29ðtheoryÞ fb:These results, along with the observed differ-
ential yields and charge asymmetries, are in agreement with next-to-leading order standard model
predictions. PACS numbers: 14.65.Ha, 13.87.-a, 14.70.Fm DOI: 10.1103/PhysRevLett.115.112001 Published by the American Physical Society under the terms of
the Creative Commons Attribution 3.0 License. Further distri-
bution of this work must maintain attribution to the author(s) and
the published article’s title, journal citation, and DOI. *Full author list given at the end of the article. week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) DOI: 10.1103/PhysRevLett.115.112001 The
signature for W þ jet events is an isolated high-pT muon
and a well-separated jet originating from the same pp
interaction. Signal events are selected by requiring a
high-pT
muon candidate and at least one jet with
ΔRðμ; jÞ > 0.5. For each event, the highest-pT muon
candidate that satisfies the trigger requirements is selected,
along with the highest-pT jet from the same pp collision. The primary background to top quark production is direct
W þ b production; however, Z þ b events, with one muon
undetected in the decay Z →μμ, and di-b-jet events also
contribute to the μ þ b-jet final state. Inclusive W þ jet production, i.e., where no SV-tag
requirement is made on the jet, is only contaminated at
the percent level by processes other than direct W þ jet
production. Therefore, W þ jet production is used to
validate both the theory predictions and the modeling of
the detector response. Furthermore, the SM prediction for
σðWbÞ=σðWjÞ has a smaller relative uncertainty than
σðWbÞ alone, since the theory uncertainties partially cancel
in the ratio. The analysis strategy is to first measure the
W þ jet yields, and then to obtain predictions for the yields
of direct W þ b production using the prediction for
σðWbÞ=σðWjÞ. To an excellent approximation, many
experimental effects, e.g., the muon reconstruction effi-
ciency, are expected to be the same for both samples and do
not need to be considered in the direct W þ b yield
prediction. The anti-kT clustering algorithm is used as implemented
in FastJet [20]. Information from all the detector subsystems
is used to create charged and neutral particle inputs to the
jet-clustering algorithm using a particle flow approach [21]. The reconstructed jets must fall within the pseudorapidity
range 2.2 < ηðjÞ < 4.2. The reduced ηðjÞ acceptance
ensures nearly uniform jet-reconstruction and heavy-flavor
tagging efficiencies. The momentum of a reconstructed jet
is corrected to obtain an unbiased estimate of the true jet
momentum. The correction factor, typically between 0.9
and 1.1, is determined from simulation and depends on the
jet pT and η, the fraction of the jet pT measured with the
tracking system, and the number of pp interactions in
the event. The W þ jet yield is determined by performing a fit to
the pTðμÞ=pTðjμÞ distribution with templates, histograms
obtained from data, as described in Ref. [19]. DOI: 10.1103/PhysRevLett.115.112001 However,
when
the
dimuon
invariant
mass
is
in
the
range
60 < Mðμþμ−Þ < 120 GeV, such events are selected as
ZðμμÞ þ jet candidates, which are used to determine the
Z þ jet background. The jets are identified (tagged) as originating from the
hadronization of a b or c quark by the presence of a
secondary vertex (SV) with ΔR < 0.5 between the jet axis
and the SV direction of flight, defined by the vector from
the pp interaction point to the SV position. Two boosted
decision trees (BDTs) [22,23], trained on the characteristics
of the SV and the jet, are used to separate heavy-flavor jets
from light-parton jets, and to separate b jets from c jets. The
two-dimensional
distribution
of
the
BDT
responses
observed in data is fitted to obtain the SV-tagged b, c,
and light-parton jet yields. The SV-tagger algorithm is
described in Ref. [24], where the heavy-flavor tagging
efficiencies and light-parton mistag probabilities are mea-
sured in data. The data samples used in Ref. [24] are too
small to validate the performance of the SV-tagger algo-
rithm in the pTðjÞ > 100 GeV region. Furthermore, the
mistag probability of light-parton jets increases with jet pT. Therefore, only jets with pT < 100 GeV are considered in
the fiducial region, which, according to simulation, retains
about 80% of all top quark events. FIG. 1 (color online). Distribution of pTðμÞ=pTðjμÞ with fit
overlaid for all W þ jet candidates. next-to-leading order (NLO) with the MCFM package [15]
and the CT10 PDF set [16], and are cross-checked using
PowhegBox [17] with hadronization simulated by Pythia. next-to-leading order (NLO) with the MCFM package [15]
and the CT10 PDF set [16], and are cross-checked using
PowhegBox [17] with hadronization simulated by Pythia. The theoretical uncertainty on the cross-section predictions
is a combination of PDF, scale, and strong-coupling (αs)
uncertainties. The PDF and scale uncertainties are evalu-
ated following Refs. [16] and [18], respectively. The αs
uncertainty is evaluated as the envelope obtained using
αsðMZÞ ∈½0.117; 0.118; 0.119 in the theory calculations. sð
ZÞ
½
;
;
y
The event selection is the same as that in Ref. [19] but a
reduced fiducial region is used to enhance the top quark
contribution relative to direct W þ b production. DOI: 10.1103/PhysRevLett.115.112001 The LHCb detector is a single-arm forward spectrometer
covering the pseudorapidity range 2 < η < 5, designed for
the study of particles containing b or c quarks. It is
described in detail in Refs. [6,7]. The trigger [8] consists
of a hardware stage, based on information from the
calorimeter and muon systems, followed by a software
stage, which applies a full event reconstruction. This
analysis requires at least one muon candidate that satisfies
the trigger requirement of pT > 10 GeV. Global event cuts
(GECs), which prevent high-occupancy events from domi-
nating the processing time of the software trigger, have an
efficiency of about 90% for W þ jet and top quark events. This Letter reports the first observation of top quark
production in the forward region. The data used correspond Simulated pp collisions are generated using Pythia [9]
with an LHCb configuration [10]. Decays of hadronic
particles are described by EvtGen [11] in which final-state
radiation is generated using Photos [12]. The interaction of
the generated particles with the detector, and its response,
are implemented using the Geant4 toolkit [13] as described
in Ref. [14]. Further theory calculations are performed at 112001-1 © 2015 CERN, for the LHCb Collaboration 0031-9007=15=115(11)=112001(10) week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) )
μ
j(
T
p
)/
μ
(
T
p
0.5
0.6
0.7
0.8
0.9
1
Candidates / 0.05
5000
10000
Data
W
Z
Jets
LHCb
+jet
μ )
μ
j(
T
p
)/
μ
(
T
p
0.5
0.6
0.7
0.8
0.9
1
Candidates / 0.05
5000
10000
Data
W
Z
Jets
LHCb
+jet
μ
FIG. 1 (color online). Distribution of pTðμÞ=pTðjμÞ with fit
overlaid for all W þ jet candidates. to as the muon jet and denoted as jμ, is used to discriminate
between W þ jet and dijet events [19]. No correction is
applied to the momentum of the muon jet. The requirement
pTðjμ þ jÞ > 20 GeV is made to suppress dijet back-
grounds, which are well balanced in pT, unlike W þ jet
events, where there is undetected energy from the neutrino. Events with a second, oppositely charged, high-pT muon
candidate from the same pp collision are vetoed. DOI: 10.1103/PhysRevLett.115.112001 The muon trigger, reconstruction, and selection
efficiencies are determined using Z →μμ events [21,25]. The GEC efficiency is obtained following Ref. [21]: an
alternative dimuon trigger requirement, which requires a
looser GEC, is used to determine the fraction of events that
are rejected. Contamination from W →τ →μ decays are
estimated to be 2.5% using both simulated W þ jet events
and inclusive W data samples [26]. The fraction of muons
that migrate out of the fiducial region due to final-state
radiation is about 1.5% [26]. Figure 4 shows a fit to the pTðμÞ=pTðjμÞ distribution
built from the b-tagged jets from the full data sample. For
pTðμÞ=pTðjμÞ > 0.9 the data are dominantly from W
decays. Figure 5 shows the yield and charge asymmetry
distributions obtained as a function of pTðμ þ bÞ. The
direct
W þ b
prediction
is
determined
by
scaling
the inclusive W þ jet distribution observed in data by
the SM prediction for σðWbÞ=σðWjÞ and by the b-tagging
efficiency measured in data [24]. As can be seen, the data
cannot be described by the expected direct W þ b con-
tribution alone. The observed yield is about 3 times larger
than the SM prediction without a top quark contribution, Migration of events in jet pT due to the detector response
is studied with a data sample enriched in b jets using SV
tagging. The pTðSVÞ=pTðjÞ distribution observed in data is
compared to templates obtained from simulation in bins of
jet pT. The resolution and scale for each jet pT bin are
varied in simulation to find the best description of the data
and to construct a detector response matrix. Figure 2 shows
that the SM predictions, obtained with all detector response
effects applied, agree with the inclusive W þ jet data. ) [GeV]
c
+
μ
(
T
p
)
c
+
W
(
N
0
50
100
150
200
Data
SM
LHCb
20
45
70
95
∞
FIG. 3 (color online). Results for W þ c compared to SM
predictions at NLO obtained using MCFM. ) [GeV]
c
+
μ
(
T
p
)
c
+
W
(
N
0
50
100
150
200
Data
SM
LHCb
20
45
70
95
∞ The yields of W þ c and W þ b, which includes t →Wb
decays, are determined using the subset of candidates with
a SV-tagged jet and binned according to pTðμÞ=pTðjμÞ. DOI: 10.1103/PhysRevLett.115.112001 The Z þ jet
contribution is fixed from the fully reconstructed ZðμμÞ þ
jet yield, where the probability for one of the muons to
escape detection is obtained using simulation. The con-
tributions of b, c, and light-parton jets are each free to vary The high-pT muon candidate is not removed from the
anti-kT inputs and so is clustered into a jet. This jet, referred 112001-2 P H Y S I C A L
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L E T T E R S week ending
11 SEPTEMBER 2015 PRL 115, 112001 (2015) ) [GeV]
j
+
μ
(
T
p
+jet)
W
(
N
0
2000
4000
6000
8000
LHCb
Data
SM
20
45
70
95
∞
) [GeV]
j
+
μ
(
T
p
Charge Asymmetry
-0.4
-0.2
0
0.2
0.4
20
45
70
95
∞
LHCb
Data
SM
FIG. 2 (color online). Results for the inclusive W þ jet yield (left) and charge asymmetry (right) versus pTðμ þ jÞ compared to SM
predictions at NLO obtained using MCFM. The data error bars are smaller than the marker size; the SM uncertainties are highly
correlated across pTðμ þ jÞ bins. Charge Asymmetry FIG. 2 (color online). Results for the inclusive W þ jet yield (left) and charge asymmetry (right) versus pTðμ þ jÞ compared to SM
predictions at NLO obtained using MCFM. The data error bars are smaller than the marker size; the SM uncertainties are highly
correlated across pTðμ þ jÞ bins. A fit to the pTðμÞ=pTðjμÞ distribution built from the c-
tagged jets from the full data sample is provided as
Supplemental Material to this Letter [27]. Figure 3 shows
that the W þ c yield versus pTðμ þ cÞ agrees with the SM
prediction. Since the W þ c final state does not have any
significant contributions from diboson or top quark pro-
duction in the SM, this comparison validates the analysis
procedures. in the fit. Figure 1 shows the fit for all candidates in the
data sample. Such a fit is performed for each muon
charge separately in bins of pTðμ þ jÞ; the differential
W þ jet
yield
and
charge
asymmetry,
defined
as
½σðWþjÞ −σðW−jÞ=½σðWþjÞ þ σðW−jÞ, are given in
Fig. 2. To compare the data to theory predictions, the detector
response must be taken into account. All significant aspects
of the detector response are determined using data-driven
techniques. DOI: 10.1103/PhysRevLett.115.112001 4 (color online). Distribution of pTðμÞ=pTðjμÞ with fit
overlaid for all W þ b candidates. while the SM prediction including both t¯t and single-top
production does describe the data well. In Ref. [19], W þ b is studied in a larger fiducial region
[pTðμÞ > 20 GeV; pTðjÞ > 20 GeV], where the top quark
contribution is expected to be about half as large as that of
direct Wþb production. The ratio ½σðWbÞþσðtopÞ=σðWjÞ
is measured in the larger fiducial region to be 1.17
0.13 ðstatÞ 0.18 ðsystÞ% at
ffiffiffis
p ¼ 7 TeV and 1.29
0.08 ðstatÞ 0.19 ðsystÞ% at
ffiffiffis
p ¼ 8 TeV. These results
agree with SM predictions, which include top quark
production, of 1.23 0.24% and 1.38 0.26%, respec-
tively. This validates the direct W þ b prediction, since
direct W þ b production is the dominant contribution to the
larger fiducial region. To determine the statistical significance of the top quark
contribution, a binned profile likelihood test is performed. The top quark distribution and charge asymmetry versus
pTðμ þ bÞ are obtained from the SM predictions. The total
top quark yield is allowed to vary freely. Systematic
uncertainties,
both
theoretical
and
experimental,
are
handled as Gaussian constraints. The profile likelihood
technique is used to compare the SM hypotheses with and
without a top quark contribution. The significance obtained
using Wilks theorem [28] is 5.4σ, confirming the obser-
vation of top quark production in the forward region. Various sources of systematic uncertainties are consid-
ered and summarized in Table I. The direct W þ b
prediction is normalized using the observed inclusive
W þ jet data yields. Therefore, most experimental system-
atic uncertainties cancel to a good approximation. The yield and charge asymmetry distributions versus
pTðμ þ bÞ observed at
ffiffiffis
p ¼ 7 and 8 TeV are each
consistent with the SM predictions. The excess of the
observed yield relative to the direct W þ b prediction at
each
ffiffiffis
p is attributed to top quark production, and used to Since the muon kinematic distributions in W þ jet and
W þ b are similar, all muon-based uncertainties are neg-
ligible with the exception of the trigger GEC efficiency. DOI: 10.1103/PhysRevLett.115.112001 In
each pTðμÞ=pTðjμÞ bin, the two-dimensional SV-tagger
BDT-response distributions are fitted to determine the
yields of c-tagged and b-tagged jets, which are used to
form the pTðμÞ=pTðjμÞ distributions for candidates with
c-tagged and b-tagged jets. These pTðμÞ=pTðjμÞ distribu-
tions are fitted to determine the SV-tagged W þ c and
W þ b yields. FIG. 3 (color online). Results for W þ c compared to SM
predictions at NLO obtained using MCFM. 112001-3 week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) )
μ
j(
T
p
)/
μ
(
T
p
0.5
0.6
0.7
0.8
0.9
1
Candidates / 0.1
100
200
300
Data
W
Z
Jets
LHCb
-tag
+b
μ )
μ
j(
T
p
)/
μ
(
T
p
0.5
0.6
0.7
0.8
0.9
1
Candidates / 0.1
100
200
300
Data
W
Z
Jets
LHCb
-tag
+b
μ
FIG. 4 (color online). Distribution of pTðμÞ=pTðjμÞ with fit
overlaid for all W þ b candidates. The data-driven GEC study discussed above shows that the
efficiencies are consistent for W þ jet and W þ b, with the
statistical precision of this study assigned as the systematic
uncertainty. Mismodeling of the pTðμÞ=pTðjμÞ distribu-
tions largely cancels, since this shifts the inclusive W þ jet
and W þ b final-state yields by the same amount, leaving
the observed excess over the expected direct W þ b yield
unaffected. The one exception is possible mismodeling of
the dijet templates, since the flavor content of the dijet
background is not the same in the two samples. Variations
of these templates are considered and a relative uncertainty
of 5% is assigned on the W boson yields. The jet-reconstruction efficiencies for heavy-flavor and
light-parton jets in simulation are found to be consistent
within 2%, which is assigned as the systematic uncertainty
for flavor dependencies in the jet-reconstruction efficiency. The SV-tagger BDT templates used in this analysis are two-
dimensional histograms obtained from the data samples
enriched in b and c jets used in Ref. [24]. Following
Refs. [19,24], a 5% uncertainty on the b-tagged yields is
assigned due to uncertainty in these templates. The pre-
cision of the b-tagging efficiency measurement (10%) in
data [24] is assigned as an additional uncertainty. FIG. DOI: 10.1103/PhysRevLett.115.112001 ) [GeV]
b
+
μ
(
T
p
)
W+b
(
N
0
100
200
Data
+top
Wb
Wb
LHCb
20
45
70
95
∞
) [GeV]
b
+
μ
(
T
p
Charge Asymmetry
-0.4
-0.2
0
0.2
0.4
Data
+top
Wb
Wb
LHCb
20
45
70
95
∞
FIG. 5 (color online). Results for the W þ b yield (left) and charge asymmetry (right) versus pTðμ þ bÞ compared to SM predictions
obtained at NLO using MCFM. ) [GeV]
b
+
μ
(
T
p
)
W+b
(
N
0
100
200
Data
+top
Wb
Wb
LHCb
20
45
70
95
∞ ) [GeV]
b
+
μ
(
T
p
Charge Asymmetry
-0.4
-0.2
0
0.2
0.4
Data
+top
Wb
Wb
LHCb
20
45
70
95
∞ Charge Asymmetry FIG. 5 (color online). Results for the W þ b yield (left) and charge asymmetry (right) versus pTðμ þ bÞ compared to SM predictions
obtained at NLO using MCFM. 112001-4 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) TABLE I. Relative systematic uncertainties. Source
Uncertainty
GEC
2%
pTðμÞ=pTðjμÞ templates
5%
Jet reconstruction
2%
SV-tag BDT templates
5%
b-tag efficiency
10%
Trigger and μ selection
2%a
Jet energy
5%a
W →τ →μ
1%a
Luminosity
1%–2%a
Total
14%
Theory
10%
aAn uncertainty that only applies to the cross-section measurement
and not the significance determination. Only the luminosity
uncertainty depends on
ffiffiffis
p : 2% at 7 TeV and 1% at 8 TeV. TABLE I. Relative systematic uncertainties. Source
Uncertainty
GEC
2%
pTðμÞ=pTðjμÞ templates
5%
Jet reconstruction
2%
SV-tag BDT templates
5%
b-tag efficiency
10%
Trigger and μ selection
2%a
Jet energy
5%a
W →τ →μ
1%a
Luminosity
1%–2%a
Total
14%
Theory
10%
aAn uncertainty that only applies to the cross-section measurement
and not the significance determination. Only the luminosity
uncertainty depends on
ffiffiffis
p : 2% at 7 TeV and 1% at 8 TeV. (France); BMBF, DFG, HGF, and MPG (Germany);
INFN
(Italy);
FOM
and
NWO
(The
Netherlands);
MNiSW and
NCN
(Poland);
MEN/IFA
(Romania);
MinES and FANO (Russia); MinECo (Spain); SNSF
and
SER
(Switzerland);
NASU
(Ukraine);
STFC
(United Kingdom); and NSF (U.S.). The Tier1 computing
centers are supported by IN2P3 (France), KIT and
BMBF (Germany), INFN (Italy), NWO and SURF (The
Netherlands), PIC (Spain), and GridPP (United Kingdom). DOI: 10.1103/PhysRevLett.115.112001 We are indebted to the communities behind the multiple
open source software packages on which we depend. We
are also thankful for the computing resources and the
access to software research and development tools provided
by Yandex LLC (Russia). Individual groups or members
have received support from EPLANET, Marie Skłodowska-
Curie Actions and ERC (European Union), Conseil général
de Haute-Savoie, Labex ENIGMASS and OCEVU, Région
Auvergne
(France),
RFBR
(Russia),
XuntaGal
and
GENCAT (Spain), and the Royal Society and Royal
Commission for the Exhibition of 1851 (United Kingdom). aAn uncertainty that only applies to the cross-section measurement
and not the significance determination. Only the luminosity
uncertainty depends on
ffiffiffis
p : 2% at 7 TeV and 1% at 8 TeV. measure the cross sections. Some additional systematic
uncertainties that apply to the cross-section measurements
do not factor into the significance determination. The
uncertainties due to the muon trigger, reconstruction, and
selection efficiencies are taken from the data-driven studies
of Refs. [21,25]. The uncertainty due to the jet energy
determination is obtained from the data-driven study used
to obtain the detector response matrix. The uncertainty due
to W →τ →μ contamination is taken as the difference
between the contamination in simulation versus that of a
data-driven study of inclusive W →μν production [26]. The luminosity uncertainty is described in detail in
Ref. [29]. The total systematic uncertainty is obtained by
adding the individual contributions in quadrature. [1] A. L. Kagan, J. F. Kamenik, G. Perez, and S. Stone, Probing
New Top Physics at the LHCb Experiment, Phys. Rev. Lett. 107, 082003 (2011). [2] R. Gauld, Leptonic Top quark asymmetry predictions at
LHCb, Phys. Rev. D 91, 054029 (2015). [3] R. Gauld, Feasibility of top quark measurements at LHCb
and constraints on the large-x gluon PDF, J. High Energy
Phys. 02 (2014) 126. [4] M. Czakon, P. Fiedler, and A. Mitov, Resolving the Tevatron
Top Quark Forward-Backward Asymmetry Puzzle: Fully
Differential
Next-to-Next-to-Leading-Order
Calculation,
Phys. Rev. Lett. 115, 052001 (2015). The resulting inclusive top production cross sections in
the
fiducial
region
defined
by
pTðμÞ > 25 GeV,
2.0 < ηðμÞ < 4.5, 50 < pTðbÞ < 100 GeV, 2.2 < ηðbÞ <
4.2, ΔRðμ; bÞ > 0.5, and pTðμ þ bÞ > 20 GeV, are [5] M. Cacciari, G. P. Salam, and G. Soyez, The anti-kt jet
clustering algorithm, J. High Energy Phys. 04 (2008)
063. DOI: 10.1103/PhysRevLett.115.112001 σðtopÞ½7 TeV ¼ 23953ðstatÞ33ðsystÞ24ðtheoryÞ fb;
σðtopÞ½8 TeV ¼ 28943ðstatÞ40ðsystÞ29ðtheoryÞ fb: [6] A. A. Alves Jr. et al. (LHCb Collaboration), The LHCb
detector at the LHC, JINST 3, S08005 (2008). [7] R. Aaij et al. (LHCb Collaboration), LHCb detector
performance, Int. J. Mod. Phys. A 30, 1530022 (2015). The systematic uncertainties are nearly 100% correlated
between the two measurements. [8] R. Aaij et al., The LHCb trigger and its performance in
2011, JINST 8, P04022 (2013). In summary, top quark production is observed for the
first time in the forward region. The cross-section results
are
in
agreement
with
the
SM
predictions
of
180þ51
−41ð312þ83
−68Þ fb at 7(8) TeV obtained at NLO using
MCFM. The differential distributions of the yield and
charge asymmetry are also consistent with SM predictions. [9] T. Sjöstrand, S. Mrenna, and P. Skands, PYTHIA 6.4
physics and manual, J. High Energy Phys. 05 (2006)
026; , A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178, 852 (2008). [10] I. Belyaev et al., Handling of the generation of primary
events in Gauss, the LHCb simulation framework, J. Phys. Conf. Ser. 331, 032047 (2011). We express our gratitude to our colleagues in the CERN
accelerator departments for the excellent performance of
the LHC. We thank the technical and administrative staff at
the LHCb institutes. We acknowledge support from CERN
and from the national agencies: CAPES, CNPq, FAPERJ,
and
FINEP
(Brazil);
NSFC
(China);
CNRS/IN2P3 [11] D. J. Lange, The EvtGen particle decay simulation package,
Nucl. Instrum. Methods Phys. Res., Sect. A 462, 152
(2001). [12] P. Golonka and Z. Was, PHOTOS Monte Carlo: A precision
tool for QED corrections in Z and W decays, Eur. Phys. J. C
45, 97 (2006). 112001-5 week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) [21] R. Aaij et al. (LHCb Collaboration), Study of forward
Z þ jet production in pp collisions at
ffiffiffis
p ¼ 7 TeV, J. High
Energy Phys. 01 (2014) 033. [13] J. Allison et al. (Geant4 Collaboration), Geant4 develop-
ments and applications, IEEE Trans. Nucl. Sci. 53, 270
(2006); S. Agostinelli et al. (Geant4 Collaboration), Geant4:
A simulation toolkit, Nucl. Instrum. Methods Phys. Res.,
Sect. A 506, 250 (2003). [22] L. Breiman, J. H. Friedman, R. A. Olshen, and C. J. R. Aaij,38 B. Adeva,37 M. Adinolfi,46 A. Affolder,52 Z. Ajaltouni,5 S. Akar,6 J. Albrecht,9 F. Alessio,38 M. Alexander,51
S. Ali,41 G. Alkhazov,30 P. Alvarez Cartelle,53 A. A. Alves Jr.,57 S. Amato,2 S. Amerio,22 Y. Amhis,7 L. An,3 L. Anderlini,17,a
J. Anderson,40 M. Andreotti,16,b J. E. Andrews,58 R. B. Appleby,54 O. Aquines Gutierrez,10 F. Archilli,38 P. d’Argent,11
A. Artamonov,35 M. Artuso,59 E. Aslanides,6 G. Auriemma,25,c M. Baalouch,5 S. Bachmann,11 J. J. Back,48 A. Badalov,36
C. Baesso,60 W. Baldini,16,38 R. J. Barlow,54 C. Barschel,38 S. Barsuk,7 W. Barter,38 V. Batozskaya,28 V. Battista,39 A. Bay,39
L. Beaucourt,4 J. Beddow,51 F. Bedeschi,23 I. Bediaga,1 L. J. Bel,41 I. Belyaev,31 E. Ben-Haim,8 G. Bencivenni,18
S. Benson,38 J. Benton,46 A. Berezhnoy,32 R. Bernet,40 A. Bertolin,22 M.-O. Bettler,38 M. van Beuzekom,41 A. Bien,11
S. Bifani,45 T. Bird,54 A. Birnkraut,9 A. Bizzeti,17,d T. Blake,48 F. Blanc,39 J. Blouw,10 S. Blusk,59 V. Bocci,25 A. Bondar,34
N. Bondar,30,38 W. Bonivento,15 S. Borghi,54 M. Borsato,7 T. J. V. Bowcock,52 E. Bowen,40 C. Bozzi,16 S. Braun,11
D. Brett,54 M. Britsch,10 T. Britton,59 J. Brodzicka,54 N. H. Brook,46 A. Bursche,40 J. Buytaert,38 S. Cadeddu,15
R. Calabrese,16,b M. Calvi,20,e M. Calvo Gomez,36,f P. Campana,18 D. Campora Perez,38 L. Capriotti,54 A. Carbone,14,g
G. Carboni,24,h R. Cardinale,19,i A. Cardini,15 P. Carniti,20 L. Carson,50 K. Carvalho Akiba,2,38 G. Casse,52 L. Cassina,20,e
L. Castillo Garcia,38 M. Cattaneo,38 Ch. Cauet,9 G. Cavallero,19 R. Cenci,23,j M. Charles,8 Ph. Charpentier,38
M. Chefdeville,4 S. Chen,54 S.-F. Cheung,55 N. Chiapolini,40 M. Chrzaszcz,40 X. Cid Vidal,38 G. Ciezarek,41
P. E. L. Clarke,50 M. Clemencic,38 H. V. Cliff,47 J. Closier,38 V. Coco,38 J. Cogan,6 E. Cogneras,5 V. Cogoni,15,k
L. Cojocariu,29 G. Collazuol,22 P. Collins,38 A. Comerma-Montells,11 A. Contu,15,38 A. Cook,46 M. Coombes,46
S. Coquereau,8 G. Corti,38 M. Corvo,16,b B. Couturier,38 G. A. Cowan,50 D. C. Craik,48 A. Crocombe,48 M. Cruz Torres,60
S. Cunliffe,53 R. Currie,53 C. D’Ambrosio,38 J. Dalseno,46 P. N. Y. David,41 A. Davis,57 K. De Bruyn,41 S. De Capua,54
M. De Cian,11 J. M. De Miranda,1 L. De Paula,2 W. De Silva,57 P. De Simone,18 C.-T. Dean,51 D. Decamp,4 M. Deckenhoff,9
L. Del Buono,8 N. Déléage,4 M. Demmer,9 D. Derkach,55 O. Deschamps,5 F. Dettori,38 A. Di Canto,38 F. Di Ruscio,24
H. Dijkstra,38 S. Donleavy,52 F. Dordei,11 M. Dorigo,39 A. Dosil Suárez,37 D. Dossett,48 A. Dovbnya,43 K. Dreimanis,52
L. Dufour,41 G. Dujany,54 F. Dupertuis,39 P. Durante,38 R. Dzhelyadin,35 A. Dziurda,26 A. Dzyuba,30 S. Easo,49,38
U. Egede,53 V. Egorychev,31 S. Eidelman,34 S. Eisenhardt,50 U. Eitschberger,9 R. Ekelhof,9 L. Eklund,51 I. El Rifai,5
Ch. Elsasser,40 S. Ely,59 S. Esen,11 H. M. Evans,47 T. Evans,55 A. Falabella,14 C. Färber,11 C. Farinelli,41 N. Farley,45 DOI: 10.1103/PhysRevLett.115.112001 Britton,59 J. Brodzicka,54 N. H. Brook,46 A. Bursche,40 J. Buytaert,38 S. Cadeddu,15
R. Calabrese,16,b M. Calvi,20,e M. Calvo Gomez,36,f P. Campana,18 D. Campora Perez,38 L. Capriotti,54 A. Carbone,14,g
24 h
19 i
15
20
50
2 38
52
20 G. Carboni,24,h R. Cardinale,19,i A. Cardini,15 P. Carniti,20 L. Carson,50 K. Carvalho Akiba,2,38 G. Casse,52 L. Cassina,20,e
L. Castillo Garcia,38 M. Cattaneo,38 Ch. Cauet,9 G. Cavallero,19 R. Cenci,23,j M. Charles,8 Ph. Charpentier,38
M. Chefdeville,4 S. Chen,54 S.-F. Cheung,55 N. Chiapolini,40 M. Chrzaszcz,40 X. Cid Vidal,38 G. Ciezarek,41
P. E. L. Clarke,50 M. Clemencic,38 H. V. Cliff,47 J. Closier,38 V. Coco,38 J. Cogan,6 E. Cogneras,5 V. Cogoni,15,k
L. Cojocariu,29 G. Collazuol,22 P. Collins,38 A. Comerma-Montells,11 A. Contu,15,38 A. Cook,46 M. Coombes,46 S. Coquereau,8 G. Corti,38 M. Corvo,16,b B. Couturier,38 G. A. Cowan,50 D. C. Craik,48 A. Crocombe,48 M. Cruz Torres,60 M. De Cian,11 J. M. De Miranda,1 L. De Paula,2 W. De Silva,57 P. De Simone,18 C.-T. Dean,51 D. Decamp,4 M. Deckenhoff,9
8
4
9
55
5
38
38
24 112001-6 DOI: 10.1103/PhysRevLett.115.112001 Stone,
Classification
and
Regression
Trees
(Wadsworth
International Group, Belmont, CA, 1984). [14] M. Clemencic,
G. Corti,
S. Easo,
C. R. Jones,
S. Miglioranzi, M. Pappagallo, and P. Robbe, The LHCb
simulation application, Gauss: Design, evolution and expe-
rience, J. Phys. Conf. Ser. 331, 032023 (2011). ¯ [23] R. E. Schapire and Y. Freund, A decision-theoretic gener-
alization of on-line learning and an application to boosting,
J. Comput. Syst. Sci. 55, 119 (1997). [24] R. Aaij et al. (LHCb Collaboration), Identification of beauty
and charm quark jets at LHCb, JINST 10, P06013 (2015). [15] J. M. Campbell and R. K. Ellis, Radiative corrections to Zb¯b
production, Phys. Rev. D 62, 114012 (2000). [16] H.-L. Lai, M. Guzzi, J. Huston, Z. Li, P. M. Nadolsky,
J. Pumplin, and C.-P. Yuan, New parton distributions
for collider physics, Phys. Rev. D 82, 074024 (2010). [25] R. Aaij et al. (LHCb Collaboration), Measurement of the
forward
Z
boson
cross-section
in
pp
collisions
at
ffiffiffis
p ¼ 7 TeV, J. High Energy Phys. 08 (2015) 039. [17] S. Alioli, P. Nason, C. Oleari, and E. Re, A general
framework for implementing NLO calculations in shower
Monte Carlo programs: The POWHEG BOX, J. High
Energy Phys. 06 (2010) 043. [26] R. Aaij et al. (LHCb Collaboration), Measurement of the
forward W boson production cross-section in pp collisions
at
ffiffiffis
p ¼ 7 TeV, J. High Energy Phys. 12 (2014) 079. [27] See
Supplemental
Material
at
http://link.aps.org/
supplemental/10.1103/PhysRevLett.115.112001 for distri-
bution of pTðμÞ=pTðjμÞ with fit overlaid for all W þ c
candidates. [18] K. Hamilton, P. Nason, E. Re, and G. Zanderighi, NNLOPS
simulation of Higgs boson production, J. High Energy Phys. 10 (2013) 222. [19] R. Aaij et al. (LHCb Collaboration), Study of W boson
production in association with beauty and charm, Phys. Rev. D 92, 052001 (2015). [28] S. S. Wilks, The large-sample distribution of the likelihood
ratio for testing composite hypotheses, Ann. Math. Stat. 9,
60 (1938). [20] M. Cacciari, G. P. Salam, and G. Soyez, FastJet user manual,
Eur. Phys. J. C 72, 1896 (2012). [29] R. Aaij et al. (LHCb Collaboration), Precision luminosity
measurements at LHCb, JINST 9, P12005 (2014). S. Bifani,45 T. Bird,54 A. Birnkraut,9 A. Bizzeti,17,d T. Blake,48 F. Blanc,39 J. Blouw,10 S. Blusk,59 V. Bocci,25 A. Bondar,34
N. Bondar,30,38 W. Bonivento,15 S. Borghi,54 M. Borsato,7 T. J. V. Bowcock,52 E. Bowen,40 C. Bozzi,16 S. Braun,11
D. Brett,54 M. Britsch,10 T. 112001-6 week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) M. Grabalosa Gándara,5 R. Graciani Diaz,36 L. A. Granado Cardoso,38 E. Graugés,36 E. Graverini,40 G. Graziani,17
A. Grecu,29 E. Greening,55 S. Gregson,47 P. Griffith,45 L. Grillo,11 O. Grünberg,63 B. Gui,59 E. Gushchin,33 Yu. Guz,35,38
T. Gys,38 T. Hadavizadeh,55 C. Hadjivasiliou,59 G. Haefeli,39 C. Haen,38 S. C. Haines,47 S. Hall,53 B. Hamilton,58 T. Hampson,46 X. Han,11 S. Hansmann-Menzemer,11 N. Harnew,55 S. T. Harnew,46 J. Harrison,54 J. He,38 T. Head,39 V. Heijne,41 K. Hennessy,52 P. Henrard,5 L. Henry,8 J. A. Hernando Morata,37 E. van Herwijnen,38 M. Heß,63 A. Hicheur,2
D. Hill,55 M. Hoballah,5 C. Hombach,54 W. Hulsbergen,41 T. Humair,53 N. Hussain,55 D. Hutchcroft,52 D. Hynds,51 M. Idzik,27 P. Ilten,56 R. Jacobsson,38 A. Jaeger,11 J. Jalocha,55 E. Jans,41 A. Jawahery,58 F. Jing C. R. Jones,47 C. Joram,38 B. Jost,38 N. Jurik,59 S. Kandybei,43 W. Kanso,6 M. Karacson,38 T. M. Karbach,38 S. Karodia,51
M. Kelsey,59 I. R. Kenyon,45 M. Kenzie,38 T. Ketel,42 B. Khanji,20,38,e C. Khurewathanakul,39 S. Klaver,54 K. Klimaszewski,28 O. Kochebina,7 M. Kolpin,11 I. Komarov,39 R. F. Koopman,42 P. Koppenburg,41,38 M. Korolev,32 M. Kozeiha,5 L. Kravchuk,33 K. Kreplin,11 M. Kreps,48 G. Krocker,11 P. Krokovny,34 F. Kruse,9 W. Kucewicz,26,n
M. Kucharczyk,26 V. Kudryavtsev,34 A. K. Kuonen,39 K. Kurek,28 T. Kvaratskheliya,31 V. N. La Thi,39 D. Lacarrere,38 P. Manning,59 A. Mapelli,38 J. Maratas,5 J. F. Marchand,4 U. Marconi,14 C. Marin Benito,36 P. Marino,23,38,j R. Märki,39
11
25
6
39
42
66
2 J. Marks,11 G. Martellotti,25 M. Martin,6 M. Martinelli,39 D. Martinez Santos,42 F. Martinez Vidal,66 D. Martins Tostes,2
A. Massafferri,1 R. Matev,38 A. Mathad,48 Z. Mathe,38 C. Matteuzzi,20 K. Matthieu,11 A. Mauri,40 B. Maurin,39
A. Mazurov,45 M. McCann,53 J. McCarthy,45 A. McNab,54 R. McNulty,12 B. Meadows,57 F. Meier,9 M. Meissner,11
D. Melnychuk,28 M. Merk,41 D. A. Milanes,62 M.-N. Minard,4 D. S. Mitzel,11 J. Molina Rodriguez,60 S. Monteil,5
22
27
6
23 j
27
50
59
50 T. Pilař,48 D. Pinci,25 A. Pistone,19 A. Piucci,11 S. Playfer,50 M. Plo Casasus,37 T. Poikela,38 F. Polci,8 A. Poluektov,48,34
I. Polyakov,31 E. Polycarpo,2 A. Popov,35 D. Popov,10,38 B. Popovici,29 C. Potterat,2 E. Price,46 J. D. Price,52 g
g
g
B. Rachwal,26 J. H. Rademacker,46 B. Rakotomiaramanana,39 M. Rama,23 M. S. Rangel,2 I. Raniuk,43 N. Rauschmayr,38 112001-7 S. Farry,52 R. Fay,52 D. Ferguson,50 V. Fernandez Albor,37 F. Ferrari,14 F. Ferreira Rodrigues,1 M. Ferro-Luzzi,38
S. Filippov,33 M. Fiore,16,38,b M. Fiorini,16,b M. Firlej,27 C. Fitzpatrick,39 T. Fiutowski,27 K. Fohl,38 P. Fol,53 M. Fontana,10
F. Fontanelli,19,i R. Forty,38 O. Francisco,2 M. Frank,38 C. Frei,38 M. Frosini,17 J. Fu,21 E. Furfaro,24,h A. Gallas Torreira,37
D. Galli,14,g S. Gallorini,22,38 S. Gambetta,50 M. Gandelman,2 P. Gandini,55 Y. Gao,3 J. García Pardiñas,37 J. Garofoli,59
J. Garra Tico,47 L. Garrido,36 D. Gascon,36 C. Gaspar,38 U. Gastaldi,16 R. Gauld,55 L. Gavardi,9 G. Gazzoni,5 A. Geraci,21,l
D. Gerick,11 E. Gersabeck,11 M. Gersabeck,54 T. Gershon,48 Ph. Ghez,4 A. Gianelle,22 S. Gianì,39 V. Gibson,47
O. G. Girard,39 L. Giubega,29 V. V. Gligorov,38 C. Göbel,60 D. Golubkov,31 A. Golutvin,53,31,38 A. Gomes,1,m C. Gotti,20,e
M. Grabalosa Gándara,5 R. Graciani Diaz,36 L. A. Granado Cardoso,38 E. Graugés,36 E. Graverini,40 G. Graziani,17
A. Grecu,29 E. Greening,55 S. Gregson,47 P. Griffith,45 L. Grillo,11 O. Grünberg,63 B. Gui,59 E. Gushchin,33 Yu. Guz,35,38
T. Gys,38 T. Hadavizadeh,55 C. Hadjivasiliou,59 G. Haefeli,39 C. Haen,38 S. C. Haines,47 S. Hall,53 B. Hamilton,58
T. Hampson,46 X. Han,11 S. Hansmann-Menzemer,11 N. Harnew,55 S. T. Harnew,46 J. Harrison,54 J. He,38 T. Head,39
V. Heijne,41 K. Hennessy,52 P. Henrard,5 L. Henry,8 J. A. Hernando Morata,37 E. van Herwijnen,38 M. Heß,63 A. Hicheur,2
D. Hill,55 M. Hoballah,5 C. Hombach,54 W. Hulsbergen,41 T. Humair,53 N. Hussain,55 D. Hutchcroft,52 D. Hynds,51
M. Idzik,27 P. Ilten,56 R. Jacobsson,38 A. Jaeger,11 J. Jalocha,55 E. Jans,41 A. Jawahery,58 F. Jing,3 M. John,55 D. Johnson,38
C. R. Jones,47 C. Joram,38 B. Jost,38 N. Jurik,59 S. Kandybei,43 W. Kanso,6 M. Karacson,38 T. M. Karbach,38 S. Karodia,51
M. Kelsey,59 I. R. Kenyon,45 M. Kenzie,38 T. Ketel,42 B. Khanji,20,38,e C. Khurewathanakul,39 S. Klaver,54
K. Klimaszewski,28 O. Kochebina,7 M. Kolpin,11 I. Komarov,39 R. F. Koopman,42 P. Koppenburg,41,38 M. Korolev,32
M. Kozeiha,5 L. Kravchuk,33 K. Kreplin,11 M. Kreps,48 G. Krocker,11 P. Krokovny,34 F. Kruse,9 W. Kucewicz,26,n
M. Kucharczyk,26 V. Kudryavtsev,34 A. K. Kuonen,39 K. Kurek,28 T. Kvaratskheliya,31 V. N. La Thi,39 D. Lacarrere,38
G. Lafferty,54 A. Lai,15 D. Lambert,50 R. W. Lambert,42 G. Lanfranchi,18 C. Langenbruch,48 B. Langhans,38 T. Latham,48
C. Lazzeroni,45 R. Le Gac,6 J. van Leerdam,41 J.-P. Lees,4 R. Lefèvre,5 A. Leflat,32,38 J. Lefrançois,7 O. Leroy,6 T. Lesiak,26
B. Leverington,11 Y. Li,7 T. Likhomanenko,65,64 M. Liles,52 R. Lindner,38 C. Linn,38 F. Lionetto,40 B. Liu,15 X. Liu,3
D. Loh,48 S. Lohn,38 I. Longstaff,51 J. H. Lopes,2 D. Lucchesi,22,o M. Lucio Martinez,37 H. Luo,50 A. Lupato,22 E. Luppi,16,b
O. Lupton,55 F. Machefert,7 F. Maciuc,29 O. Maev,30 K. Maguire,54 S. Malde,55 A. Malinin,64 G. Manca,7 G. Mancinelli,6
P. Manning,59 A. Mapelli,38 J. Maratas,5 J. F. Marchand,4 U. Marconi,14 C. Marin Benito,36 P. Marino,23,38,j R. Märki,39
J. Marks,11 G. Martellotti,25 M. Martin,6 M. Martinelli,39 D. Martinez Santos,42 F. Martinez Vidal,66 D. Martins Tostes,2
A. Massafferri,1 R. Matev,38 A. Mathad,48 Z. Mathe,38 C. Matteuzzi,20 K. Matthieu,11 A. Mauri,40 B. Maurin,39
A. Mazurov,45 M. McCann,53 J. McCarthy,45 A. McNab,54 R. McNulty,12 B. Meadows,57 F. Meier,9 M. Meissner,11
D. Melnychuk,28 M. Merk,41 D. A. Milanes,62 M.-N. Minard,4 D. S. Mitzel,11 J. Molina Rodriguez,60 S. Monteil,5
M. Morandin,22 P. Morawski,27 A. Mordà,6 M. J. Morello,23,j J. Moron,27 A. B. Morris,50 R. Mountain,59 F. Muheim,50
J. Müller,9 K. Müller,40 V. Müller,9 M. Mussini,14 B. Muster,39 P. Naik,46 T. Nakada,39 R. Nandakumar,49 A. Nandi,55
I. Nasteva,2 M. Needham,50 N. Neri,21 S. Neubert,11 N. Neufeld,38 M. Neuner,11 A. D. Nguyen,39 T. D. Nguyen,39
C. Nguyen-Mau,39,p V. Niess,5 R. Niet,9 N. Nikitin,32 T. Nikodem,11 D. Ninci,23 A. Novoselov,35 D. P. O’Hanlon,48
A. Oblakowska-Mucha,27 V. Obraztsov,35 S. Ogilvy,51 O. Okhrimenko,44 R. Oldeman,15,k C. J. G. Onderwater,67
B. Osorio Rodrigues,1 J. M. Otalora Goicochea,2 A. Otto,38 P. Owen,53 A. Oyanguren,66 A. Palano,13,q F. Palombo,21,r
M. Palutan,18 J. Panman,38 A. Papanestis,49 M. Pappagallo,51 L. L. Pappalardo,16,b C. Pappenheimer,57 C. Parkes,54
G. Passaleva,17 G. D. Patel,52 M. Patel,53 C. Patrignani,19,i A. Pearce,54,49 A. Pellegrino,41 G. Penso,25,s M. Pepe Altarelli,38
S. Perazzini,14,g P. Perret,5 L. Pescatore,45 K. Petridis,46 A. Petrolini,19,i M. Petruzzo,21 E. Picatoste Olloqui,36 B. Pietrzyk,4 112001-7 Vollhardt,40
10
46
30
34
63
41
63
48 q
q
g
q
G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. Vollhardt,40
10
46
30
34
63
41
63
48 G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardon R. Wallace,12 J. Walsh,23 S. Wandernoth,11 J. Wang,59 D. R. Ward,47 N. K. Watson,45 D. Web T. Williams,45 F. F. Wilson,49 J. Wimberley,58 J. Wishahi,9 W. Wislicki,28 M. Witek,26 G. Wormser,7 S. A. Wotton,47
S. Wright,47 K. Wyllie,38 Y. Xie,61 Z. Xu,39 Z. Yang,3 J. Yu,61 X. Yuan,34 O. Yushchenko,35 M. Zangoli,14 M. Zavertyaev,10,v L. Zhang,3 Y. Zhang,3 A. Zhelezov,11 A. Zhokhov,31 and L. Zhong3 (LHCb Collaboration) 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil
2 2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
3 3Center for High Energy Physics, Tsinghua University, Beijing, China 4LAPP, Université Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, Fran
5 y
t Université, Université Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 7LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France NHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France
9 et Marie Curie, Université Paris Diderot, CNRS/IN 9Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany nstitut für Kernphysik (MPIK), Heidelberg, Germany Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Ger
12 12School of Physics, University College Dublin, Dublin, Ireland 13Sezione INFN di Bari, Bari, Italy 14Sezione INFN di Bologna, Bologna, Italy
15 15Sezione INFN di Cagliari, Cagliari, Italy
16 16Sezione INFN di Ferrara, Ferrara, Italy
17 17Sezione INFN di Firenze, Firenze, Italy 18Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy
19 19Sezione INFN di Genova, Genova, Italy 20Sezione INFN di Milano Bicocca, Milano, Italy
21 21Sezione INFN di Milano, Milano, Italy
22 22Sezione INFN di Padova, Padova, Italy
23 23Sezione INFN di Pisa, Pisa, Italy 24Sezione INFN di Roma Tor Vergata, Roma, Italy
25 25Sezione INFN di Roma La Sapienza, Roma, Italy k Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland ty of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, Poland 28National Center for Nuclear Research (NCBJ), Warsaw, Poland
29 ei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania
30 30Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 31Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia
2 f
p
y
32Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia
33 33Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russia 112001-8 M.-H. Schune,7 R. Schwemmer,38 B. Sciascia,18 A. Sciubba,25,s A. Semennikov,31 I. Sepp,53 N. Serra,40 J. Serrano,6
L. Sestini,22 P. Seyfert,20 M. Shapkin,35 I. Shapoval,16,43,b Y. Shcheglov,30 T. Shears,52 L. Shekhtman,34 V. Shevchenko,64
A. Shires,9 R. Silva Coutinho,48 G. Simi,22 M. Sirendi,47 N. Skidmore,46 I. Skillicorn,51 T. Skwarnicki,59 E. Smith,55,49
E. Smith,53 I. T. Smith,50 J. Smith,47 M. Smith,54 H. Snoek,41 M. D. Sokoloff,57,38 F. J. P. Soler,51 D. Souza,46
B. Souza De Paula,2 B. Spaan,9 P. Spradlin,51 S. Sridharan,38 F. Stagni,38 M. Stahl,11 S. Stahl,38 O. Steinkamp,40
O. Stenyakin,35 F. Sterpka,59 S. Stevenson,55 S. Stoica,29 S. Stone,59 B. Storaci,40 S. Stracka,23,j M. Straticiuc,29
U. Straumann,40 L. Sun,57 W. Sutcliffe,53 K. Swientek,27 S. Swientek,9 V. Syropoulos,42 M. Szczekowski,28 P. Szczypka,39,38
T. Szumlak,27 S. T’Jampens,4 T. Tekampe,9 M. Teklishyn,7 G. Tellarini,16,b F. Teubert,38 C. Thomas,55 E. Thomas,38
J. van Tilburg,41 V. Tisserand,4 M. Tobin,39 J. Todd,57 S. Tolk,42 L. Tomassetti,16,b D. Tonelli,38 S. Topp-Joergensen,55
N. Torr,55 E. Tournefier,4 S. Tourneur,39 K. Trabelsi,39 M. T. Tran,39 M. Tresch,40 A. Trisovic,38 A. Tsaregorodtsev,6
P. Tsopelas,41 N. Tuning,41,38 A. Ukleja,28 A. Ustyuzhanin,65,64 U. Uwer,11 C. Vacca,15,k V. Vagnoni,14 G. Valenti,14
A. Vallier,7 R. Vazquez Gomez,18 P. Vazquez Regueiro,37 C. Vázquez Sierra,37 S. Vecchi,16 J. J. Velthuis,46 M. Veltri,17,u
G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. Vollhardt,40
D. Volyanskyy,10 D. Voong,46 A. Vorobyev,30 V. Vorobyev,34 C. Voß,63 J. A. de Vries,41 R. Waldi,63 C. Wallace,48
R. Wallace,12 J. Walsh,23 S. Wandernoth,11 J. Wang,59 D. R. Ward,47 N. K. Watson,45 D. Websdale,53 A. Weiden,40
M. Whitehead,48 D. Wiedner,11 G. Wilkinson,55,38 M. Wilkinson,59 M. Williams,38 M. P. Williams,45 M. Williams,56
T. Williams,45 F. F. Wilson,49 J. Wimberley,58 J. Wishahi,9 W. Wislicki,28 M. Witek,26 G. Wormser,7 S. A. Wotton,47
S. Wright,47 K. Wyllie,38 Y. Xie,61 Z. Xu,39 Z. Yang,3 J. Yu,61 X. Yuan,34 O. Yushchenko,35 M. Zangoli,14 M. Zavertyaev,10,v
L Zh
3 Y Zh
3 A Zh l
11 A Zh kh
31
d L Zh
3 112001-7 week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) M.-H. Schune,7 R. Schwemmer,38 B. Sciascia,18 A. Sciubba,25,s A. Semennikov,31 I. Sepp,53 N. Serra,40 J. Serrano,6
L. Sestini,22 P. Seyfert,20 M. Shapkin,35 I. Shapoval,16,43,b Y. Shcheglov,30 T. Shears,52 L. Shekhtman,34 V. Shevchenko,64
A. Shires,9 R. Silva Coutinho,48 G. Simi,22 M. Sirendi,47 N. Skidmore,46 I. Skillicorn,51 T. Skwarnicki,59 E. Smith,55,49
E. Smith,53 I. T. Smith,50 J. Smith,47 M. Smith,54 H. Snoek,41 M. D. Sokoloff,57,38 F. J. P. Soler,51 D. Souza,46
B. Souza De Paula,2 B. Spaan,9 P. Spradlin,51 S. Sridharan,38 F. Stagni,38 M. Stahl,11 S. Stahl,38 O. Steinkamp,40
O. Stenyakin,35 F. Sterpka,59 S. Stevenson,55 S. Stoica,29 S. Stone,59 B. Storaci,40 S. Stracka,23,j M. Straticiuc,29
U. Straumann,40 L. Sun,57 W. Sutcliffe,53 K. Swientek,27 S. Swientek,9 V. Syropoulos,42 M. Szczekowski,28 P. Szczypka,39,38
T. Szumlak,27 S. T’Jampens,4 T. Tekampe,9 M. Teklishyn,7 G. Tellarini,16,b F. Teubert,38 C. Thomas,55 E. Thomas,38
J. van Tilburg,41 V. Tisserand,4 M. Tobin,39 J. Todd,57 S. Tolk,42 L. Tomassetti,16,b D. Tonelli,38 S. Topp-Joergensen,55
N. Torr,55 E. Tournefier,4 S. Tourneur,39 K. Trabelsi,39 M. T. Tran,39 M. Tresch,40 A. Trisovic,38 A. Tsaregorodtsev,6
P. Tsopelas,41 N. Tuning,41,38 A. Ukleja,28 A. Ustyuzhanin,65,64 U. Uwer,11 C. Vacca,15,k V. Vagnoni,14 G. Valenti,14
A. Vallier,7 R. Vazquez Gomez,18 P. Vazquez Regueiro,37 C. Vázquez Sierra,37 S. Vecchi,16 J. J. Velthuis,46 M. Veltri,17,u
G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. Vollhardt,40
D. Volyanskyy,10 D. Voong,46 A. Vorobyev,30 V. Vorobyev,34 C. Voß,63 J. A. de Vries,41 R. Waldi,63 C. Wallace,48
R. Wallace,12 J. Walsh,23 S. Wandernoth,11 J. Wang,59 D. R. Ward,47 N. K. Watson,45 D. Websdale,53 A. Weiden,40
M. Whitehead,48 D. Wiedner,11 G. Wilkinson,55,38 M. Wilkinson,59 M. Williams,38 M. P. Williams,45 M. Williams,56
T. Williams,45 F. F. Wilson,49 J. Wimberley,58 J. Wishahi,9 W. Wislicki,28 M. Witek,26 G. Wormser,7 S. A. Wotton,47
S. Wright,47 K. Wyllie,38 Y. Xie,61 Z. Xu,39 Z. Yang,3 J. Yu,61 X. Yuan,34 O. Yushchenko,35 M. Zangoli,14 M. Zavertyaev,10,v
L Zhang 3 Y Zhang 3 A Zhelezov 11 A Zhokhov 31 and L Zhong3 A. Vallier,7 R. Vazquez Gomez,18 P. Vazquez Regueiro,37 C. Vázquez Sierra,37 S. Vecchi,16 J. J. Velthuis,46 M. Veltri,17,u
G. Veneziano,39 M. Vesterinen,11 B. Viaud,7 D. Vieira,2 M. Vieites Diaz,37 X. Vilasis-Cardona,36,f A. 112001-8 f eAlso at Università di Milano Bicocca, Milano, Italy. f fAlso at LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. fAlso at LIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. gAlso at Università di Bologna, Bologna, Italy. h hAlso at Università di Roma Tor Vergata, Roma,
i iAlso at Università di Genova, Genova, Italy. j iAlso at Università di Genova, Genova, Italy. j jAlso at Scuola Normale Superiore, Pisa, Italy. jAlso at Scuola Normale Superiore, Pisa, Italy. k kAlso at Università di Cagliari, Cagliari, Italy. l kAlso at Università di Cagliari, Cagliari, Italy. l lAlso at Politecnico di Milano, Milano, Italy. lAlso at Politecnico di Milano, Milano, Italy. mAlso at Universidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. nAlso at AGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków,
Poland. o oAlso at Università di Padova, Padova, Italy. oAlso at Università di Padova, Padova, Italy. pAlso at Hanoi University of Science, Hanoi, Viet Nam. pAlso at Hanoi University of Science, Hanoi, Viet Nam. pAlso at Hanoi University of Science, Hanoi, V g
nAlso at AGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków,
Poland.
o 112001-8 week ending
11 SEPTEMBER 2015 week ending
11 SEPTEMBER 2015 P H Y S I C A L
R E V I E W
L E T T E R S PRL 115, 112001 (2015) 34Budker Institute of Nuclear Physics (SB RAS) and Novosibirsk State University, Novosibirsk, Russia
35Institute for High Energy Physics (IHEP), Protvino, Russia Imperial College London, London, United Kingdom ool of Physics and Astronomy, University of Manchester, Manchester, United Kingdom
55 55Department of Physics, University of Oxford, Oxford, United Kingdom
56 56Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
57 57University of Cincinnati, Cincinnati, Ohio, USA
8 58University of Maryland, College Park, Maryland, USA
59 59Syracuse University, Syracuse, New York, USA 60Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil
(associated with Institution Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil)
61Institute of Particle Physics Central China Normal University Wuhan Hubei China 60Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil
(associated with Institution Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
61 61Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China (associated with Institution Center for High Energy Physics, Tsinghua University, Beijing, China)
62 Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia 62Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia 63Institut für Physik, Universität Rostock, Rostock, Germany associated with Institution Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, He
64 stitution Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany)
64 64National Research Centre Kurchatov Institute, Moscow, Russia (associated with Institution Institute of Theoretical and Experimental Physics (ITEP), Moscow,
65 tion Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia)
65 65Yandex School of Data Analysis, Moscow, Russia (associated with Institution Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russi
66 66Instituto de Fisica Corpuscular (IFIC), Universitat de Valencia-CSIC, Valencia, Spain (associated with Institution Universitat de Barcelona, Barcelona, Spain) 67Van Swinderen Institute, University of Groningen, Groningen, The Netherlands (associated with Institution Nikhef National Institute for Subatomic Physics, Amsterdam, The Netherlands) aAlso at Università di Firenze, Firenze, Italy. b bAlso at Università di Ferrara, Ferrara, Italy. c bAlso at Università di Ferrara, Ferrara, Italy. cAlso at Università della Basilicata, Potenza, Italy. d cAlso at Università della Basilicata, Potenza, Italy. d dAlso at Università di Modena e Reggio Emilia, Modena, Italy. dAlso at Università di Modena e Reggio Emilia, Modena, Italy. eAlso at Università di Milano Bicocca, Milano, Italy. 112001-9 week ending
11 SEPTEMBER 2015 vAlso at P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia. PRL 115, 112001 (2015) qAlso at Università di Bari, Bari, Italy. so at Università degli Studi di Milano, Milano, Ital sAlso at Università di Roma La Sapienza, Roma, Italy. tAlso at Università di Pisa, Pisa, Italy. uAlso at Università di Urbino, Urbino, Italy. uAlso at Università di Urbino, Urbino, Italy. vAlso at P.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russ 112001-10
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https://openalex.org/W2767015251
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English
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Isolation and identification of halotolerant soil bacteria from coastal Patenga area
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BMC research notes
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cc-by
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© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Objective: Halotolerant bacteria have multiple uses viz. fermentation with lesser sterility control and industrial pro‑
duction of bioplastics. Moreover, it may increase the crop productivity of coastal saline lands in Bangladesh by trans‑
ferring the salt tolerant genes into the plants. The study focused on the isolation and identification of the halotolerant
bacteria from three soil samples, collected from coastal Patenga area. The samples were inoculated in nutrient media
containing a wide range of salt concentrations. Results: All the samples showed 2, 4 and 6% (w/v) salt tolerance. The isolates from Patenga soil (4, 6%) and beach
soil (2%) showed catalase activity and all the isolates showed negative results for oxidase activity, indole produc‑
tion, lactose and motility. All the samples provided positive results for dextrose fermentation. Other tests provided
mixed results. Based on the morphological characteristics, biochemical tests and ABIS software analysis the isolates
fall within the Enterobacteriaceae, Clostridium and Corynebacterium, with a predominance of Vibrios. Overall the
isolates can be considered as mild halotolerant, with the best growth observed at lower salinities and no halophilism
detected. Among many possibilities, the genes responsible for the salt tolerant trait in these species can be identified,
extracted and inserted into the crop plants to form a transgenic plant to result in higher yield for the rest of the year. Keywords: Halotolerant, Enterobacteriaceae, Clostridium, Corynebacterium, Salinity Keywords: Halotolerant, Enterobacteriaceae, Clostridium, Corynebacterium, Salinity halotolerant. Non-halotolerant which can grow in low
salt concentration about 1% w/v. Slightly tolerant as pseu-
domonads, enterobacteria, and vibrios, can survive in
up to 2–8%, moderately tolerant 18–20% and extremely
tolerant microbes can grow over the whole range of salt
concentrations from zero to saturation. The halotolerant
organisms maintain a low level of ionic concentrations
to synthesize compatible solutes to balance the osmotic
level inside the cytoplasm with the outer medium. These
maintenance mechanisms of the internal environment
and the properties of the cytoplasmic membrane help
them to adapt to changes in the saline environment as
salt lakes, saline soils, and salted food products [4]. Isolation and identification
of halotolerant soil bacteria from coastal
Patenga area Shafkat Shamim Rahman1,2* , Romana Siddique1 and Nafisa Tabassum1 *Correspondence: shafkatshamimrahman@gmail.com
1 Biotechnology Program, Department of Mathematics and Natural
Sciences, BRAC University, 66, Mohakhali, Dhaka 1212, Bangladesh
Full list of author information is available at the end of the article Rahman et al. BMC Res Notes (2017) 10:531
DOI 10.1186/s13104-017-2855-7 Rahman et al. BMC Res Notes (2017) 10:531
DOI 10.1186/s13104-017-2855-7 BMC Research Notes Biochemical tests Total 18 primarily screened isolates (2 each from 2, 4
and 6% plates) were sub-cultured on NA plates without
salt with the same dilution factor and nine inocula were
tested further. After 24-h of incubation at 37 °C, the
plates were observed [19]. p
Standard gram staining protocols were followed and
then slide observed under a microscope. The presence or
absence of bubbles or foam was observed to determine
the capability of catalase activity [20]. Two drops of oxi-
dase reagent p-aminodimethylaniline oxalate were added
to the surface of test organisms’ growth in oxidase test
[21]. Nitrate broth (beef extract 3 g/L, peptone 5 g/L,
potassium nitrate 5 g/L) [22] and MR-VP broth (pep-
tone, dextrose and potassium phosphate) was prepared
and incubated at 24-h at 37 °C for Methyl Red test and
VP test [23]. The triple sugar iodine agar was prepared
and after inoculation, incubated for about 24-h at 35 °C
for TSI test [24]. After inoculation, MIU media (MIU test
[25]) and Simmon’s agar slants were also incubated for
24-h at 37 °C [22, 23]. Trypticase 10 g/L, NaCl 5 g/L, phe-
nol red 0.018/L, sugar (glucose, lactose or sucrose) 5 g/L
contained broth incubated at 37 °C for 24-h to determine
the capability of fermenting carbohydrate substrate with
acid and gas production [26]. Autoclaved starch agar
(beef extract 3 g/L, soluble starch 10 g/L and agar 15 g/L)
plates were incubated for 24-h to indicate the presence
of α-amylase [27]. Sterile tubes containing inoculum was
transferred in 6.5% NaCl solution and kept in 24-h incu-
bation to assess salt hindrance. Halophilic and halotolerant bacteria are essential for
salty foods production as Thai fish sauce, pickling brines
and salt-cured bacon [10]. Isolates from effluents of tex-
tile industries also showed the ability to decolorize the
utilized azo dyes [11]. Halotolerant bacteria, recovered
from the composting process, were able to produce
hydrolases, lipases, proteases, amylases, cellulases and
biopolymers [12]. Four halotolerant species (Bacillus
atrophaeus, Halomonas shengliensis, Halomonas kore-
ensis and Virgibacillus salarius) showed the ability to
metabolize hydrocarbons and isolates as V. salarius and
Brevibacillus sp. KUMAs1 has the potential to be used
for bioremediation [13, 14]. Nevertheless, Corynebacte-
rium xerosis was the potent degraders of hydrocarbons
(petrol and diesel) [15]. Isolation and screening containing
aminocyclopropane-1-carboxylate
(ACC)
deaminase, [6] examined their effect on salinity stress tol-
erance in okra through the induction of ROS-scavenging
enzyme activity. PGPR inoculated plants exhibited higher
germination percentage, growth parameters, and chloro-
phyll content than control. 5 g of each of the soil samples were taken to prepare a
suspension. Then serially diluted (10−3, 10−5 and 10−7,
100 μL) samples were taken and incubated on the nutri-
ent agar plates containing 2% (w/v), 4% (w/v), 6% (w/v),
8% (w/v) and 10% (w/v) NaCl for 24-h at 37 °C. No bac-
terial growth observed for 8% (w/v) and 10% (w/v) NaCl
plates. In a salt stress improvement research for red pepper
plants by 1-aminocyclopropane-1-carboxylic acid (ACC)
deaminase producing halotolerant bacteria was studied
[7, 8]. The result showed salt stress ethylene production
by increasing enzyme activities of a biosynthetic pathway. It was also reported that the growth promotion in inoc-
ulated red pepper plants under inhibitory levels of salt
stress is due to ACC deaminase activity present in the
halotolerant bacteria [9]. Biochemical tests Halophilic and halotolerant bac-
teria can be used for the production of enzymes with dif-
ferent immunological properties [16] and also essential
for nutrient recycling and for maintaining the soil health
in a salty environment [17].h The goal of the research was to isolate and identify
halotolerant bacteria from natural sources [18]. Multiple
uses of these species viz. fermentation with lesser control
on sterility and industrial production of bioplastics, can
be beneficial for different sectors in Bangladesh. Besides
that, it may provide the salt tolerant genes for plants in
future. Therefore, plants can uptake and store salt as a
nutrient and result in a good yield throughout the year. Introduction Salinization is one of the root reasons for the crop
destruction in Bangladesh [1, 2]. Almost 20% of the coun-
try is covered as the coastal area from which about 53%
of lands are affected by very slight to very strong salinity
[3]. The upward movement of the saline ground water in
the dry season (November–May) is the factor that initi-
ates the development of saline soil. Nevertheless, the
intrusion of the seawater also increases the degree of
salinity of the coastal drinking water and cause severe
health problems viz. hypertension or high blood pres-
sure, stroke, heart diseases, pre-eclampsia. Bacteria that grow in the absence of salt and in the
presence of high salt concentrations are known as It was reported that amelioration of salt stress inhibi-
tory effect on the canola seed germination was attrib-
uted to the inoculation of ACC deaminase-producing
halotolerant bacteria modulating ethylene emission and
inducing hydrolytic enzymes [5]. A research was exe-
cuted on plant growth-promoting rhizobacteria (PGPR) Rahman et al. BMC Res Notes (2017) 10:531 Page 2 of 6 Results All the samples showed growth in NA media contain-
ing 2% (w/v), 4% (w/v) and 6% (w/v) NaCl. But failed to
grow in media containing 8% (w/v) and 10% (w/v) NaCl
(Table 1). Main text
Methods
Sample collectionhf The isolates were all gram positive (Fig. 1; Table 2). Pat-
enga 4, 6% were cocci shaped and rest were rod shaped. Sample collectionhf Patenga area 4% and Beach
soil 2, 4, 6% hydrolyzed starch. Patenga 2, 4%, Beach soil
2, 6%, Land soil 2, 4 and 6% showed the ability to grow at
6.5% NaCl solution. Sample collectionhf soil 2%—Vibrio metschnikovii; Beach soil 4%—Vibrio
metschnikovii; Beach soil 6%—Volucribacter psittaci-
cida; Land soil 2%—Aggregatibacter (Haemophilus)
segnis; Land soil 4%—Aggregatibacter (Haemophilus) seg-
nis; Land soil 6%—Clostridium innocuum/Clostridium
spiroforme. soil 2%—Vibrio metschnikovii; Beach soil 4%—Vibrio
metschnikovii; Beach soil 6%—Volucribacter psittaci-
cida; Land soil 2%—Aggregatibacter (Haemophilus)
segnis; Land soil 4%—Aggregatibacter (Haemophilus) seg-
nis; Land soil 6%—Clostridium innocuum/Clostridium
spiroforme. became yellow for Patenga 2%, Beach soil 2, 4 and 6%. The only slant was acidic for Land soil 2 and 4% and Land
soil 6% for the butt. Patenga 2%, Beach soil 2, 4 and 6%
recorded as MR test positive. Patenga 4, 6%, Beach soil 2,
4%, Land soil 2, 4, and 6% found VP test positive because
the color did not change to pink. All negative results
recorded at MIU test, except non-motile Patenga 6%
sample was urease positive. Simmon’s citrate test showed
a positive result for Beach soil 2, 4% and land soil 4, 6%. Land soil 4, 6%, Patenga area 2, 4, 6%, Beach soil 2, 4, and
6% found positive for sucrose metabolism. No positive
result observed for lactose fermentation. All the sam-
ple result was positive for dextrose fermentation, except
Land soil 2%. Patenga area 2% and Beach soil 4% pro-
duced gas in fermentation. Patenga area 4% and Beach
soil 2, 4, 6% hydrolyzed starch. Patenga 2, 4%, Beach soil
2, 6%, Land soil 2, 4 and 6% showed the ability to grow at
6.5% NaCl solution. became yellow for Patenga 2%, Beach soil 2, 4 and 6%. The only slant was acidic for Land soil 2 and 4% and Land
soil 6% for the butt. Patenga 2%, Beach soil 2, 4 and 6%
recorded as MR test positive. Patenga 4, 6%, Beach soil 2,
4%, Land soil 2, 4, and 6% found VP test positive because
the color did not change to pink. All negative results
recorded at MIU test, except non-motile Patenga 6%
sample was urease positive. Simmon’s citrate test showed
a positive result for Beach soil 2, 4% and land soil 4, 6%. Land soil 4, 6%, Patenga area 2, 4, 6%, Beach soil 2, 4, and
6% found positive for sucrose metabolism. No positive
result observed for lactose fermentation. All the sam-
ple result was positive for dextrose fermentation, except
Land soil 2%. Patenga area 2% and Beach soil 4% pro-
duced gas in fermentation. Sample collectionhf Three different soil samples (Patenga Beach soil
22°15′06.8″N, 91°45′29.5″E; Land soil 22°14′31.6″N,
91°47′15.9″E and Patenga area soil 22°14′25.4″N,
91°48′59.7″E) were collected from 10 to 12-in. depth
at different locations of the coastal Chittagong area;
these were kept in a sterile polythene packet at room
temperature. In catalase test, hydrogen peroxide was used and bro-
ken down to water and oxygen. Patenga 2, 6% and Beach
soil 2% were positive. Oxidase test resulted as negative
for all (Table 2). Only Land soil 2 and 4% was reduced
nitrate by the change of color (red) (Table 2). Most of the
samples were responsive to TSI test. Both slant and butt Page 3 of 6
Rahman et al. BMC Res Notes (2017) 10:531
Table 1 Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl
Conc. Patenga soil
Beach soil
Land soil
2%
4%
6%
8%
10%
2%
4%
6%
8%
10%
2%
4%
6%
8%
10%
Growth
✓
✓
✓
x
x
✓
✓
✓
x
x
✓
✓
✓
x
x
Fig. 1 Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Rahman et al. BMC Res Notes (2017) 10:531 Page 3 of 6 Table 1 Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl
Conc. Patenga soil
Beach soil
Land soil
2%
4%
6%
8%
10%
2%
4%
6%
8%
10%
2%
4%
6%
8%
10%
Growth
✓
✓
✓
x
x
✓
✓
✓
x
x
✓
✓
✓
x
x Table 1 Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Table 1 Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl Table 1 Results of growth in NA media containing 2% (w/v) to 10% (w/v) NaCl
Conc. Patenga soil
Beach soil
Land soil
2%
4%
6%
8%
10%
2%
4%
6%
8%
10%
2%
4%
6%
8%
10%
Growth
✓
✓
✓
x
x
✓
✓
✓
x
x
✓
✓
✓
x
x Fig. 1 Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Fig. 1 Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Fig. 1 Gram positive bacterial isolates from a Patenga area soil, b Beach soil and c Land soil sample Fig. Discussion In this study, three soil samples were collected from the
coastal area of Chittagong [29–31]. The isolated bacteria
from the samples had successfully survived in a limited
range of salinities and no halophilism detected. All the iso-
lates showed tolerance to 2% (w/v), 4% (w/v) and 6% (w/v)
of NaCl (Table 1). The dilution factor was inversely pro-
portional to the number of colonies. The study divulged
the abundance of gram positive bacteria (Table 2). The
isolates from Patenga area (2, 6%) and Beach (2%) showed
catalase activity and all the isolates showed negative
results for oxidase activity, indole production, lactose and
motility. Aerobic and facultative aerobes exhibit oxidase
activity whereas Enterobacteriaceae are oxidase nega-
tive. This provided the evidence of Enterobacteriaceae in
the samples. In the MIU test, only the isolates extracted
from Patenga area that is 6% (w/v) NaCl tolerant showed
urease positive. In addition to this, all the isolates pro-
vided positive results for dextrose fermentation. Vibrios Finally, the test results were analyzed in ABIS online
software [28], which is used to analyze the genus of
the organism. Based on the morphology characteris-
tics, Biochemical test and ABIS software the follow-
ing results were predicted as Patenga 2%—Brevibacillus
agri; Patenga 4%—Pantoea stewartii subsp. stewartii;
Patenga
6%—Corynebacterium
xerosis/Corynebacte-
rium minutissimum/Corynebacterium kutscheri; Beach Rahman et al. BMC Res Notes (2017) 10:531 Page 4 of 6 Table 2 Results of biochemical and sugar tests of the isolates collected from nutrient agar
K alkaline reaction, A acidic reaction, + positive reaction, − negative reaction
Isolate
no
Sample
isolate
name
Gram stain
TSI
MIU
Catalase Oxidase Sim-
mon’s
citrate
MR
VP
Nitrate
reduc-
tion
Starch
hydroly-
sis
Carbohydrate
6.5%
NaCl
solution
Presumptive
organism
±
Shape
BUTT
SLANT
H2S
Gas
Motility
Indole
Urease
Sucrose Lactose Dextrose
1. Patenga
area
soil
2%
+
Rod
A
A
−
−
−
−
−
+
−
−
+
−
−
−
+
−
+
+
Brevibacillus
agri
2. Patenga
area
soil
4%
+
Cocci
A
K
−
−
−
−
−
−
−
−
−
+
−
+
+
−
+
+
Pantoea stew-
artii subsp. stewartii
3. Patenga
area
soil
6%
+
Cocci
K
A
−
−
−
−
+
+
−
−
−
+
−
−
+
−
+
−
Corynebacte-
rium xerosis/
Corynebacte-
rium minutis-
simum/
Corynebacte-
rium kutscheri
4. Discussion Beach
soil
2%
+
Rod
A
A
−
−
−
−
−
+
−
+
+
+
−
+
+
−
+
+
Vibrio
metschnikovii
5. Beach
soil
4%
+
Rod
A
A
−
−
−
−
−
−
−
+
+
+
−
+
+
−
+
−
Vibrio
metschnikovii
6. Beach
soil
6%
+
Rod
A
A
−
−
−
−
−
−
−
−
+
−
−
+
+
−
+
+
Volucribacter
psittacicida
7. Land
soil
2%
+
Rod
K
A
−
−
−
−
−
−
−
−
−
+
+
−
−
−
−
+
Aggregatibacter
(Haemophi-
lus) segnis
8. Land
soil
4%
+
Rod
K
A
−
−
−
−
−
−
−
+
−
+
+
−
+
−
+
+
Aggregatibacter
(Haemophi-
lus) segnis
9. Land
soil
6%
+
Rod
A
K
−
−
−
−
−
−
−
+
−
+
−
−
+
−
+
+
Clostridium
innocuum/
Clostridium
spiroforme Rahman et al. BMC Res Notes (2017) 10:531 Page 5 of 6 were predominating in the investigated soil along with
Corynebacterium, Clostridium and Enterobacteriaceae. were predominating in the investigated soil along with
Corynebacterium, Clostridium and Enterobacteriaceae. References 1. Elaine Colligan. Salinity issues in Bangladesh. Georget J Int Aff. 2011. http://journal.georgetown.edu/guide-to-salinity-issues-in-bangladesh/. Accessed 26 June 2017. Abbreviations
MIU
tilit i d MIU: motility indole urease test; et al.: and others; NA: nutrient agar; Mg: mil‑
ligram; sp.: species; mL: milliliter; TSI: triple sugar iodine; MR: methyl red; VP:
Voges Proskauer; w/v: weight by volume; g/L: gram per liter. y
8. Glick BR. Bacteria with ACC deaminase can promote plant growth and
help to feed the world. Microbiol Res. 2014;169(1):30–9. 8. Glick BR. Bacteria with ACC deaminase can promote plant growth and
help to feed the world. Microbiol Res. 2014;169(1):30–9. 9. Nabti E, Schmid M, Hartmann A. Application of halotolerant bacteria
to restore plant growth under salt stress. In: Halophiles. Berlin: Springer
International Publishing; 2015. p. 235–59. 9. Nabti E, Schmid M, Hartmann A. Application of halotolerant bacteria
to restore plant growth under salt stress. In: Halophiles. Berlin: Springer
International Publishing; 2015. p. 235–59. Limitations 2. Rasel HM, Hasan MR, Ahmed B, Miah MS. Investigation of soil and water
salinity, its effect on crop production and adaptation strategy. Int J Water
Res Environ Eng. 2013;5(8):475–81. Extreme halotolerant species as Halomonas elongata
CHR63, Thioalkalivibrio versutus or Sporosarcina pas-
teurii wasn’t detected in the study [32]. The future pros-
pect can be the plantation of transgenic plants in the
coastal area for better agricultural use. The research also
provides insights for adaption and use of soil microor-
ganisms as natural fertilizers after natural calamity struck
the coastal region. Before that, the success of this investi-
gation is not properly accomplished. 3. Haque SA. Salinity problems and crop production in coastal regions of
Bangladesh. Pak J Bot. 2006;38(5):1359–65. 4. Roberts MF. Organic compatible solutes of halotolerant and halophilic
microorganisms. Saline Syst. 2005;1(1):5. 5. Siddikee MA, Sundaram S, Chandrasekaran M, Kim K, Selvakumar G, Sa
T. Halotolerant bacteria with ACC deaminase activity alleviate salt stress
effect in canola seed germination. Appl Biol Chem. 2015;2(58):237–41. f
6. Habib SH, Kausar H, Saud HM. Plant growth-promoting rhizobacteria
enhance salinity stress tolerance in okra through ROS-scavenging
enzymes. Biomed Res Int. 2016;2016:1–10. 7. Siddikee MA, Glick BR, Chauhan PS, Jong Yim W, Sa T. Enhancement of
growth and salt tolerance of red pepper seedlings (Capsicum annuum
L.) by regulating stress ethylene synthesis with halotolerant bacteria
containing 1-aminocyclopropane-1-carboxylic acid deaminase activity. Plant Physiol Biochem. 2011;49(4):427–34. Author details
1 12. Oliveira LC, Ramos PL, Marem A, Kondo MY, Rocha R, Bertolini T, Silveira
MA, Cruz JB, Vasconcellos SP, Juliano L, Okamoto DN. Halotolerant bac‑
teria in the São Paulo Zoo composting process and their hydrolases and
bioproducts. Braz J Microbiol. 2015;46(2):347–54. 1 Biotechnology Program, Department of Mathematics and Natural Sciences,
BRAC University, 66, Mohakhali, Dhaka 1212, Bangladesh. 2 Present Address:
United Surgical (BD) Ltd, Plot# 659‑661, Islampur, Kadda, Gazipur 1702,
Bangladesh. 13. Kothari V, Panchal M, Srivastava N. Hydrocarbon degradation potential
of halotolerant bacteria. In: Scientific study. 2014. http://www.grin.com/
en/e-book/268982/hydrocarbon-degradation-potential-of-halotolerant-
bacteria. Accessed 27 June 2017. Authors’ contributions NT carried out experimental studies; analyzed, interpreted data and drafted
the dissertation. SSR carried out the experimental studies partially and drafted,
edited, finalized and revised the article and coordinated to the publisher. RS
participated in designing, supervising and coordinating the study. All authors
read and approved the final manuscript. 10. Margesin R, Schinner F. Potential of halotolerant and halophilic microor‑
ganisms for biotechnology. Extremophiles. 2001;5(2):73–83. 11. Asad S, Amoozegar MA, Pourbabaee A, Sarbolouki MN, Dastgheib SM. Decolorization of textile azo dyes by newly isolated halophilic and halo‑
tolerant bacteria. Bioresour Technol. 2007;98(11):2082–8. Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data presented within the manuscript.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Funding
This study was self-funded by authors and their institution Department of
Mathematics and Natural Sciences, BRAC University.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
Received: 13 July 2017 Accepted: 23 October 2017 Competing interests
The authors declare that they have no competing interests. Availability of data and materials
All data presented within the manuscript. Consent for publication
Not applicable. Ethics approval and consent to participate
Not applicable. Funding
This study was self-funded by authors and their institution Department of
Mathematics and Natural Sciences, BRAC University. Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. Received: 13 July 2017 Accepted: 23 October 2017 Ethics approval and consent to participate
Not applicable. 19. Aditi FY, Rahman SS, Hossain MM. A study on the microbiological status
of mineral drinking water. Open Microbiol J. 2017;11:31–44. https://doi. org/10.2174/1874285801711010031. Funding This study was self-funded by authors and their institution Department of
Mathematics and Natural Sciences, BRAC University. 20. Reiner K. Catalase test protocol. Washington: American Society
for Microbiology; 2010. http://www.microbelibrary.org/library/
laboratorytest/3226-catalase-test-protocol.htm. Accessed 5 July 2017. Acknowledgements Authors are grateful to Prof. Dr. AAZ Ahmad (Chairperson) and Prof. Naiyyum
Choudhury of Department of Mathematics and Natural Sciences, BRAC
University, Dhaka, for motivation. Their sincere appreciation goes to Dr. M. Mahboob Hossain and Jebunnesa Chowdhury for drawing novel thoughts. And, grateful to Asma Binte Afzal, Nahreen Mirza and Salman Khan Promon for
their unremitting help. 14. Mallick I, Hossain ST, Sinha S, Mukherjee SK. Use of indigenous bacteria
from arsenic contaminated soil for arsenic bioremediation. In: Manage‑
ment of natural resources in a changing environment. Berlin: Springer
International Publishing; 2015. p. 155–65. 15. Jyothi K, Babu KS, Clara NK, Kumar A. Identification and isolation of
hydrocarbon degrading bacteria by molecular characterization. Helix. 2012;2:105–11. Consent for publication 18. Grattieri M, Suvira M, Hasan K, Minteer SD. Halotolerant extremophile
bacteria from the Great Salt Lake for recycling pollutants in microbial fuel
cells. J Power Sources. 2017;356:310–8. Competing interests g
The authors declare that they have no competing interests. 16. Shirazian P, Asad S, Amoozegar MA. The potential of halophilic and
halotolerant bacteria for the production of antineoplastic enzymes:
l-asparaginase and l-glutaminase. EXCLI J. 2016;15:268. 17. Lavik G, Stührmann T, Brüchert V, Van der Plas A, Mohrholz V, Lam P,
Mußmann M, Fuchs BM, Amann R, Lass U, Kuypers MM. Detoxification of
sulphidic African shelf waters by blooming chemolithotrophs. Nature. 2009;457(7229):581–4. Publisher’s Note Physi‑
ological characterization of a halotolerant anoxygenic phototrophic
Fe(II)-oxidizing green-sulfur bacterium isolated from a marine sediment. FEMS Microbiol Ecol. 2017;93(5):fix054. i
32. Aljohny BO. Halophilic bacterium—a review of new studies. Biosci Bio‑
technol Res Asia. 2015;12(3):2061–9. https://doi.org/10.13005/bbra/1874. i
32. Aljohny BO. Halophilic bacterium—a review of new studies. Biosci Bio‑
technol Res Asia. 2015;12(3):2061–9. https://doi.org/10.13005/bbra/1874. 27. Starch hydrolysis test. http://www.vumicro.com/vumie/help/VUMICRO/
Starch_Hydrolysis_Test.htm. Accessed 5 July 2017. 27. Starch hydrolysis test. http://www.vumicro.com/vumie/help/VUMICRO/
Starch_Hydrolysis_Test.htm. Accessed 5 July 2017. Publisher’s Note 21. Shields P, Cathcart L. Oxidase test protocol. Washington: American
Society for Microbiology; 2010. http://www.microbelibrary.org/library/
laboratory-test/3229-oxidase-test-protocol.htm. Accessed 5 July 2017. Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. Received: 13 July 2017 Accepted: 23 October 2017 Received: 13 July 2017 Accepted: 23 October 2017 22. Ferdous TA, Kabir SML, Amin MM, Hossain KMM. Identification and anti‑
microbial susceptibility of salmonella species isolated from washing and
rinsed water of broilers in pluck shops. Int J Anim Vet Adv. 2013;5(1):1–8. Rahman et al. BMC Res Notes (2017) 10:531 Page 6 of 6 23. McDevitt S. Methyl red and Voges–Proskauer test protocols 2009 ASM
microbe library. 2013. http://www.microbelibrary.org/component/
resource/laboratory-test/3204-methyl-red-andvoges-proskauer-test-
protocols.htm. Accessed 5 July 2017. 24. Cappuccino JG, Sherman N. Microbiology a laboratory manual. Benjamin
Cummings: New York; 2005. p. 125–79. 25. Acharya T. Tests for bacterial motility: procedure and results 2015. http://
microbeonline.com/tests-bacterial-motilityprocedure-results/. Accessed
5 July 2017. 26. Rahman SS, Hossain MM, Choudhury N. Effect of various parameters
on the growth and ethanol production by yeasts isolated from natural
sources. Bangladesh J Microbiol. 2013;30(1–2):49–54. https://doi. org/10.3329/bjm.v30i1-2.28453. 27. Starch hydrolysis test. http://www.vumicro.com/vumie/help/VUMICRO/
Starch_Hydrolysis_Test.htm. Accessed 5 July 2017. 28. Analysis of bacteria identification software. http://www.tgw1916.net/
bacteria_logare_desktop.html. Accessed 1 July 2017. 23. McDevitt S. Methyl red and Voges–Proskauer test protocols 2009 ASM
microbe library. 2013. http://www.microbelibrary.org/component/
resource/laboratory-test/3204-methyl-red-andvoges-proskauer-test-
protocols.htm. Accessed 5 July 2017. 29. Arias D, Cisternas LA, Rivas M. Biomineralization of calcium and mag‑
nesium crystals from seawater by halotolerant bacteria isolated from
Atacama Salar (Chile). Desalination. 2017;405:1–9. 24. Cappuccino JG, Sherman N. Microbiology a laboratory manual. Benjamin
Cummings: New York; 2005. p. 125–79. 30. Khansha J, Ranjbaran M, Amoozegar MA. Isolation and identification of
halophilic and halotolerant bacteria from Badab-e Surt Travertine Spring,
Kiasar, Iran, and investigation of calcite biomineralization induction. Geomicrobiol J. 2017:1. 25. Acharya T. Tests for bacterial motility: procedure and results 2015. http://
microbeonline.com/tests-bacterial-motilityprocedure-results/. Accessed
5 July 2017. 26. Rahman SS, Hossain MM, Choudhury N. Effect of various parameters
on the growth and ethanol production by yeasts isolated from natural
sources. Bangladesh J Microbiol. 2013;30(1–2):49–54. https://doi. org/10.3329/bjm.v30i1-2.28453. 31. Laufer K, Niemeyer A, Nikeleit V, Halama M, Byrne JM, Kappler A. Physi‑
ological characterization of a halotolerant anoxygenic phototrophic
Fe(II)-oxidizing green-sulfur bacterium isolated from a marine sediment. FEMS Microbiol Ecol. 2017;93(5):fix054. 31. Laufer K, Niemeyer A, Nikeleit V, Halama M, Byrne JM, Kappler A. Submit your next manuscript to BioMed Central
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Table of Contents Vol 55 (2021)
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In die skriflig/In die Skriflig
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Page i of iv
Table of Contents
Table of Contents
In die Skriflig / In Luce Verbi
ISSN: 1018-6441 (print) | ISSN: 2305-0853 (online)
Vol 55, No 1 (2021)
Book Review
1
A new commentary in the vernacular: Zephaniah: A review
with a translation in isiZulu by Riens de Haan and Thulani Hlela
Bob Wielenga
Original Research
51
Science in service of theology: Gender and sexual orientation
Pieter H. Labuschagne
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2665 | 22 February 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2643 | 22 April 2021
Book Review
3
Original Research
With Calvin, beyond Calvin
Robert Vosloo
A two-tier protestant evaluative framework for cults applied to
KwaSizabantu Mission
Elfrieda Fleischmann, Ignatius W. Ferreira, Francois Muller
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2745 | 29 April 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2686 | 29 March 2021
Book Review
5
Original Research
Leadership according to God’s model: Is it possible to use the
Bible to define God’s leadership?
Marius Nel
Shifting negative migrant categories to encourage embrace and
inclusivity: Perspective from Matthew 22:34–40
Christopher Magezi
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2757 | 26 May 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2664 | 30 March 2021
Book Review
7
Original Research
61
70
81
Nudged by Nathan Following a Lessing line in interfaith theology?
Petrus P. Kruger
Partnering for development: German-South African perspectives
on religion and state cooperation
Alfred R. Brunsdon
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2685 | 22 April 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2768 | 28 May 2021
Book Review
8
Original Research
Listen to these voices!
Elma M. Cornelius
Marriage as a choice or duty: Considering Nigerian Christians’
attitude to singlehood from the biblical perspective
Solomon O. Ademiluka
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2793 | 10 November 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2674 | 22 April 2021
Book Review
10
Original Research
The phenomenon of multilingualism between ancient
perspectives and contemporary developments
Isabella Bonati
The Christian scholar today and Bonhoeffer’s legacy of the
transformative gospel
Aleksandar S. Santrac
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2799 | 10 November 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2678 | 26 April 2021
Original Research
12
Original Research
Church decline: A comparative investigation assessing more than numbers
Ignatius W. Ferreira, Wilbert Chipenyu
In the same boat? Jonah and Jesus as wave-beaten heralds
Alistair I. Wilson
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2645 | 18 January 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2679 | 28 April 2021
Original Research
22
Original Research
White fragility, white supremacy and white normativity make
theological dialogue on race difficult
Kelebogile T. Resane
Introducing a re-reading of Lamentations through the lens of
trauma studies: The challenge of the COVID-19 pandemic
Johan L. Serfontein
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2661 | 25 January 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2688 | 28 April 2021
Original Research
32
Original Research
‘For the husband is the head of the wife’: A contextual re-reading
of Ephesians 5:22–33 among Nigerian Yoruba Christians
Solomon O. Ademiluka
Die Algemene Sinode van die Nederduitse Gereformeerde Kerk
van 1990 en die Gelofte van 1838
Piet J. Strauss
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2613 | 04 February 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2717 | 18 May 2021
Original Research
42
The role of spirituality in facilitating personal development
according to the Pauline corpus
Frederick J. de Beer, Jan A. du Rand
Original Research
Rethinking violence through the narrative of Genesis 4:1–16
Blessing O. Boloje
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2715 | 19 May 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2677 | 17 February 2021
http://www.indieskriflig.org.za
Open Access
90
99
105
113
121
130
Page ii of iv
Original Research
138
Table of Contents
Original Research
Ἐπιφάνεια as wederkomswoord in die Nuwe Testament
H.P. Malan van Rhyn
Critical evaluation of Theonomist eschatology
Morne Diedericks
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2705 | 20 May 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2753 | 01 September 2021
Original Research
148
Original Research
236
243
Living as a diakonos of Christ and pastoral care to the
narcissistically entitled person
Gert Breed
Guidelines towards plausible interpretation of gospel parables
Aniedi M. Akpan, Francois P. Viljoen
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2701 | 27 May 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2742 | 13 September 2021
Original Research
157
‘I Am the LORD your Healer’ Exodus 15:26 ()אנייהוהרפאך: Healing
in the Old Testament and the African (Yoruba) context
David T. Adamo
Original Research
253
Theology and culture in dialogue towards harmonious multireligious and multi-cultural South Africa
Kelebogile T. Resane
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2689 | 27 May 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2764 | 14 September 2021
Original Research
165
Hannah’s prayer for a male child: Interpreting 1 Samuel 1:11 in
the Nigerian context
Solomon O. Ademiluka
Original Research
262
Exploring possibilities of the United Nations High Commissioner
for Refugees’ integration with churches in refugee response
Christopher Magezi
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2719 | 15 June 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2750 | 29 September 2021
Original Research
173
Practical theological perspectives on preaching to listeners
experiencing angst or nothingness within the present reality of
a post-pandemic world
Ferdi P. Kruger
Original Research
274
The insignificant impact of the historical Jesus
Andre van Oudtshoorn
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2756 | 30 September 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2736 | 17 June 2021
Original Research
182
Homiletical perspectives on preaching the truth to post-pandemic
postmodernist listeners with reference to the emotional appeal
of the text
Ferdi P. Kruger, Ben J. de Klerk
Original Research
192
Jy moet jou naaste asook jou vyand liefhê: Liefde in Lukas
6:27–38 en 10:25–37
Jan G. van der Watt
Original Research
200
The lame man at the pool of Bethesda: Christological and doxological
significance of characterisation in John 5
Nathan Hahn, Ernest van Eck
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2774 | 27 October 2021
Original Research
Original Research
Key questions on the outmoded Bernese policy on religion
Matthias G. Inniger, Jacobus M. Vorster, Riaan Rheeder
209
Women in the Bible: What can they teach us about gender equality?
Christin E. Bøsterud
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2754 | 24 August 2021
218
311
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2763 | 29 November 2021
Original Research
Calvin, Bucer and missionary opportunities in times of crises
Stéphan van der Watt
302
Ἀποκάλυψισ en ἀποκαλύπτω as wederkomswoorde in die Nuwe
Testament
H.P. Malan van Rhyn
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2713 | 23 July 2021
Original Research
292
Die tempel-metafoor in Efesiërs 2:11–22 as deel van
identiteitsvorming van die gelowige in-groep
Aletta Vrey
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2760 | 27 October 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2744 | 21 July 2021
Original Research
284
Contextualisation within context: A pedagogical spectrum of six
methodologies
Brian A. DeVries
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2751 | 26 October 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2737 | 23 June 2021
Original Research
Original Research
322
Calvin’s exposition of the sixth commandment as a trajectory in
his catechetical works
Rudolph M. Britz
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2773 | 30 November 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2761 | 27 August 2021
Original Research
227
The Deuteronomic roots of postexilic prophetic eschatology in Malachi
Bob Wielenga
Original Research
Die koninkryk van God in die Ou Testament: ’n Kort oorsig
Daniel F. O’Kennedy
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2787 | 15 December 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2759 | 01 September 2021
http://www.indieskriflig.org.za
Open Access
331
Page iii of iv
Correction
341
Erratum: Auto-memorialisation: Augustus’ Res Gestae as
slanted narrative
Johanna Maria Claassen
Table of Contents
Correction
342
Erratum: Background perspectives on infinity and God
Danie F.M. Strauss
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2789 | 16 November 2021
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2788 | 16 November 2021
Vol 55, No 2 (2021) Special Collection: Gert Breed Festschrift
Editorial
343
Original Research
Gert Breed Festschrift Foreword
Albert J. Coetsee, Francois P. Viljoen
A call for peacemaking: A perspective from the Sermon on the Mount
Francois P. Viljoen
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2806 | 30 November 2021
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2708 | 17 May 2021
Original Research
346
Original Research
Contextual pastoral counselling: Paradigm shifts in practical
theological development since the middle 20th century
Amanda L. du Plessis
Breed se verstaan van ’n bybelse pastorale model: ’n Verkennende,
beskrywende perspektief
Rineé Pretorius
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2696 | 18 March 2021
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2707 | 24 June 2021
Original Research
355
Die behoefte aan apologetiese toerusting reeds by voorskoolse kategese
Henk G. Stoker, G. Jonker Venter
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2722 | 29 October 2021
Original Research
363
Anagnorisis (processing forgiveness): The mystical praxis-space
of diaconal reaching out to the Other/others (the hopeful case
of Joseph and his brothers)
Daniël J. Louw
Original Research
410
418
427
Reconstructing communities and individuals after conflict and
violence: An avant-garde quest for a forgiveness process that
includes koinonia and diakonia
Rudy A. Denton
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2724 | 20 July 2021
Original Research
436
Bekentenisse van ’n YouTube-prediker: Voorlopige outoetnografiese refleksies oor die opname en lei van ’n digitale erediens
Jan-Albert van den Berg
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2651 | 31 March 2021
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2740 | 23 August 2021
Original Research
373
Breed se bybelse pastorale model Skriftuurlik begrond in 2 Petrus
1:3–11: ’n Eksegetiese toeligting
Douw G. Breed
443
Liturgy as an anti-racist praxis for Reformed Churches in South Africa
Eugene Baron, Rantoa Letšosa
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2673 | 31 March 2021
Original Research
Original Research
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2709 | 27 August 2021
382
A more comprehensive comprehension and appropriate
application: An answer to dwindling faith commitment from
the book of Hebrews
Albert J. Coetsee
Original Research
453
Peace is not the absence of war but the presence of a
relationship founded by God – שׁלֹום
ָ (shalom) in Isaiah and Micah
Chris van der Walt
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2728 | 02 September 2021
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2704 | 26 April 2021
Original Research
392
Fight, flight or faith: A pastoral model for spiritual coping
Steve le Roux, George Lotter
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2700 | 29 April 2021
Original Research
461
Verspreiding van persoonlikheidstipes by predikante in die
gereformeerde Kerke van Suid-Afrika – om risikogroepe vir
diensverlating te identifiseer
Gerhardus J. Niemann
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2727 | 03 November 2021
Original Research
401
Mentoring fathers who grapple with fatherhood issues in a
faith-based context: A pastoral-theological review
Fazel E. Freeks
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2698 | 30 April 2021
Correction
Erratum: Breed se bybelse pastorale model Skriftuurlik begrond
in 2 Petrus 1:3–11: ’n Eksegetiese toeligting
Douw G. Breed
In die Skriflig/In Luce Verbi | Vol 55, No 2 | a2795 | 23 November 2021
http://www.indieskriflig.org.za
Open Access
472
Page iv of iv
Table of Contents
Vol 55, No 3 (2021) Special Collection: Bible 200
Editorial
473
Foreword Bible 200
Francois P. Viljoen, Albert J. Coetsee
Original Research
538
Eksegese van 2 Petrus 3:1–2 en die hermeneuse daarvan met
spesifieke verwysing na standpunte van die sogenaamde Nuwe Ateïste
Douw G. Breed
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2808 | 15 December 2021
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2720 | 17 June 2021
Original Research
476
The impact of a Biblical Fatherhood Programme for faith communities
in the Christiana district: A reflective and community engagement strategy
Fazel E. Freeks
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2680 | 30 March 2021
Original Research
485
By everyone and for everyone: The principles underlying ‘justice’
in Deuteronomy 16:18–20
Albert J. Coetsee
Original Research
548
To what extent did the Bible translations into indigenous
languages of Southern Africa produced since 1966 reflect the
purpose of providing meaning-based translations?
Jacobus A. van Rooy
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2747 | 24 June 2021
Original Research
555
Die 2020-direkte vertaling in Afrikaans: Waarom en hoe?
Gert J.C. Jordaan
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2654 | 31 March 2021
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2758 | 25 June 2021
Original Research
496
The (mis)interpretation of the Bible in South Africa: Towards a
better hermeneutic
Bradley M. Trout
Original Research
561
‘Ek is Kain’: ’n Hermeneutiek van weerloosheid as ’n antwoord op
die dekoloniale diskoers
Gerrie F. Snyman
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2748 | 05 August 2021
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2738 | 13 July 2021
Original Research
504
Oral-based Bible translation: A contextualised model for the
nomadic Himba people of southern Africa
Karen J. Floor
Original Research
571
Translation types and niche translations: Comparing five
Afrikaans translations
Sebastian J. Floor
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2752 | 23 September 2021
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2743 | 24 November 2021
Original Research
510
Die betroubaarheid van die apostels en hulle getuienis volgens
2 Petrus 1:3-4 en die betekenis daarvan vir die geskrifte van die
Nuwe Testament: ‘n Eksegetiese studie
Douw G. Breed
Original Research
582
Die invloed van die lojaliteite en onderliggende vertaalfilosofie
van die vertaler op die vertaalkeuses, met verwysing na die
Afr2020 Bybelvertaling
Roelie van der Spuy
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2703 | 17 May 2021
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2755 | 15 December 2021
Original Research
519
Skrifgebruik en Skrifgesag in die postmodernistiese konteks
Johan Janse van Rensburg
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2687 | 18 May 2021
Original Research
529
Translating psalms for Africa today: Involving the community and
transmitting through performance
June F. Dickie
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2726 | 26 May 2021
http://www.indieskriflig.org.za
Correction
594
Erratum: The impact of a Biblical Fatherhood Programme for
faith communities in the Christiana district: A reflective and
community engagement strategy
Fazel E. Freeks
In die Skriflig/In Luce Verbi | Vol 55, No 3 | a2794 | 23 November 2021
Reviewer Acknowledgement
In die Skriflig/In Luce Verbi | Vol 55, No 1 | a2826 | 22 December 2021
Open Access
595
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https://link.springer.com/content/pdf/10.1007/s10853-022-07325-2.pdf
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English
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Corrosion performance of a steel surface modified by a robust graphene-based superhydrophobic film with hierarchical roughness
|
Journal of materials science
| 2,022
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cc-by
| 8,607
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Corrosion performance of a steel surface modified
by a robust graphene-based superhydrophobic film
with hierarchical roughness M. E. Mohamed1,*
and B. A. Abd-El-Nabey1 M. E. Mohamed1,*
and B. A. Abd-El-Nabey1 1Faculty of Science, Chemistry Department, Alexandria University, P. O. Box 426, Alexandria 21321, Egypt Received: 31 January 2022
Accepted: 10 May 2022
Published online:
4 June 2022
The Author(s) 2022 Received: 31 January 2022
Accepted: 10 May 2022
Published online:
4 June 2022
The Author(s) 2022 METALS & CORROSION
J Mater Sci (2022) 57:11376–11391
Metals & corrosion METALS & CORROSION
J Mater Sci (2022) 57:11376–11391
Metals & corrosion J Mater Sci (2022) 57:11376–11391 Handling Editor: Maude Jimenez. ABSTRACT Potentiostatic deposition of cobalt film and cobalt-graphene, Co-G, composite,
followed by modification with low surface energy stearic acid (SA), was used to
fabricate superhydrophobic films on a steel substrate successfully. A scanning
electron microscope was used to analyze the surface morphology of the pre-
pared superhydrophobic cobalt film modified by stearic acid, Co-SA, and the
cobalt-graphene film modified by stearic acid, Co-G-SA. The findings show that
both the fabricated films have micro-nanostructures. The Co-G-SA film shows a
higher roughness due to the network structures of graphene and so exhibits
higher superhydrophobicity. The Fourier transform infrared spectrophotometer,
FTIR, results confirm the formation of Co-SA and Co-G-SA films on the steel
surface. The wettability of the prepared films shows that they exhibit super-
hydrophobicity, where the Co-SA and Co-G-SA films have contact angles of
155 and 158, respectively. The Potentiodynamic polarization results show that
the value of the corrosion current density for steel coated with Co-SA
(0.7094 lA) is lower than that of bare steel (0.1457 mA), while the coated steel
with Co-G-SA film has the lowest value (0.1732 lA). The electrochemical
impedance spectroscopy, EIS, results show that the charge transfer resistance for
steel coated with Co-SA is 38 times that of bare steel, while steel coated with Co-
SA is 57 times that of bare steel. Potentiodynamic polarization and EIS results
show that the prepared Co-G-SA film superhydrophobic films exhibit higher
corrosion resistance. Co-G-SA film has higher mechanical stability (maintains
superhydrophobicity until 900 abrasion cycles), chemical stability (has super-
hydrophobicity in the pH range 1–13), and long-term stability (retains super-
hydrophobicity after 30 days in a 0.5 M NaCl solution) in 0.5 M NaCl solution. Handling Editor: Maude Jimenez. https://doi.org/10.1007/s10853-022-07325-2 https://doi.org/10.1007/s10853-022-07325-2 Address correspondence to E-mail: elshahatchemist93@gmail.com Introduction films typically show low substrate bonding strength
and low hydrophobic efficiency, which decrease their
applications significantly. Fortunately, doping with
metals or non-metals also enhanced substrate adhe-
sion and uniformity; moreover, some novel charac-
teristics can be found based on retaining the original
excellent efficiency [29]. Because of their significance in fundamental science
and
commercial
applications,
superhydrophobic
surfaces have sparked great interest [1–5]. Superhy-
drophobic surfaces have applications in diverse
fields, such as anti-icing [6], oil–water separation [7],
corrosion
resistance
[8],
self-cleaning
[9],
drag
reduction [10], and antifouling technologies [11]. Superhydrophobic surfaces have been fabricated
using various techniques, such as electrospinning
[12], etching [6], sol–gel process [13], electrodeposi-
tion [14], laser fabrication [15], and electrochemical
anodization [16]. However, most of these approaches
require extreme conditions that restrict their practical
aspects, such as complicated chemical treatments,
high-cost materials, and multi-step manufacturing. Due to its low cost, flexibility, ambient temperature
operation, and ability to monitor electrodeposition
parameters, electrodeposition is an excellent tech-
nique for creating artificial superhydrophobic sur-
faces. Thus
far,
the
commercialization
of
superhydrophobic surfaces has been significantly
reduced due to their low surface chemical and
mechanical
stabilities
and
the
fragility
of
their
microscopic nanostructures characteristics [17]. The
mechanical
strength
and
durability
of
superhy-
drophobic coatings have been a major focus of recent
research [17, 18]. It is essential to improve the
mechanical abrasion resistance and chemical stability
of superhydrophobic surfaces in order to use them in
industrial applications. y
Steel materials are used in a wide range of indus-
tries because of their high mechanical strength. However, steel structures have poor corrosion resis-
tance due to their thermodynamic instability when
exposed to extreme temperature, humidity, and pH
[30]. Many techniques have been used to protect the
steel surfaces; an important of them is the fabrication
of superhydrophobic films, which greatly improve
the
corrosion
resistance
of
steel
[31]. Superhy-
drophobic coatings were fabricated on different
substrates using different techniques [32–37]. Philip
et al. studied the fabrication of robust superhy-
drophobic coating on ferritic steel with the self-
cleaning ability and superior corrosion resistance
using
a
template-free
one-step
electrodeposition
method [32]. Siddaiah et al. studied the steel coatings
by nickel (Ni) as well as Ni–graphene (Ni–Gr) [36]. On a Si substrate, Yan et al. created carbon-based
films
with
excellent
self-cleaning
and
corrosion
resistance [37]. Dong et al. J Mater Sci (2022) 57:11376–11391 11377 Chemical stability A water droplet with various pH values (pH = 1–13)
was placed on the prepared superhydrophobic sur-
faces, and the CAs and SAs at each pH were mea-
sured. Sodium hydroxide and sulphuric acid have
been used to control the pH value of the water
droplet. After electrodeposition, the Co and Co-G films
were washed with distilled water and dried for 24.0 h
at room temperature, immersed in 0.01 M ethanolic
solution of stearic acid for 30 min, and then removed
from the solution and left to dry at room conditions. After that, the as-prepared Co film modified by
stearic acid, Co-SA, and the Co-G film modified by Characterization Sodium
hydroxide,
anhydrous
ethanol,
sodium
chloride, sulfuric acid, cobalt chloride, boric acid,
sodium carbonate, sodium dodecyl sulfate and stea-
ric acid were purchased from Sigma-Aldrich. The surface morphology of prepared films was
studied using a scanning electron microscope, SEM
(model JSM-IT 200). A Fourier transform infrared
spectrophotometer, FTIR (FTIR LX 18–5255 Perkin
Elmer), was used to investigate the chemical com-
position of the prepared films. The spectra were
recorded
in
the
wave
number
range
of
4000–500 cm-1. X-ray diffraction (XRD) was done
with
monochromatic
Cu
K
radiation
(k = 0.154056 nm) by an X-ray diffractometer (Bruker
D2 phaser). An optical contact angle meter (OCALS
plus) was used to calculate the water contact angle,
CA, and sliding angle, SA, with 5 ll water droplets. The recorded CAs and SAs are the averages of three
measurements taken at various locations on the
sample’s surface. A coating thickness gauge (FN
Type CT-100) measured the thickness of the as-pre-
pared superhydrophobic films. The reported film
thickness is the average of three measurements done
at different positions of the prepared sample. Sample preparation A steel plate with dimensions of 2.0 9 2.0 9 0.1 cm
and chemical composition of (wt%): S, 0.04; C, 0.21;
Mn, 2.5; Si, 0.35; P, 0.04 and Fe, 96.86 was used as a
working electrode. The substrate was rubbed with
emery paper of various grades before electrodeposi-
tion, beginning with coarse (150 grade) and pro-
gressing in steps to the finest (1200 grade). Then it
was degreased for 30.0 min in an aqueous solution
containing 20 g L-1 Na2CO3, 15 g L-1 NaOH and 8 g
L-1 sodium dodecyl sulfate, followed by pickling and
oxide removal in 2.0 M H2SO4 for 1.0 min, and finally
rinsing with distilled water and ethanol. The elec-
trodeposition was done in an aqueous solution con-
taining CoCl2 (200 g L-1) and H3BO3 (30 g L-1) using
a potential of 3.0 V and deposition time equals
20.0 min; then, the potential was increased to 7.0 V
for a time of 1.0 min. The two-step electrodeposition
was used to fabricate various superhydrophobic
films [39–41]. In the first deposition step (at low
potential), the crystal growth rate of cobalt is faster
than the nucleation rate, so a coarse deposit of Co is
formed. In the second electrodeposition step (at high
potential), the nucleation rate of cobalt is faster than
the crystal growth rate, so a nano deposit of Co is
formed. For the manufacture of Co film, a platinum
rod was used as the anode, and for the manufacture
of Co film doped with graphene, Co-G film, a gra-
phite rod was used as the anode, where the graphite
is
electrochemically
exfoliated,
giving
graphene,
which incorporated into the deposited layer of Co
[42, 43]. Experimental stearic acid, Co-G-SA, were exposed to different
evaluations and characterization techniques. Introduction [4] studied the fabrication
of Ni–B4C superhydrophobic composite coatings at
Q235 steel by electrodeposition and investigated its
corrosion performance. Zhang et al. [38] investigated
the preparation and performance of a biomimetic
flower-like superhydrophobic coating on X80 pipe-
line steel using a static self-assembly method. Abd-
El-Nabey et al. [14] studied the construction of robust
superhydrophobic films on steel surfaces and studied
their corrosion performance, mechanical and chemi-
cal stability. Cobalt (Co) is commonly used in aerospace, ship-
building wear-resistant, automotive, corrosion resis-
tance, high-strength alloys, catalysis, solar energy
absorption and magnetic recording [19, 20]. The
material’s internal characteristics and morphology
decide these different characteristics [21, 22]. As a
result, controlling the development of unique cobalt
nanostructures has become a critical issue in the
materials fabrication industry. Because of its strength,
single atomic layer thickness, chemical inertness, and
impermeability to most gases, graphene is thought to
be a good material for coatings [23–26]. Graphene can
be
processed
as
nanoplatelets,
nanosheets,
and
functionalized graphene using various methodolo-
gies, such as chemical or electrochemical exfoliation,
chemical vapor deposition, and single-crystal SiC
crystal cleavage and annealing [27, 28]. Carbon-based This study aims to fabricate Co film and Co film
doped with in situ prepared graphene by electro-
chemical exfoliation method on the steel surface. Then, the as-prepared films were modified with
stearic acid to fabricate superhydrophobic surfaces. To the best of our knowledge, this is the first study on
constructing a superhydrophobic Co film doped with
in situ produced graphene. The effect of graphene
doping on the wettability, mechanical and chemical
stability, long-term stability in 0.5 M NaCl, and cor-
rosion resistance properties of a superhydrophobic
cobalt
film
modified
by
stearic
acid
were
investigated. 11378 J Mater Sci (2022) 57:11376–11391 Mechanical abrasion The abrasion test has been used to analyze the
mechanical stability of the as-prepared films. Sand-
paper (1800 mesh and length of 30 mm) has been
used as an abrasion surface. The prepared superhy-
drophobic film is oriented to face the sand surface,
and a pressure of 5.0 kPa was applied to the super-
hydrophobic film. Corrosion tests All electrochemical experiments were carried out in a
three-electrode cell containing a 0.5 M NaCl aqueous
solution at room temperature using a frequency SEM and wettability results SEM and wettability results One of the most outstanding parameters for investi-
gating superhydrophobic properties is the surface
morphology, so the SEM technique has been used to
study the topography of the prepared superhy-
drophobic films on the steel substrate. Figure 1a
shows the micrograph of steel coated by Co; it is clear
that the electrodeposited cobalt has micro-nanos-
tructures of papillae structure. Figure 1b shows the
micrograph of the magnified papillae structure,
which has a hierarchical roughness. Figure 1c shows
the micrograph of steel coated by Co-SA; it shows
stearic acid’s white structures covering the elec-
trodeposited micro-nano-papillae structure. Results and discussion The thickness of the Co-SA and Co-G-SA films are
19 lm
and
25 lm,
respectively. These
results
demonstrate that doping of Co film with graphene
increases the thickness of the prepared film and
improves the roughness and so shows higher super-
hydrophobicity after modification with the stearic
acid as a low surface energy material. Based on the
Cassie–Baxter state [46], much air can be easily stored
in the micro-nanostructures of the Co-G-SA film. Furthermore, the wettability of the superhydrophobic
coated steel by Co-G-SA is superior to several pre-
viously recorded values [47, 48]. 11379 J Mater Sci (2022) 57:11376–11391 response analyzer potentiostat (PARSTAT, USA). The
reference and counter electrodes were an Ag/AgCl
electrode and a platinum rod. The bare steel and steel
covered by superhydrophobic Co-SA and Co-G-SA
films have been used as working electrodes. The
operating procedure was given in previous work
[44]. The working electrode was placed in a cell
containing 0.5 M NaCl solution that was opened to
the environment at room temperature and left for
30 min before electrochemical measurements were
taken to establish the equilibrium potential. The fre-
quency range of the electrochemical impedance
spectroscopy
(EIS)
measurements
was
0.01 B f B 1.0 9 105 with an applied potential signal
amplitude of 10 mV around the equilibrium poten-
tial. The polarization curves were measured starting
from cathodic potential (-300 mV) to anodic potential
(? 450 mV) around the equilibrium potential at a
0.5 mV/sec scan rate. respectively. The value of contact and sliding angles
for Co-SA film are 155 and 3, respectively, while the
contact and sliding angles for Co-G-SA are 158 and
2, respectively. The contact angle image of the water
droplet on the prepared superhydrophobic surfaces
is shown in Fig. 2. These results indicate that the grafting of the Co
film at the steel surfaces leads to an enhancement of
the hydrophilic character of the bare steel, and the
contact angle is decreased. While, when the Co film is
doped with graphene, Co-G film, the contact angle
shows a greater decrease reflecting higher hydro-
philic characteristics. When the prepared Co and Co-
G films are modified with stearic acid, superhy-
drophobic
surfaces
were
obtained
with
greater
superhydrophobic characteristics of Co-G-SA film
due to the presence of graphene, which enhances the
surface roughness. These results can be discussed on
the basis that as the surface roughness increases, the
hydrophilic surfaces become more hydrophilic, while
the hydrophobic surfaces show higher hydrophobic-
ity [45]. FTIR and XRD results Figure 3 and Table 1 show the FTIR spectra and band
assignments of stearic acid powder and steel coated
by Co, Co-SA, Co-G, and Co-G-SA. The spectrum for
steel coated with cobalt shows a peak at 576 cm-1
characteristics of the Co–O stretching vibration. In
addition, the weak absorptions peaks at 3342 and
1660 cm-1 correspond to the stretching and bending
vibration modes of the –OH group; this will prove the
absorption of water by the deposited nanostructure
of cobalt at the steel surface[49, 50]. The spectrum for
the powder of stearic acid shows the characteristic
peaks of the stearic acid. The peaks at 2866 cm-1 and
2944 cm-1 are assigned to the stretching vibrations of Figure 1d shows the micrograph of steel coated by
Co-G; it is clear that the graphene forms a network
layer of micro-nano-papillae structure; the porosity of
the network gives a higher roughness, hierarchical
roughness, to the formed layer. Figure 1e depicts the
micrograph of steel coated by Co-G-SA; it is clear that
stearic acid, white structures, graft the prepared film. The values of the contact angles of bare steel and steel
coated with Co and Co-G films are 58, 21 and 15, 11380 J Mater Sci (2022) 57:11376–11391 gure 1 SEM images of the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and
Co-G-SA film. Figure 1 SEM images of the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and
e C
G SA fil the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and Figure 1 SEM images of the prepared films a Co film, b magnified image for papillae structure in Co film, c Co-SA film, d Co-G film and
e Co-G-SA film. peak at 3363 cm-1 is attributed to the bond tension
vibration of O–H; the peak at 1151 cm-1 corresponds
to the hydroxyl groups of the graphene. The peak at
1651 cm-1 is characteristic of the double bond’s gra-
phene ring. The peak at 1078 cm-1 is attributed to the
C–O–C group [53]. –CH2 groups. The peak at 1719 cm-1 is due to the
stretching vibration of the carbonyl group of stearic
acid. FTIR and XRD results The peak at 1314 cm-1 is attributed to the
bending
vibration
of
-OH,
while
the
peak
at
1477 cm-1 is due to the bending vibration of C–H
[51, 52]. The spectrum for steel coated by Co-G
depicts the characteristic peaks of the graphene. The 11381 J Mater Sci (2022) 57:11376–11391 Figure 2 Contact angle graphs of steel coated with a Co-SA film, and b Co-G-SA film. Figure 2 Contact angle graphs of steel coated with a Co-SA film, and b Co-G-SA film. Co3O4, and its sharpness indicates that the deposited
cobalt has good crystallinity [54, 55]. For Co-SA
coating, there are six diffraction peaks at 2h values of
30.7, 36.4, 38.1, 44.5, 59.1 and 64.9, which cor-
respond to faced cubic centered, fcc, of Co3O4 (JCPDS
No. 00-042-1467)[56]. The (311) plane has the highest
intensity of the three peaks, indicating the preferred
crystal orientation, with higher periodicity than the
other orientations [57]. The Co-G-SA film has the
same six diffraction peaks as the Co-SA film, with one
greater diffraction peak at 2h values of 24.2, corre-
sponding to graphene [58]. The graphene peak is
broad, showing that graphene has a small particle
size. Co3O4, and its sharpness indicates that the deposited
cobalt has good crystallinity [54, 55]. For Co-SA
coating, there are six diffraction peaks at 2h values of
30.7, 36.4, 38.1, 44.5, 59.1 and 64.9, which cor-
respond to faced cubic centered, fcc, of Co3O4 (JCPDS
No. 00-042-1467)[56]. The (311) plane has the highest
intensity of the three peaks, indicating the preferred
crystal orientation, with higher periodicity than the
other orientations [57]. The Co-G-SA film has the
same six diffraction peaks as the Co-SA film, with one
greater diffraction peak at 2h values of 24.2, corre-
sponding to graphene [58]. The graphene peak is
broad, showing that graphene has a small particle
size. Figure 3 FTIR spectra of stearic acid powder and steel coated by
Co, Co-G, Co-G-SA, and Co-SA. Mechanical abrasion resistance Figure 3 FTIR spectra of stearic acid powder and steel coated by
Co, Co-G, Co-G-SA, and Co-SA. The superhydrophobic surfaces are susceptible to
mechanical
abrasion. Improvement
of
superhy-
drophobic coatings’ abrasion resistance has become
the primary concern for their industrial applications
[59]. The steel coated by Co-SA spectrum shows two
peaks at 2919 cm-1 and 2850 cm-1 attributed to
asymmetry and symmetry vibration of –CH2– of the
stearic acid. The stretching vibration of C=O is
responsible for the peak at 1702 cm-1, while the
bending vibration of C-H is responsible for the peak
at 1468 cm-1. The bending vibration absorption peak
of –OH is at 1330 cm-1. The stearic acid peaks at
around 2922, 2852, and 1703 cm-1 in the spectrum for
steel coated with Co-G-SA suggest that the deposited
graphene is modified by stearic acid. Abrasion testing was used to assess the resistance
of the prepared superhydrophobic films to mechan-
ical abrasion. The variations in water contact and
sliding angles of the prepared superhydrophobic
films as a function of the number of abrasion cycles
are shown in Fig. 5. Increased abrasion cycles result in a decrease in
contact angle values and an increase in sliding angle
values, as seen in the graph. The superhydrophobic
Co-SA film exhibits superhydrophobicity until 500
abrasion cycles; however, the superhydrophobic Co-
G-SA film maintains superhydrophobicity until 900 The composition and crystal orientation of steel
coated with Co-SA and Co-G-SA films were deter-
mined using the XRD technique. The XRD patterns of
Co-SA and Co-G-SA films are depicted in Fig. 4. Mechanical abrasion resistance The
diffraction peaks imply that the deposited film is J Mater Sci (2022) 57:11376–11391 Table 1 FTIR band assignments for stearic acid powder and steel coated by Co, Co-G, Co-SA, and Co-G –SA films
Film type
Band wave numbers, cm-1
Assignment
Co film
3342
stretching vibration of-OH group
1660
bending vibration of-OH group
576
Co–O stretching vibration
Stearic acid powder
2944
asymmetric stretching vibration of CH2
2866
symmetric stretching vibration of CH2
1719
stretching vibration of the carbonyl group of stearic acid
1477
bending vibration of C-H
1314
bending vibration of -OH
Co-G film
3363
bond tension vibration of O–H
2925
asymmetric stretching vibration of CH2
2854
symmetric stretching vibration of CH2
1651
the double bond’s graphene ring
1460
Bending Vibrations of CH
1151
hydroxyl groups of the graphene
1078
C–O–C group of the graphene
Co-SA film
2919
asymmetric stretching vibration of CH2
2850
symmetric stretching vibration of CH2
1702
stretching vibration of C = O
1468
bending vibration of C-H
1330
bending vibration absorption peak of -OH
Co-G –SA
2922
asymmetric stretching vibration of CH2
2852
symmetric stretching vibration of CH2
1703
stretching vibration of C=O
1471
bending vibration of C–H
1336
bending vibration absorption peak of –OH Figure 4 XRD patterns of steel coated by a Co-SA film and
b Co-G-SA film. Figure 6 shows the SEM micrographs of steel
coated with Co-SA and Co-G-SA films after the
abrasion test. The micro-nano-papillae structure was
destroyed, and the density of stearic acid (white
structures) on the surface was decreased, so the sur-
face lost its superhydrophobic characteristics. The
contact angle shape of the water droplet on the pre-
pared superhydrophobic surfaces after the abrasion
test is shown in Fig. 7. The abrasion resistance of the
superhydrophobic coated steel by Co-G-SA is supe-
rior
to
several
previously
recorded
values
[32–35, 47, 60, 61]. Chemical stability Figure 4 XRD patterns of steel coated by a Co-SA film and
b Co-G-SA film. To demonstrate that the prepared superhydrophobic
film can be used in the industrial sector, a chemical
stability test must be conducted. Figure 8 shows the
relationship between each contact and sliding angles
and the pH of the water droplets. abrasion cycles, which may be due to the higher
adhesion of the low surface energy stearic acid to the
rough structure of the Co-G composite than that of
the Co film alone. J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 The Co-SA film has superhydrophobicity only in
the pH range 3–11, while the Co-G-SA film has
superhydrophobicity in the pH range 1–13, where the
CAs are always greater than 150, and the SAs are less than 10. The two essential factors necessary for the
fabrication of superhydrophobic films are low surface
energy and surface roughness. So, the aggressive
acidic and basic liquids could reduce the density of
hydrophobic groups on the surface and destroys the
micro/nanostructures of the surface, and so the sur-
face
loses
its
superhydrophobic
characteristics
[9, 45, 62, 63]. The chemical stability of the superhy-
drophobic coated steel by Co-G-SA is superior to
several previously recorded values [47, 64]. Figure 5 CAs and SAs as a function of the number of abrasion
cycles for coated steel by a Co-SA film, and b Co-G-SA film. 11383 Long term stability The
manufacture
of
surfaces
with
long-term
stable superhydrophobicity remains a major problem
that restricts superhydrophobic surface industrial
applications. By measuring the contact angle every
2 days for 30 days, the long-term stability of the
prepared Co-SA and Co-G-SA superhydrophobic
films on steel substrate in 0.5 M NaCl solution was
investigated. Figure 9 shows that the Co-SA film
exhibit superhydrophobicity and has a contact angle
greater than 150; after immersion for 20 days, the
contact angle becomes smaller than 150, and the
surface loses the superhydrophobic property. The Figure demonstrates that the Co-G-SA film
retains superhydrophobicity after 30 days in a 0.5 M
NaCl solution, implying that doping the Co-SA film
with graphene improves the long-term stability of the
superhydrophobic
film
prepared. The
prepared
superhydrophobic films will lose their superhy-
drophobic characteristics after a definite immersion
time in a 0.5 M NaCl solution as the Cl- ions attack Figure 5 CAs and SAs as a function of the number of abrasion
cycles for coated steel by a Co-SA film, and b Co-G-SA film. Figure 6 SEM images of the superhydrophobic films a Co-SA film and b Co-G-SA film after abrasion test. Figure 6 SEM images of the superhydrophobic films a Co-SA film and b Co-G-SA film after abrasion test. 11384 J Mater Sci (2022) 57:11376–11391 Figure 7 Contact angle images of steel coated with a Co-SA film and b Co-G-SA film after abrasion test. Figure 7 Contact angle images of steel coated with a Co-SA film and b Co-G-SA film after abrasion test. Figure 7 Contact angle images of steel coated with a Co-SA film and b Co-G-SA film after abrasion test. the
films
and
could
decrease
the
hydrophobic
groups’ density on the surface and destroy the
micro/nanostructures of the surface, and so the sur-
face loses its superhydrophobicity. The enhanced
mechanical, chemical, and long-term stability of Co-
G-SA layer in a 0.5 M NaCl solution is due to the
synergistic effect of superhydrophobicity and the
high chemical and mechanical stability, imperme-
ability, hydrophobicity, and chemical inertness of
graphene [58, 65–69]. The long-term stability of the
superhydrophobic
coated
steel
by
Co-G-SA
is
Figure 8 Variation of pH values of a water droplet with the CAs
and SAs of the coated steel by a Co-SA film and b Co-G-SA film. Figure 9 CAs and SAs as a function of immersion time of coated
steel by a Co-SA film, and b Co-G-SA film. Corrosion resistance behaviour It is clear that the icorr. value for steel coated with
Co-SA (0.7094 lA) is lower than that of bare steel
(0.1457 mA); this can be attributed to coated steel’s
superhydrophobic
behaviour. The
trapped
air
around the microstructures can reduce the contact
area between the coated steel and the solution,
resulting in a significant reduction in the icorr [75]. The steel coated with Co-G-SA film has a greater
reduction in both the contact area between the coated
steel and the medium and the icorr. value (0.1732 lA)
because the presence of graphene increases the
superhydrophobicity of the prepared Co-G-SA film
as well as the high mechanical and chemical stability,
hydrophobicity, impermeability, and chemical inert-
ness of graphene [58, 65–69]. So, the inhibition effi-
ciency of steel coated by Co-G-SA is higher than that
of Co-SA. The value of Ecorr for steel coated by Co-G-
SA is nobler than Co-SA, which is extremely noble
than bare steel. The corrosion resistance of bare steel and superhy-
drophobic coated steel by Co-SA and Co-G-SA has
been investigated using the potentiodynamic polar-
ization technique. Figure 10 shows the potentiody-
namic
polarization
curves
of
bare
steel
and
superhydrophobic coated steel in a 0.5 M NaCl
aqueous solution. It is obvious that the cathodic polarization plots
show a limiting diffusion current, IL, due to the
reduction of oxygen according to Eq. (1). O2 þ 2H2O þ 4e ! 4OH
ð1Þ ð1Þ O2 þ 2H2O þ 4e ! 4OH
ð1Þ Thus, the cathodic process is controlled by mass
transport. The rapid formation of corrosion products,
in the case of bare steel, or formation of passive layer,
in the case of the prepared superhydrophobic coated
steel, on the electrode surface hinders the develop-
ment of an ideal anodic Tafel region [72, 73]. J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 superior
to
several
previously
recorded
values
[5, 18, 70, 71]. superior
to
several
previously
recorded
values
[5, 18, 70, 71]. %P ¼
io
corr: icorr:
nio
corr:
100
ð2Þ %P ¼
io
corr: icorr:
nio
corr:
100 ð2Þ io
corr. and icorr. are the corrosion current density for
bare steel and superhydrophobic coated steel. io
corr. and icorr. are the corrosion current density for
bare steel and superhydrophobic coated steel. Corrosion resistance behaviour
Potentiodynamic polarization results Corrosion resistance behaviour Long term stability Figure 8 Variation of pH values of a water droplet with the CAs
and SAs of the coated steel by a Co-SA film and b Co-G-SA film. Figure 9 CAs and SAs as a function of immersion time of coated
steel by a Co-SA film, and b Co-G-SA film. the
films
and
could
decrease
the
hydrophobic
groups’ density on the surface and destroy the
micro/nanostructures of the surface, and so the sur-
face loses its superhydrophobicity. The enhanced
mechanical, chemical, and long-term stability of Co-
G-SA layer in a 0.5 M NaCl solution is due to the synergistic effect of superhydrophobicity and the
high chemical and mechanical stability, imperme-
ability, hydrophobicity, and chemical inertness of
graphene [58, 65–69]. The long-term stability of the
superhydrophobic
coated
steel
by
Co-G-SA
is synergistic effect of superhydrophobicity and the
high chemical and mechanical stability, imperme-
ability, hydrophobicity, and chemical inertness of
graphene [58, 65–69]. The long-term stability of the
superhydrophobic
coated
steel
by
Co-G-SA
is 11385 Electrochemical impedance spectroscopy results Table 2 displays the result of the bare steel and
superhydrophobic coated steel’s potentiodynamic
polarization parameters, including corrosion current
density, icorr., corrosion potential, Ecorr., and protec-
tion efficiency, % P. Equation 2 is used to measure
the protection efficiency [74] The Nyquist and Bode plots of bare steel and
superhydrophobic coated steel in 0.5 M NaCl solu-
tion are shown in Fig. 11. At high frequency, the
Nyquist plots show a depressed capacitive semicir-
cle, accompanied by a diffusion tail at low frequency. The interfacial charge transfer reaction is responsible
for the depressed capacitive semicircle of the Nyquist
plots at high frequencies [76]. The diffusion tail at low
frequency is due to the mass transport process. These
results indicate that steel coated by Co-SA, which
shows high charge transfer resistance compared to
bare steel, has larger charge transfer resistance due to
the presence of a protective superhydrophobic layer. Steel coated by Co-G-SA shows the highest capacitive
semicircle, so it has the highest protection efficiency. The superhydrophobic coated steel blocks the active
corrosion sites and limits the diffusion of the corro-
sive species, such as Cl- and H2O, into the surface of
steel metal. Figure 10 Potentiodynamic polarization curves for bare steel and
superhydrophobic coated steel in 0.5 M NaCl solution. According to Fig. 11b, the Bode plots for prepared
superhydrophobic coated steel in 0.5 M NaCl solu-
tion show higher impedance magnitudes at the low
frequency than bare steel. This indicates the protec-
tive action of the prepared superhydrophobic coats Figure 10 Potentiodynamic polarization curves for bare steel and
superhydrophobic coated steel in 0.5 M NaCl solution. 11386 J Mater Sci (2022) 57:11376–11391 Deposit
-Ecorr (mV)
ba (mV/decade)
-bc (mV/decade)
icorr (mA cm-2)
%P
Bare steel
473.5
155.5
183.5
0.1457064
–
Steel ? Co-SA film
259.1
62.9
138.9
0.0007094
99.5
Steel ? Co-G-SA film
222.5
61.4
132.7
0.0001732
99.6 Table 2 The potentiodynamic
polarization parameters for the
bare steel and the prepared
superhydrophobic coated steel
in 0.5 M NaCl solution polarization parameters for the
bare steel and the prepared
superhydrophobic coated steel
in 0.5 M NaCl solution
p
cor
Bare steel
473.5
Steel ? Co-SA film
259.1
Steel ? Co-G-SA film
222.5
Figure
11 Nyquist
and
Bode
plots
of
bare
steel
and
superhydrophobic coated steel in 0.5 M NaCl solution. Steel ? Co-G-SA film
222.5 on the steel substrate. The phase angle plot, Fig. Conclusion 1. superhydrophobic Co-SA and Co-G-SA films
were fabricated on the steel substrate. 1. superhydrophobic Co-SA and Co-G-SA films
were fabricated on the steel substrate. 2. The doping of the superhydrophobic Co film
with graphene greatly enhances the superhy-
drophobicity, and the contact angle increases
from 155 to 158. On the contrary, the steel coated by superhy-
drophobic films has a micro-nanostructure covered
by adsorbed hydrophobic material. The roughness of
the superhydrophobic coatings allows air to be
trapped easily within the valleys between the peaks
of the rough surface. Consequently, the aggressive
ion species such as Cl- in the electrolyte or corrosive
environments can rarely attack the underlying sur-
face
due
to
trapped
air’s
obstructive
influence
[32, 45]. In fact, the air trapped on the superhy-
drophobic surface acts as a passivation layer between
the substrate and the corrosive environment. The
enhanced corrosion resistance for steel coated by Co-
G-SA films is due to the synergistic effect of super-
hydrophobicity and the high mechanical and chemi-
cal stability, impermeability, hydrophobicity, and
chemical
inertness
of
graphene. The
schematic 3. The doping of the superhydrophobic Co film
with
graphene
also
greatly
improves
steel’s
chemical, mechanical, long-term stability and
corrosion resistance behaviour in 0.5 M NaCl
solution. J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 Table 3 The impedance parameters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution
Deposit
Rs (X cm2)
CPEf 9 10–6
(sn X-1 cm2)
n1
Rf (X cm2)
CPEdl 9 10–6
(sn X-1 cm2)
n2
Rct (X cm2)
%P
Bare steel
2.28
281
0.78
56
278
0.77
73.2
–
Steel ? Co-SA
3.95
91
0.76
214
88
0.74
2795
97.38
Steel ? Co-G-SA
4.22
59
0.68
273
53
0.72
4165
98.24
Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. Table 3 The impedance parameters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution
Deposit
Rs (X cm2)
CPEf 9 10–6
(sn X-1 cm2)
n1
Rf (X cm2)
CPEdl 9 10–6
(sn X-1 cm2)
n2
Rct (X cm2)
%P
Bare steel
2.28
281
0.78
56
278
0.77
73.2
–
Steel ? Co-SA
3.95
91
0.76
214
88
0.74
2795
97.38
Steel ? Co-G-SA
4.22
59
0.68
273
53
0.72
4165
98.24
Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. meters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution le 3 The impedance parameters for the bare steel and superhydrophobic coated steel in 0.5 M NaCl solution Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared superhydrophobic films. Figure 13 Schematic representation of the proposed mechanism for corrosion protection of the prepared Mechanism of anti-corrosion performance representation of the proposed mechanism for cor-
rosion protection of the prepared superhydrophobic
films is shown in Fig. 13. representation of the proposed mechanism for cor-
rosion protection of the prepared superhydrophobic
films is shown in Fig. 13. Bare steel freely interacts with surrounding water
molecules; the water molecules can be adsorbed to
the steel surface. Along with water molecules, chlo-
ride ions can also get adsorbed to the steel surface
and form [FeClOH]-, which will lead to severe cor-
rosion of the uncoated steel. So, water and Cl- ions
easily reach the metal surface and initiate corrosion
[32]. Electrochemical impedance spectroscopy results 11c,
shows two times constant at low and moderate fre-
quencies. The time constant appearing in the low-
frequency range was due to the protective superhy-
drophobic film or the unprotective corrosion prod-
ucts in the case of bare steel. The time constant
appearing at the moderate frequency was attributed
to the electrical double layer [77–79]. The impedance parameters were determined using
the Zsimpwin software to fit the Nyquist plots to the
equivalent circuit shown in Fig. 12. The equivalent
circuit includes solution resistance, Rs, film resis-
tance, Rf, film constant phase element, CPEf, charge
transfer resistance, Rct, and double-layer constant
phase element, CPEdl. Table 3 shows the EIS param-
eters of bare steel and superhydrophobic coated steel. Equation (3) is used to calculate the protection effi-
ciency [74] %P ¼
Rct Ro
ct
=Rct
100
ð3Þ ð3Þ Rct
o and Rct are the charge transfer resistance for
the bare steel and superhydrophobic coated steel. It is
clear that each of Rct, and %P increase in the fol-
lowing order, bare steel \ steel ? Co-SA \ steel ? Co-G-SA, and so increasing the corrosion resistance
in the same order. The corrosion resistance of the
superhydrophobic coated steel by Co-G-SA compos-
ite is superior to several previously reported values
[5, 71, 80]. Figure
12 The
equivalent
circuit
model
used
to
fit
the
experimental Nyquist plots for steel in 0.5 M NaCl solution. Figure
11 Nyquist
and
Bode
plots
of
bare
steel
and
superhydrophobic coated steel in 0.5 M NaCl solution. Figure
12 The
equivalent
circuit
model
used
to
fit
the
experimental Nyquist plots for steel in 0.5 M NaCl solution. 11387 Funding Open access funding provided by The Science,
Technology & Innovation Funding Authority (STDF)
in cooperation with The Egyptian Knowledge Bank
(EKB). This research did not receive any specific 11388 J Mater Sci (2022) 57:11376–11391 grant from funding agencies in the public, commer-
cial, or not-for-profit sectors. Appl Surf Sci 562:150192. https://doi.org/10.1016/j.apsusc. 2021.150192 Appl Surf Sci 562:150192. https://doi.org/10.1016/j.apsusc. 2021.150192 [6]
Hou W, Shen Y, Tao J et al (2020) Anti-icing performance of
the superhydrophobic surface with micro-cubic array struc-
tures fabricated by plasma etching. Colloids Surfaces A
Physicochem Eng Asp 586:124180. https://doi.org/10.1016/
j.colsurfa.2019.124180 Declarations Conflict of interest
The authors declare that there is
no conflict of interest. [7]
Chu Z, Feng Y, Seeger S (2015) Oil/water separation with
selective superantiwetting/superwetting surface materials. Angew Chemie-Int Ed 54:2328–2338. https://doi.org/10.10
02/anie.201405785 Open Access
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directly from the copyright holder. To view a copy of
this licence, visit http://creativecommons.org/licen
ses/by/4.0/. [8]
Mohamed ME, Abd-El-Nabey BA (2022) Superhydrophobic
cobalt-graphene composite for the corrosion protection of
copper bipolar plates in proton exchange membrane fuel
cells. J Electrochem Energy Convers Storage 19:031007. h
ttps://doi.org/10.1115/1.4053782 [9]
Xu S, Wang Q, Wang N, Zheng X (2019) Fabrication of
superhydrophobic green surfaces with good self-cleaning,
chemical stability and anti-corrosion properties. J Mater Sci
54:13006–13016. https://doi.org/10.1007/s10853-019-0378
9-x [10] Li Z, Marlena J, Pranantyo D et al (2019) A porous super-
hydrophobic surface with active air plastron control for drag
reduction and fluid impalement resistance. J Mater Chem A
7:16387–16396. https://doi.org/10.1039/c9ta02745a [11] Qin L, Hafezi M, Yang H et al (2019) Constructing a dual-
function surface by microcasting and nanospraying for effi-
cient drag reduction and potential antifouling capabilities. Micromachines 10:490. https://doi.org/10.3390/mi10070490 J Mater Sci (2022) 57:11376–11391 11389 J Mater Sci (2022) 57:11376–11391 blocks for carbon-based materials. Small 6:711–723. http
s://doi.org/10.1002/smll.200901934 [17] Verho T, Bower C, Andrew P et al (2011) Mechanically
durable superhydrophobic surfaces. Adv Mater 23:673–678. https://doi.org/10.1002/adma.201003129 [30] Musa AY, Kadhum AAH, Mohamad AB et al (2010) On the
inhibition of mild steel corrosion by 4-amino-5-phenyl-4H-
1, 2, 4-trizole-3-thiol. Corros Sci 52:526–533. https://doi. org/10.1016/j.corsci.2009.10.009 [18] Liang T, Yuan H, Li C et al (2020) Corrosion inhibition
effect of nano–SiO2 for galvanized steel superhydrophobic
surface. Surf Coat Technol. https://doi.org/10.1016/j.surfcoa
t.2020.126673 [31] Kumar CMP, Venkatesha TV, Chandrappa KG (2012) Effect
of surfactants on co-deposition of B 4C nanoparticles in Zn
matrix by electrodeposition and its corrosion behavior. Surf
Coat Technol 206:2249–2257. https://doi.org/10.1016/j.surfc
oat.2011.09.075 [19] Vazquez-Arenas J, Altamirano-Garcia L, Treeratanaphitak T
et al (2012) Co-Ni alloy electrodeposition under different
conditions of pH, current and composition. Electrochim Acta
65:234–243. https://doi.org/10.1016/j.electacta.2012.01.050 [32] Rasitha TP, Vanithakumari SC, George RP, Philip J (2019)
Template-free one-step electrodeposition method for fabri-
cation of robust superhydrophobic coating on ferritic steel
with self-cleaning ability and superior corrosion resistance. Langmuir 35:12665–12679. https://doi.org/10.1021/acs.lang
muir.9b02045 [20] Yang J, Bao C, Zhu K et al (2014) High catalytic activity and
stability of nickel sulfide and cobalt sulfide hierarchical
nanospheres on the counter electrodes for dye-sensitized
solar cells. Chem Commun 50:4824–4826. https://doi.org/
10.1039/c4cc00001c [21] An ZG, Zhang JJ, Pan SL (2010) Simple synthesis and
characterization of highly ordered sisal-like cobalt super-
structures. Mater Chem Phys 123:795–800. https://doi.org/
10.1016/j.matchemphys.2010.05.062 [33] Rasitha TP, Vanithakumari SC, Nanda Gopala Krishna D
et al (2022) Facile fabrication of robust superhydrophobic
aluminum surfaces with enhanced corrosion protection and
antifouling properties. Prog Org Coatings 162:106560. http
s://doi.org/10.1016/j.porgcoat.2021.106560 [22] Cheng
G
(2014)
Facile
synthesis
of
leaf-like
cobalt
microstructures
at low temperature. Micro Nano Lett
9:312–314. https://doi.org/10.1049/mnl.2014.0027 [34] Tp R, Philip J (2022) Optimal condition for fabricating
mechanically durable superhydrophobic titanium surface by
rapid breakdown anodization: self cleaning and bouncing
characteristics. Appl Surf Sci 585:152628. https://doi.org/10. 1016/j.apsusc.2022.152628 [23] Montemor MF (2014) Functional and smart coatings for
corrosion protection: a review of recent advances. Surf
Coatings Technol 258:17–37. https://doi.org/10.1016/j.surfc
oat.2014.06.031 [24] Chen-Yang YW, Yang HC, Li GJ, Li YK (2005) Thermal
and anticorrosive properties of polyurethane/clay nanocom-
posites. J Polym Res 11:275–283. https://doi.org/10.1007/
s10965-005-3982-8 [35] Manoj TP, Rasitha TP, Vanithakumari SC et al (2020) A
simple, rapid and single step method for fabricating super-
hydrophobic titanium surfaces with improved water bounc-
ing
and
self
cleaning
properties. Appl
Surf
Sci
512:145636. References [1]
Khadka A, Subeshan B, Asmatulu R (2021) Studies on de-
icing and anti-icing of carbon fiber-reinforced composites for
aircraft surfaces using commercial multifunctional perma-
nent
superhydrophobic
coatings. J
Mater
Sci
56(4):3078–3094. https://doi.org/10.1007/s10853-02 [1]
Khadka A, Subeshan B, Asmatulu R (2021) Studies on de-
icing and anti-icing of carbon fiber-reinforced composites for
aircraft surfaces using commercial multifunctional perma-
nent
superhydrophobic
coatings. J
Mater
Sci
56(4):3078–3094. https://doi.org/10.1007/s10853-02 [12] Zhang G, Wang P, Zhang X et al (2019) Preparation of
hierarchically structured PCL superhydrophobic membrane
via
alternate
electrospinning/electrospraying
techniques. J Polym Sci Part B Polym Phys 57:421–430. https://doi.org/
10.1002/polb.24795 [2]
Zhu G, Zhao Y, Liu L et al (2021) Facile fabrication and
evaluation of self-healing Zn-Al layered double hydroxide
superhydrophobic coating on aluminum alloy. J Mater Sci
56:14803–14820. https://doi.org/10.1007/s10853-021-0624
7-9 [2]
Zhu G, Zhao Y, Liu L et al (2021) Facile fabrication and
evaluation of self-healing Zn-Al layered double hydroxide
superhydrophobic coating on aluminum alloy. J Mater Sci
56:14803–14820. https://doi.org/10.1007/s10853-021-0624
7-9 [13] Vidal K, Go´mez E, Goitandia AM et al (2019) The synthesis
of a superhydrophobic and thermal. Coatings 9:1–33 [14] Abd-El-Nabey BA, Ashour M, Aly A, Mohamed M (2022)
Fabrication of robust superhydrophobic nickel films on steel
surface with high corrosion resistance mechanical and
chemical stability. J Eng Mater Technol 144:021007. http
s://doi.org/10.1115/1.4052768 [3]
Mohamed ME, Mahgoub FM, Ragheb DM, Abdel-Gaber
AM (2021) Novel and facile method for fabrication of robust
superhydrophobic film on copper surface and its chemical,
mechanical, and corrosion performance. Surf Eng. https://d
oi.org/10.1080/02670844.2021.1951502 [3]
Mohamed ME, Mahgoub FM, Ragheb DM, Abdel-Gaber
AM (2021) Novel and facile method for fabrication of robust
superhydrophobic film on copper surface and its chemical,
mechanical, and corrosion performance. Surf Eng. https://d
oi.org/10.1080/02670844.2021.1951502 [15] Yang Z, Tian Y, Zhao Y, Yang C (2019) Study on the fab-
rication of superhydrophobic surface on Inconel Alloy via
nanosecond laser ablation. Materials (Basel). https://doi.org/
10.3390/ma12020278 [4]
Dong S, Yang Y, Liang T et al (2021) Construction and
corrosion resistance of Ni-B4C superhydrophobic composite
coatings on Q235 steel. Surf Coat Technol. https://doi.org/
10.1016/j.surfcoat.2021.127551 [16] Saji VS (2020) Superhydrophobic surfaces and coatings by
electrochemical anodic oxidation and plasma electrolytic
oxidation. Adv Colloid Interface Sci 283:102245. https://doi. org/10.1016/j.cis.2020.102245 [5]
Lv XS, Qin Y, Liang H et al (2021) A facile method for
constructing a superhydrophobic zinc coating on a steel
surface with anti-corrosion and drag-reduction properties. J Mater Sci (2022) 57:11376–11391 https://doi.org/10.1016/j.apsusc.2020.145636 [25] Jena G, Anandkumar B, Vanithakumari SC et al (2020)
Graphene oxide-chitosan-silver composite coating on Cu-Ni
alloy with enhanced anticorrosive and antibacterial proper-
ties suitable for marine applications. Prog Org Coatings
139:105444. https://doi.org/10.1016/j.porgcoat.2019.105444 [36] Siddaiah A, Kumar P, Henderson A et al (2019) Surface
energy and tribology of electrodeposited Ni and Ni-graphene
coatings on steel. Lubricants. https://doi.org/10.3390/lubrica
nts7100087 [26] Jena G, George RP, Philip J (2021) Fabrication of a robust
graphene oxide-nano SiO2-polydimethylsiloxane composite
coating on carbon steel for marine applications. Prog Org
Coat 161:106462. https://doi.org/10.1016/j.porgcoat.2021. 106462 [37] Yan Q, Zhou S, Ma L et al (2019) Approach to excellent
superhydrophobicity and corrosion resistance of carbon-
based films by graphene and cobalt synergism. Surf Interface
Anal 51:152–163. https://doi.org/10.1002/sia.6546 [38] Zhang W, Fan S, Wang Y et al (2021) Preparation and per-
formance of biomimetic superhydrophobic coating on X80
pipeline steel for inhibition of hydrate adhesion. Chem Eng J
419:129651. https://doi.org/10.1016/j.cej.2021.129651 [27] Mattevi C, Kim H, Chhowalla M (2011) A review of
chemical vapour deposition of graphene on copper. J Mater
Chem 21:3324–3334. https://doi.org/10.1039/c0jm02126a [39] Hang T, Hu A, Ling H et al (2010) Super-hydrophobic nickel
films with micro-nano hierarchical structure prepared by
electrodeposition. Appl Surf Sci 256:2400–2404. https://doi. org/10.1016/j.apsusc.2009.10.074 [28] Lee DS, Riedl C, Krauss B et al (2008) Raman spectra of
epitaxial graphene on SiC and of epitaxial graphene trans-
ferred to SiO2. Nano Lett 8:4320–4325. https://doi.org/10. 1021/nl802156w [29] Compton OC, Nguyen ST (2010) Graphene oxide, highly
reduced graphene oxide, and graphene: versatile building [40] Esmailzadeh S, Khorsand S, Raeissi K, Ashrafizadeh F
(2015) Microstructural evolution and corrosion resistance of 11390 J Mater Sci (2022) 57:11376–11391 organic solvents. J Nanopart Res. https://doi.org/10.1007/s1
1051-014-2561-5 organic solvents. J Nanopart Res. https://doi.org/10.1007/s1
1051-014-2561-5 superhydrophobic electrodeposited nickel films. Surf Coat
Technol 283:337–346. https://doi.org/10.1016/j.surfcoat.201
5.11.005 [53] Valencia C, Valencia CH, Zuluaga F et al (2018) Synthesis
and application of scaffolds of chitosan-graphene oxide by
the freeze-drying method for tissue regeneration. Molecules
23:2651. https://doi.org/10.3390/molecules23102651 [41] Khorsand S, Raeissi K, Ashrafizadeh F (2014) Corrosion
resistance and long-term durability of superhydrophobic
nickel film prepared by electrodeposition process. Appl Surf
Sci 305:498–505. https://doi.org/10.1016/j.apsusc.2014.03. 123 [54] Prabaharan DDM, Sadaiyandi K, Mahendran M, Sagadevan
S (2017) Precipitation method and characterization of cobalt
oxide nanoparticles. Appl Phys A Mater Sci Process. http
s://doi.org/10.1007/s00339-017-0786-8 [42] Yu P, Lowe SE, Simon GP, Zhong YL (2015) Electro-
chemical exfoliation of graphite and production of functional
graphene. Curr Opin Colloid Interface Sci 20:329–338. J Mater Sci (2022) 57:11376–11391 h
ttps://doi.org/10.1016/j.cocis.2015.10.007 [55] Jadhav CH, Pisal KB, Chavan AR et al (2020) Electro-
chemical supercapacitive performance study of spray pyr-
olyzed cobalt oxide film. Mater Today Proc 43:2742–2746. h
ttps://doi.org/10.1016/j.matpr.2020.06.477 [43] Su CY, Lu AY, Xu Y et al (2011) High-quality thin graphene
films from fast electrochemical exfoliation. ACS Nano
5:2332–2339. https://doi.org/10.1021/nn200025p [56] Ikhuoria EU, Omorogbe SO, Sone BT, Maaza M (2018)
Bioinspired shape controlled antiferromagnetic Co3O4 with
prism like-anchored octahedron morphology: a facile green
synthesis using manihot esculenta crantz extract. Sci Technol
Mater. https://doi.org/10.1016/j.stmat.2018.02.003 [44] Mohamed ME, Abd-El-Nabey BA (2020) Facile and eco-
friendly method for fabrication of superhydrophobic surface
on copper metal. ECS J Solid State Sci Technol. https://doi. org/10.1149/2162-8777/ab9dc7 [57] Zhou J, Wang H (2003) The physical meanings of 5 basic
parameters for an X-ray diffraction peak and their applica-
tion. Chin J Geochem 22:38–44. https://doi.org/10.1007/bf
02831544 [45] Barati Darband G, Aliofkhazraei M, Khorsand S et al (2020)
Science and engineering of superhydrophobic surfaces:
review of corrosion resistance, chemical and mechanical
stability. Arab J Chem 13:1763–1802. https://doi.org/10.10
16/j.arabjc.2018.01.013 [58] Shang W, Li J, Rabiei Baboukani A et al (2020) Study on the
relationship between graphene dispersion and corrosion
resistance of graphene composite film. Appl Surf Sci
511:145518. https://doi.org/10.1016/j.apsusc.2020.145518 [46] Cassie ABD, Baxter S (1944) Wettability of porous surfaces. Trans Faraday Soc 40:546–551 [47] Ma L, Wang J, Zhang Z et al (2021) Preparation of a
superhydrophobic TiN/PTFE composite film toward self-
cleaning and corrosion protection applications. J Mater Sci
56:1413–1425. https://doi.org/10.1007/s10853-020-05364-1 [59] Nguyen-Tri P, Tran HN, Plamondon CO et al (2019) Recent
progress in the preparation, properties and applications of
superhydrophobic nano-based coatings and surfaces: a
review. Prog Org Coat 132:235–256. https://doi.org/10.101
6/j.porgcoat.2019.03.042 [48] Forooshani HM, Aliofkhazraei M, Bagheri H (2019) Fabri-
cation of hierarchical dual structured (HDS) nickel surfaces
and their corrosion behavior. J Alloys Compd 784:556–573. https://doi.org/10.1016/j.jallcom.2019.01.079 [60] Wang H, Zhu Y, Hu Z et al (2016) A novel electrodeposition
route for fabrication of the superhydrophobic surface with
unique self-cleaning, mechanical abrasion and corrosion
resistance properties. Chem Eng J 303:37–47. https://doi. org/10.1016/j.cej.2016.05.133 [49] Zhang F, Yuan C, Lu X et al (2012) Facile growth of
mesoporous Co 3O 4 nanowire arrays on Ni foam for high
performance electrochemical capacitors. J Power Sources
203:250–256. https://doi.org/10.1016/j.jpowsour.2011.12. 001 [61] Tan C, Li Q, Cai P et al (2015) Fabrication of color-con-
trollable superhydrophobic copper compound coating with
decoration performance. Appl Surf Sci 328:623–631. J Mater Sci (2022) 57:11376–11391 [64] Du C, He X, Tian F et al (2019) Preparation of superhy-
drophobic steel surfaces with chemical stability and corro-
sion. Coatings
9:398. https://doi.org/10.3390/coating
s9060398 [73] McCafferty E (2005) Validation of corrosion rates measured
by
the
Tafel
extrapolation
method. Corros
Sci
47:3202–3215. https://doi.org/10.1016/j.corsci.2005.05.046 [74] Fetouh HA, Abd-El-Nabey B, Goher YM, Karam MS (2018)
An Electrochemical investigation in the anticorrosive prop-
erties of silver nanoparticles for the acidic corrosion of
aluminium. J Electrochem 24:89–100 [65] Zhu H, Yue L, Zhuang C et al (2016) Fabrication and
characterization of self-assembled graphene oxide/silane
coatings
for
corrosion
resistance. Surf
Coat
Technol
304:76–84. https://doi.org/10.1016/j.surfcoat.2016.07.002 [75] Ou J, Liu M, Li W et al (2012) Corrosion behavior of
superhydrophobic surfaces of Ti alloys in NaCl solutions. Appl Surf Sci 258:4724–4728. https://doi.org/10.1016/j.aps
usc.2012.01.066 [66] Zhong F, He Y, Wang P et al (2019) Self-assembled gra-
phene oxide-graphene hybrids for enhancing the corrosion
resistance of waterborne epoxy coating. Appl Surf Sci
488:801–812. https://doi.org/10.1016/j.apsusc.2019.05.321 [76] Ghiamati Yazdi E, Ghahfarokhi ZS, Bagherzadeh M (2017)
Protection of carbon steel corrosion in 3.5% NaCl medium
by aryldiazonium grafted graphene coatings. J Chem
41:12470–12480. https://doi.org/10.1039/c7nj01655g [67] Chu JH, Tong LB, Zhang JB et al (2019) Bio-inspired gra-
phene-based coatings on Mg alloy surfaces and their inte-
grations of anti-corrosive/wearable performances. Carbon N
Y
141:154–168. https://doi.org/10.1016/j.carbon.2018.09. 047 [77] Nady H, El-Rabiei MM, Samy M (2017) Corrosion behavior
and electrochemical properties of carbon steel, commercial
pure titanium, copper and copper–aluminum–nickel alloy in
3.5% sodium chloride containing sulfide ions. Egypt J Pet
26:79–94. https://doi.org/10.1016/j.ejpe.2016.02.008 [68] Singh BP, Nayak S, Nanda KK et al (2013) The production
of a corrosion resistant graphene reinforced composite
coating on copper by electrophoretic deposition. Carbon N Y
61:47–56. https://doi.org/10.1016/j.carbon.2013.04.063 [78] Mahdavian M, Naderi R (2011) Corrosion inhibition of mild
steel in sodium chloride solution by some zinc complexes. Corros Sci 53:1194–1200. https://doi.org/10.1016/j.corsci.2
010.12.013 [69] Ding S, Xiang T, Li C et al (2017) Fabrication of self-
cleaning superhydrophobic nickel/graphene hybrid film with
improved corrosion resistance on mild steel. Mater Des
117:280–288. https://doi.org/10.1016/j.matdes.2016.12.084 [79] Berrani A, Benassaoui H, Zouarhi M et al (2018) Analytical
& bioanalytical electrochemistry. Anal Bioanal Electrochem
10:1299–1316 [70] Polyakov NA, Botryakova IG, Glukhov VG et al (2021)
Formation and anticorrosion properties of superhydrophobic
zinc coatings on steel. Chem Eng J 421:127775. https://doi. J Mater Sci (2022) 57:11376–11391 http
s://doi.org/10.1016/j.apsusc.2014.12.025 [50] Allaedini G, Muhammad A (2013) Study of influential fac-
tors in synthesis and characterization of cobalt oxide
nanoparticles. J Nanostruct Chem. https://doi.org/10.1186/
2193-8865-3-77 [62] Wang J, Wu Y, Cao Y et al (2020) Influence of surface
roughness on contact angle hysteresis and spreading work. Colloid Polym Sci 298:1107–1112. https://doi.org/10.1007/
s00396-020-04680-x [51] Zeng YX, Zhong XW, Liu ZQ et al (2013) Preparation and
enhancement of thermal conductivity of heat transfer oil-
based MoS2 nanofluids. J Nanomater. https://doi.org/10.11
55/2013/270490 [63] Guo M, Kang Z, Li W, Zhang J (2014) A facile approach to
fabricate a stable superhydrophobic film with switchable
water adhesion on titanium surface. Surf Coat Technol
239:227–232. https://doi.org/10.1016/j.surfcoat.2013.11.047 [52] Luo S, Yang D, Feng J, Ng KM (2014) Synthesis and
application
of non-agglomerated
ITO nanocrystals via
pyrolysis of indium-tin stearate without using additional 11391 J Mater Sci (2022) 57:11376–11391 J Mater Sci (2022) 57:11376–11391 org/10.1016/j.cej.2020.127775 [80] Qu JE, Yu C, Nie C et al (2021) A new environmentally
friendly approach to prepare superhydrophobic colored
stainless steel surface for decoration, anti-corrosion and self-
cleaning. J Mater Sci 56:854–869. https://doi.org/10.1007/
s10853-020-05293-z [71] Varshney P, Mohapatra SS, Kumar A (2021) Durable and
regenerable superhydrophobic coating on steel surface for
corrosion protection. J Bio-Tribo-Corrosion 7:1–11. https://d
oi.org/10.1007/s40735-021-00518-3 Publisher’s Note
Springer Nature remains neutral with
regard to jurisdictional claims in published maps and
institutional affiliations. [72] Flitt HJ, Schweinsberg DP (2005) Evaluation of corrosion
rate from polarisation curves not exhibiting a Tafel region. Corros Sci 47:3034–3052. https://doi.org/10.1016/j.corsci.2
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Hubungan Faktor-faktor Alih Fungsi Lahan Padi Sawah dan Perbedaan Tingkat Penerimaan Usahatani Petani Di Kecamatan Keliling Danau Kabupaten Kerinci
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Jurnal ilmiah sosio ekonomika bisnis/Jurnal ilmiah sosio-ekonomika bisnis
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1) Alumni Jurusan Agribisnis Program Studi Agribisnis Fakultas Pertanian Universitas Jambi
2) Staf Pengajar Jurusan Agribisnis Universitas Jambi
Email: peni.agri@yahoo.co.id
ABSTRAK Tujuan penelitian ini adalah untuk mengetahui hubungan faktor-faktor yang mempengaruhi alih fungsi
lahan sawah pada tingkat petani dan mengetahui perbedaan penerimaan usahatani petani yang
mengalihkan lahan sebelum dan sesudah alih fungsi di Desa Koto Dian dan Koto Tuo. Faktor-faktor alih
fungsi adalah tingkatusia, lama pendidikan, luaslahan, jumlah tanggungan, dan pengalaman bertani. Metode pengambilan data diambil dari data primer dan sekunder. Data diolah dan dianalisis
menggunakan analisis deskriptif untuk menggambarkan hubungan faktor-faktor alih fungsi lahan sawah,
kemudian digunakan analisis uji beda dua rata-rata untuk mengetahui perbedaan penerimaan usahatani
petani yang mengalih fungsikan lahan sawah sebelum dan sesudah alih fungsi lahan. Dengan jumlah
responden sebanyak 37 petani yang mengalihkan sebagian lahan dan 20 petani responden yang
mengalihkan semua lahan. Hasil analisis deskriptif yang menggambarkan hubungan faktor-faktor alih
fungsi lahan adalah semakin tinggi tingkat usia, maka semakin tinggi tingkat alih fungsi lahan; semakin
rendah tingkat pendidikan seorang petani, maka semakin tinggi tingkat alih fungsi lahan; semakin luas
kepemilikan lahan, maka peluang petani untuk mengalih fungsikan lahannya lebihkecil; semakin banyak
jumlah tanggungan yang harus ditanggung, maka alih fungsilahan akan semakin tinggi; dan semakin
lama pengalaman dalam berusahatani, maka akan semakin berat dalam pengambilan keputusan untuk
alih fungsi lahan. Kata kunci: hubungan faktor-faktor, alih fungsi lahan sawah, penerimaan usahatani. Key words: relationship factors, diversion of paddy fields, farming acceptance. Peniarti1), Rosyani2), Elwamendri3) 1) Alumni Jurusan Agribisnis Program Studi Agribisnis Fakultas Pertanian Universitas Jambi
2) Staf Pengajar Jurusan Agribisnis Universitas Jambi
Email: peni.agri@yahoo.co.id
ABSTRAK JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 PENDAHULUAN Indonesia merupakan negara agraris dimana pertanian merupakan basis utama perekonomian
nasional.Sebagian besar masyarakat Indonesia masih menggantungkan hidupnya pada sektor pertanian. Sektor pertanian telah memberikan sumbangan besar dalam pembangunan nasional, seperti
peningkatan ketahanan nasional, penyerapan tenaga kerja, peningkatan pendapatan masyarakat,
peningkatan Pendapatan Domestik Regional Bruto (PDRB) , perolehan devisa melalui ekspor-impor, dan
penekanan inflasi. Sektor pertanian, peternakan, kehutanan, dan perikanan merupakan sektor kedua
setelah sektor industri pengolahan yang memberikan kontribusi besar terhadap peningkatan PDRB
Indonesia.PDRB merupakan salah satu indikator yang menggambarkan pertumbuhan ekonomi suatu
wilayah atau negara.Dalam menghadapi pembangunan, sektor pertanian masih terdapat banyak
persoalan besar yang harus diselesaikan, salah satu diantaranya adalah permasalahan alih fungsi lahan
pertanian menjadi lahan non-pertanian yang saat ini terus mengalami peningkatan. Menurut Utomo
(1992) Alih fungsi lahan atau konversi lahan adalah berubahnya satu penggunanaan lahan ke
penggunanaan lahan lainnya.Banyak faktor baik internal maupun eksternal yang mempengaruhi
terjadinya alih fungsi lahan.Alih fungsi lahan pertanian sebenarnya bukan masalah baru. Sejalan dengan
adanya peningkatan jumlah penduduk serta meningkatnya kebutuhan infrastruktur seperti, perumahan,
jalan, industri, perkantoran, dan bangunan lain menyebabkan kebutuhan akan lahan meningkat. Selain
itu, pertumbuhan ekonomi yang tinggi menyebabkan pertumbuhan yang sangat cepat di beberapa
sektor ekonomi. Pertumbuhan tersebut juga membutuhkan lahan yang lebih luas sehingga terjadi peningkatan
kebutuhan lahan untuk pembangunan, sementara ketersediaan lahan relatif tetap menyebabkan
persaingan dalam pemanfaatan lahan. Kebanyakan lahan yang dialih fungsikan umumnya adalah lahan-
lahan pertanian karena land rent (sewa lahan). Sewa ekonomi lahan (land rent) mengandung pengertian
nilai ekonomi yang diperoleh oleh satu bidang lahan bila lahan tersebut digunakan untuk kegiatan
proses produksi. Land rent lahan pertanian relatif lebih tinggi penggunaannya untuk non-pertanian
dibandingkan dengan lahan pertanian yang dikelola oleh petani. Kabupaten Kerinci merupakan wilayah
di Provinsi Jambi yang penghasil komuditas padi tertinggi dibanding dengan kabupaten–kabupaten lain. Hampir sebagian luas wilayah Kabupaten Kerinci merupakan lahan sawah. Namun Kabupaten Kerinci
merupakan wilayah yang rawan akan masalah lahan, terutama karena adanya pemukiman penduduk. Adanya pertambahan jumlah penduduk Kabupaten Kerinci setiap tahun serta pemekaran wilayah
menyebabkan kebutuhan baik pemukiman maupun perumahan terus meningkat ( Badan Pusat Statistik
Kabupaten Kerinci 2015). Fenomena alih fungsi lahan pertanian merupakan dampak dari transformasi sruktur ekonomi
(pertanian ke industri), dan demografi (pedesaan ke perkotaan) yang pada akhirnya mendorong
transformasi sumberdaya lahan dari pertanian ke non-pertanian. Persoalan ini harus dicarikan
solusipemecahannya karena melihat juga dampak yang ditimbulkan dari alih fungsi lahan ini dapat
merugikan petani khususnya dan masyarakat Indonesia pada umumnya. ABSTRACT The purpose of this study was to determine the relationship of the factors that affect the
transfer of paddy fields function at the farm level and determine differences in farming
acceptance of farmers who divert land before and after conversion in Koto Dian and Koto Tuo
village. Diversion factors are age, length of education, land area, number of dependents, and
farming experience. The method of collecting data taken from primary and secondary data. Data were processed and analyzed using descriptive analysis to describe the relationship
factors over paddy field function, and then used the analysis of two different test average to
determine differences in farming acceptance of farmers who divert paddy field before and
after land diversion. The number of respondents as many as 37 farmers who divert some of
the land and 20 farmers respondent divert all land.Descriptive analysis that describes the
relationship of the factors of land diversion is the higher the age, the higher the rate of land
diversion; the lower the level of education of a farmer, the higher the rate of land diversion;
more extensive land holdings, then the smaller chances of farmers to divert their land; more
the number of dependents must be assured, then land diversion will be higher; and the longer
experience in farming, more difficult in the decision to land diversion. - 1 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 METODE PENELITIAN Penelitian dilaksanakan didua desa, yakni Desa Koto Dian dan Desa Koto Tuo Kecamatan Keliling
Danau Kabupaten Kerinci. Objek penelitian ini adalah petani yang mengalihkan sebagian lahan sawah
dan yang mengalihkan semua lahan sawahnya.Penelitian dilakukan dengan mengambil sampel Desa
Koto Dian dan Desa Koto Tuo. Pemilihan lokasi dilakukan secara sengaja (purposive) atau disebut juga
judgemental samplingkarena wilayah tersebut merupakan wilayah yang mengalami alih fungsi lahan
tertinggi di Kecamatan Keliling Danau . Sumber data Jenis data yang digunakan dalam penelitian ini adalah data primer dan data
sekunder.Data primer digunakan untuk mengetahui faktor-faktor yang mempengaruhi alih fungsi lahan
di tingkat petani, serta dampak alih fungsi lahan pertanian terhadap pendapatan usahatani petani.Data
primer diperoleh dari hasil wawancara langsung dari pemilik lahan baik melalui kusioner maupun
melalui wawancara mendalam. Data sekunder digunakan untuk faktor-faktor yang mempengaruhi alih
fungsi lahan di tingkat wilaayah. Pengumpulan Data yang dilakukan kepada petani pemilik lahan yang
mengalami alih fungsi lahan dan tidak mengalami alih fungsi lahan dilakukan secara purposive sampling. Penelitian dilaksanakan menggunakan metode sampling non-probability disebabkan oleh jumlah
masing-masing populasi yang akan diteliti tidak diketahui secara pasti. Sampel pada samplingtidak acak
akan menyebabkan populasi yang akan diteliti tidak memiliki kesempatan yang sama untuk dipilih
sebagai sampel.Populasi di desa Koto Dian sebanyak 385 kk sedangkan di desa Koto Tuo sebanyak 444
kk. Responden dalam penelitian ini adalah petani setempat yang lahan usaha taninya pernah mengalami
alih fungsi lahan. Penelitian yang dilaksanakan mengambil responden berjumlah 70 responden untuk
petani di Desa Koto Dian dan 90 Responden di Desa Koto Tuo.Penetapan sampel ini disasarkan pada
pendapat Bailey dalam Hasan (2002) yang menyatakan bahwa ukuran sampel minimum yang
menggunakan analisis data statistik ialah 30 responden dimana populasi menyebar normal. Sampel
merupakan bagian dari populasi yang diambil melalui cara-cara tertentu yang juga mewakili karateristik
tertentu, jelas, dan lengkap yang bisa dianggap bisa mewakili populasi. Pengambilan data primer dilakukan melalui teknik wawancara dengan bantuan kuisioner kepada
responden.Responden merupakan pihak yang memberikan informasi dan dapat mewakili dalam
menjawab permasalahan penelitian. Petani sampel pada Penelitian ini adalah petani yang mengalih
fungsikan lahan sawahnya dengan kata lain petani yang pada awalnya menanam komuditi padi pada
lahan sawahnya kemudian pada saat ini sudah melakukan alih fungsi atau mengubah sebagian atau
keseluruhan dari lahannya. Kecamatan Keliling Danau dipilih secara sengaja (purposive). PENDAHULUAN Adanya alih fungsi lahan
pertanian khususnya lahan sawah akan mempengaruhi produksi beras yang mana merupakan makanan
pokok masyarakat Indonesia sehingga akan berpengaruh terhadap ketahanan pangan. Fenomena alih
fungsi lahan pertanian ke penggunaan non-pertanian saat ini terjadi sangat pesat di beberapa wilayah di
Indonesia terutama di Pulau Sumatra. Satu wilayah penyumbang beras tertinggi khususnya di Provinsi
Jambi sampai saat ini tetap mengalami alih fungsi lahan pertanian khususnya lahan sawah.Salah satu
Kabupaten yang mengalami alih fungsi lahan pertanian di Provinsi Jambi adalah kabupaten
Kerinci.Wilayah ini juga terkenal sebagai lumbung padi Provinsi karena merupakan daerah dataran
tinggi. Kabupaten Kerinci menjadi salah satu penghasil padi terbesar di Provinsi Jambi. Dalam lima tahun terakhir Kecamatan Keliling Danau mengalami tingkat penurunan luas lahan
padi sawah yang cukup tinggi, Tahun 2011 Luas panen sebesar 3.209 Ha namun terjadi penurunan
sehingga pada Tahun 2013 luas panen hanya mencapai 2.949 Ha. Sementara pada Tahun 2014 - 2 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 penurunan luas panen semakin menungkat yaitu 2.888 ha dan kembali menurun menjadi 2.859 ha di
Tahun 2015. Desa Koto dian dan Koto Tuo bukan merupaakan desa dengan Luas lahan sawah tertinggi di
Kecamatan Keliling Danau namun tingkat pengalihan fungsi lahan tertinggi terletek didua desa
tersebut.Lahan yang dialih fungsikan berupa lahan sawah produktif yang berada dipinggir jalan raya
utama Desa Koto Dian dan Koto Tuo. Pembangunan di wilayah ini lebih banyak untuk perumahan. Banyak Masyarakat yang memilih membangun Pemukiman tepat disisi Jalan utama meskipun lahan
tersebut merupakan lahan produktif bagi pertanian khususnya padi sawah. Tujuan dari penelitian ini
adalah untuk mengidentifikasi faktor-faktor yang mempengaruhi alih fungsi lahan sawah pada tingkat
petani dikedua desa serta hubungannya terhadap tingkat alih fungsi lahan sawah dan mengetahui
perbedaan penerimaan usahatani Petani yang mengalihkan lahan sebelum dan sesudah alih fungsi lahan
sawah kedua desa tersebut. 1.
Tingkat Usia Usia merupakan salah satu faktor yang berpengaruh dalam suatu tindakan alih fungsi lahan. Faktor usia dianggap faktor utama pendorong bagi petani untuk mengalihkan lahan terutama dalam hal
ini lahan sawah. Rentang usia petani sampelpun menjadi bahasan dalam proses penelitian ini. Berikut
tabel yang menunjukkan Tingkat usia petani sampel yang mengalihkan sebagian lahan sawah dan semua
lahan sebelum dan sesudah alih fungsi lahan. Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian bel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian
Lahan Sawah dan Semua Lahan Sawah. Tabel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian
Lahan Sawah dan Semua Lahan Sawah. Tabel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian
Lahan Sawah dan Semua Lahan Sawah. Rentang
Usia
Frekuensi Petani yang
Mengalihkan Sebagian
Lahan Sawah
Persentase
Frekuensi Petani yang
Mengalihkan Semua
Lahan Sawah
Persentase
29
–
35
tahun
5 jiwa
13,51 %
2 jiwa
10
36
–
42
tahun
12 jiwa
32,43 %
6 jiwa
30
43
-
49
tahun
9 jiwa
24,32 %
1 jiwa
5
50
-
56
tahun
4 jiwa
10,81 %
4 jiwa
20
57
–
63
tahun
5 jiwa
13,51 %
2 jiwa
10
64
–
70
tahun
1
2,70 %
3 jiwa
15
71
–
77
tahun
-
-
2 jiwa
10
Jumlah
37 jiwa
100 %
20 jiwa
100
Pada petani sampel yang mengalihkan sebagian lahan sawahnya terlihat jelas bahwa rentangan
usia yang mengalihkan sebagian lahan sawah tergolong masih dalam usia yang produktif yaitu pada
rentang usia 29- 35 tahun dengan jumlah persentase sekitar 13,51 % dari 37 sampel yang ada, 36- 42
tahun 32,43 %, 43- 49 tahun 24,32%, 50- 56 tahun 10,81 % , 57- 63 tahun 13,51 % sedangkan pada
rentang usia 64 - 70 tahun persentase jumlah petani yang mengalihkan sebagian lahan yakni 2,70 % dari
37 sampel. Sementara itu petani yang mengalihkan semua lahan sawahnya memiliki persentase rentang
usia yang berbeda pula yaitu pada rentang usia 29- 35 tahun 10 %, 36- 42 tahun 30 %, 43- 49 tahun 5 %,
50- 56 tahun 20 %, 57- 63 tahun 10 %, 64- 70 tahun 15 %, dan pada rentang usia 71- 77 tahun 10 % dari
20 sampel petani yang mengalihkan semua lahan. 1.
Tingkat Usia Petani sampel yang berada pada usia rentan/ rawan
dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan sementara
sebesar 60% pada tingkat petani yang mengalihkan semua lahan. Petani sampel yang berada pada usia
rentan/ rawan dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan
sementara sebesar 60% pada tingkat petani yang mengalihkan semua lahan. Hal ini mengindikasikan
b h
i
k
i b
h
d
lih
f
i l h
di K
K lili
D
K b Tabel 1 . Distribusi Frekuensi dan Persentase Usia Petani Sampel yang Mengalihkan Sebagian
Lahan Sawah dan Semua Lahan Sawah. Rentang
Usia
Frekuensi Petani yang
Mengalihkan Sebagian
Lahan Sawah
Persentase
Frekuensi Petani yang
Mengalihkan Semua
Lahan Sawah
Persentase
29
–
35
tahun
5 jiwa
13,51 %
2 jiwa
10
36
–
42
tahun
12 jiwa
32,43 %
6 jiwa
30
43
-
49
tahun
9 jiwa
24,32 %
1 jiwa
5
50
-
56
tahun
4 jiwa
10,81 %
4 jiwa
20
57
–
63
tahun
5 jiwa
13,51 %
2 jiwa
10
64
–
70
tahun
1
2,70 %
3 jiwa
15
71
–
77
tahun
-
-
2 jiwa
10
Jumlah
37 jiwa
100 %
20 jiwa
100
Pada petani sampel yang mengalihkan sebagian lahan sawahnya terlihat jelas bahwa rentangan Pada petani sampel yang mengalihkan sebagian lahan sawahnya terlihat jelas bahwa rentangan
usia yang mengalihkan sebagian lahan sawah tergolong masih dalam usia yang produktif yaitu pada
rentang usia 29- 35 tahun dengan jumlah persentase sekitar 13,51 % dari 37 sampel yang ada, 36- 42
tahun 32,43 %, 43- 49 tahun 24,32%, 50- 56 tahun 10,81 % , 57- 63 tahun 13,51 % sedangkan pada
rentang usia 64 - 70 tahun persentase jumlah petani yang mengalihkan sebagian lahan yakni 2,70 % dari
37 sampel. Sementara itu petani yang mengalihkan semua lahan sawahnya memiliki persentase rentang
usia yang berbeda pula yaitu pada rentang usia 29- 35 tahun 10 %, 36- 42 tahun 30 %, 43- 49 tahun 5 %,
50- 56 tahun 20 %, 57- 63 tahun 10 %, 64- 70 tahun 15 %, dan pada rentang usia 71- 77 tahun 10 % dari
20 sampel petani yang mengalihkan semua lahan. Petani sampel yang berada pada usia rentan/ rawan
dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan sementara
sebesar 60% pada tingkat petani yang mengalihkan semua lahan. JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS uji beda rata-rata bertujuan untuk mengetahui pengaruhnya terhadap tingkat penerimaan usahata
petani. 2.
Lama Pendidikan METODE PENELITIAN Dari sebanyak
24 desa di Kecamatan Keliling Danau ini dipilih secara Proposive Desa Koto Dian dan Desa Koto Tuo
karena ke 2 desa tersebut merupakan salah satu Desa yang paling tinggi tingkat penurunan luas lahan
(Keliling Danau Dalam Angka). Berdasarkan imformasi langsung yang peneliti dapatkan dilapangan,
kedua desa di Kecamatan Keliling Danau Kabupaten Kerinci jumlah Petani yang melakukan alih fungsi
dari lahan sawah menjadisebanyak 70 Kk di Desa Koto Dian (Kantor Kepala Desa Koto Dian tahun 2015),
sedangkan di Desa Koto Tuo sebanyak 90 Kk (Kantor Kepala Desa Koto Tuo tahun 2015) yang mengalih
fungsikan lahan sawahnya ke Pemukiman. Dalam hal ini Formula yang digunakan untuk menentukan
besarnya ukuran sampel adalah sebagai berikut (Slovin,1964 dan Nazir,2005). Metode analisis deskriftif
digunakan dengan tujuan untuk memberikan penjelasan dan interpretasi atas data dan informasi pada
tabulasi data serta faktor-faktor yang mempengaruhi alih fungsi lahan sawah. Kemudian metode analisis - 3 - 1.
Tingkat Usia Tingkat pendidikan merupakan hal yang paling penting dalam usaha meningkatkan produksi dan
kesejahteraan petani, maka diharapkan pola pikir petani akan rasional dalam mengambil keputusan
sehingga usahatani yang dikelola akan meningkat baik dalam hal jumlah dan mutu produksi yang akan
dihasilkan serta dengan memiliki pendidikan yang tinggi petani juga dapat memajukan dan mengelola
kelompok tani yang telah ada. Pada Petani yang mengalihkan sebagian lahan sebanyak 8,10% yang
berada pada tingkat sekolah dasar. Sedangkan lulusan SMP dan SMA persentasenya masing-masing
29,72% dan 37,83%, sementara pada petani yang mengalihkan semua lahan sawahnya sebanyak 30%
berada pada tingkat SD dan pada tingkat SMP sebesar 20% serta tingkat SMA sebanyak 25%. Dari
angka tersebut dapat dinyatakan bahwa rata-rata tingkat pendidikan formal petani sampel tergolong
masih rendah. Rendahnya tingkat pendididkan ini dapat mempengaruhi pola pemikiran dalam
mengelola usahataninya, sehingga pada akhirnya petani akan cenderung mengalihkan lahan usahatani
padi sawah. Semakin rendah tingkat pendidikan seorang petani maka semakin tinggi tingkat alih fungsi
lahan, terutama dalam hal ini lahan padi sawah.Petani yang memiliki pendidikan rendah cenderung
gampang mengalih fungsikan lahan sawah disebakan petani tersebut hanya memikirkan keuntungan
jangka pendek tanpa memikirkan kerugian dikemudian hari. Sedangkan petani yang memiliki tingkat
pendidikan tinggi akan memiliki pola pikir kedepan dengan mempertimbangkan lagi kerugian jangka
panjang apabila ia melakukan pengalihan fungsi lahan sawah. 1.
Tingkat Usia Petani sampel yang berada pada usia
rentan/ rawan dan non produktif sebesar 54,06 % pada tingkat petani yang mengalihkn sebagian lahan
sementara sebesar 60% pada tingkat petani yang mengalihkan semua lahan. Hal ini mengindikasikan
bahwa tingkat usia berpengaruh pada pengalihan fungsi lahan di Kecamatan Keliling Danau Kabupaten
Kerinci. Semakin tinggi tingkat usia maka semakin tinggi tingkat alih fungsi lahan. Ini terjadi disebabkan
karena semakin tinggi tingkat usia seseorang maka kondisi fisik akan semakin lemah. Mereka sudah
tidak kuat lagi bekerja di sektor pertanian yang membutuhkan tenaga yang kuat. Kondisi ini membatasi
kemampuan responden untuk menghasilkan sesuatu sehingga akan cenderung mengalih fungsikan
lahan yang dimilikinya. Apalagi dengan melihat kondisi saat ini dimana anak-anak mereka yang tidak lagi
mengikuti jejak orang tua mereka untuk bekerja di sektor pertanian. Dengan mengalih fungsikan lahan,
mereka dapat bekerja disektor lain yang tidak membutuhkan tenaga lebih. - 4 - - 4 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 Pendidikan
adalah
bagian
yang
dinilai
paling
penting
dalam
kehidupan
social
masyarakat.Pendidikan menunjukkan tingkat pengetahuan, wawasan, pola pikIr dan perilaku seseorang. Tingkat pendidikan formal yang dimiliki petani memberikan gambaran sumberdaya manusia petani pada
aspek formal.Pada masa sekarang lama seseorang berpendidikan menjadi suatu indikator dalam
menetukan sejahtera atau tidak sejahteranya suatu individu. Tabel 2 . Distribusi frekuensi dan Persentase Pendidkan Petani sampel yang mengalihkan
sebagian dan semua lahan sawah. Tingkat Pendidikan
Sebagian Lahan sawah
Semua Lahan Sawah
Frekuensi
Persentase (%)
Frekuensi
Persentase (%)
SD
3
8,10
6
30
SMP
11
29,72
4
20
SMA
14
37,83
5
25
Perguruan Tinggi
9
24,32
5
25
Jumlah
37
100
20
100
Tingkat pendidikan merupakan hal yang paling penting dalam usaha meningkatkan produksi dan
kesejahteraan petani, maka diharapkan pola pikir petani akan rasional dalam mengambil keputusan
sehingga usahatani yang dikelola akan meningkat baik dalam hal jumlah dan mutu produksi yang akan
dihasilkan serta dengan memiliki pendidikan yang tinggi petani juga dapat memajukan dan mengelola
kelompok tani yang telah ada. Pada Petani yang mengalihkan sebagian lahan sebanyak 8,10% yang
berada pada tingkat sekolah dasar. Sedangkan lulusan SMP dan SMA persentasenya masing-masing
29,72% dan 37,83%, sementara pada petani yang mengalihkan semua lahan sawahnya sebanyak 30%
berada pada tingkat SD dan pada tingkat SMP sebesar 20% serta tingkat SMA sebanyak 25%. Dari
angka tersebut dapat dinyatakan bahwa rata-rata tingkat pendidikan formal petani sampel tergolong
masih rendah. Luas lahan yang dimaksud dalam penelitian ini adalah luas lahan sawah yang digarap petani
selama melakukan kegiatan usahataninya. Dari hasil penelitian luas lahan yang dimiliki petani yang
mengalihkan sebagian lahan sawahnya sebanyak 43,24% ≤ 0,31 ha dan 56,76% petani yang memiliki 1.
Tingkat Usia Rendahnya tingkat pendididkan ini dapat mempengaruhi pola pemikiran dalam
mengelola usahataninya, sehingga pada akhirnya petani akan cenderung mengalihkan lahan usahatani
padi sawah. Semakin rendah tingkat pendidikan seorang petani maka semakin tinggi tingkat alih fungsi
lahan, terutama dalam hal ini lahan padi sawah.Petani yang memiliki pendidikan rendah cenderung
gampang mengalih fungsikan lahan sawah disebakan petani tersebut hanya memikirkan keuntungan
jangka pendek tanpa memikirkan kerugian dikemudian hari. Sedangkan petani yang memiliki tingkat
pendidikan tinggi akan memiliki pola pikir kedepan dengan mempertimbangkan lagi kerugian jangka
panjang apabila ia melakukan pengalihan fungsi lahan sawah. 3
h Tabel 2 . Distribusi frekuensi dan Persentase Pendidkan Petani sampel yang mengalihkan
sebagian dan semua lahan sawah. Tingkat Pendidikan
Sebagian Lahan sawah
Semua Lahan Sawah
Frekuensi
Persentase (%)
Frekuensi
Persentase (%)
SD
3
8,10
6
30
SMP
11
29,72
4
20
SMA
14
37,83
5
25
Perguruan Tinggi
9
24,32
5
25
Jumlah
37
100
20
100 bel 2 . Distribusi frekuensi dan Persentase Pendidkan Petani sampel yang mengalihkan
sebagian dan semua lahan sawah. Jumlah
37
100
20
100
Tingkat pendidikan merupakan hal yang paling penting dalam usaha meningkatkan produksi dan
kesejahteraan petani, maka diharapkan pola pikir petani akan rasional dalam mengambil keputusan
sehingga usahatani yang dikelola akan meningkat baik dalam hal jumlah dan mutu produksi yang akan
dihasilkan serta dengan memiliki pendidikan yang tinggi petani juga dapat memajukan dan mengelola
kelompok tani yang telah ada. Pada Petani yang mengalihkan sebagian lahan sebanyak 8,10% yang
berada pada tingkat sekolah dasar. Sedangkan lulusan SMP dan SMA persentasenya masing-masing
29,72% dan 37,83%, sementara pada petani yang mengalihkan semua lahan sawahnya sebanyak 30%
berada pada tingkat SD dan pada tingkat SMP sebesar 20% serta tingkat SMA sebanyak 25%. Dari
angka tersebut dapat dinyatakan bahwa rata-rata tingkat pendidikan formal petani sampel tergolong
masih rendah. Rendahnya tingkat pendididkan ini dapat mempengaruhi pola pemikiran dalam
mengelola usahataninya, sehingga pada akhirnya petani akan cenderung mengalihkan lahan usahatani
padi sawah. Semakin rendah tingkat pendidikan seorang petani maka semakin tinggi tingkat alih fungsi
lahan, terutama dalam hal ini lahan padi sawah.Petani yang memiliki pendidikan rendah cenderung
gampang mengalih fungsikan lahan sawah disebakan petani tersebut hanya memikirkan keuntungan
jangka pendek tanpa memikirkan kerugian dikemudian hari. Sedangkan petani yang memiliki tingkat
pendidikan tinggi akan memiliki pola pikir kedepan dengan mempertimbangkan lagi kerugian jangka
panjang apabila ia melakukan pengalihan fungsi lahan sawah. 3.
Luas Lahan Dalam hal ini luas lahan yang dilihat adalah luas lahan yang dimiliki petani dan luas lahan yang
dialih fungsikan baik pada petani yang mengalihkan sebagian lahan dan yang mengalihkan semua lahan
sawahnya.Berikut tabel luas lahan petani yang mengalihkan sebagian lahan dan semua lahan sawah. Dalam hal ini luas lahan yang dilihat adalah luas lahan yang dimiliki petani dan luas lahan yang
dialih fungsikan baik pada petani yang mengalihkan sebagian lahan dan yang mengalihkan semua lahan
sawahnya.Berikut tabel luas lahan petani yang mengalihkan sebagian lahan dan semua lahan sawah. Tabel 3 . Distribusi dan Frekuensi Luas Lahan Petani yang Mengalihkan Sebagian dan Semua Lahan
Luas lahan
Sebagian Lahan sawah
Semua Lahan Sawah
Frekuensi
Persentase (%)
Frekuensi
Persentase (%)
≤ 0,31 ha
16
43,24
11
55
≥ 0,31 ha
21
56,76
9
45
Jumlah
37
100
20
100
L
l h
di
k
d d l
liti
i i
d l h l
l h
h
di
t
i sawahnya.Berikut tabel luas lahan petani yang mengalihkan sebagian lahan dan semua lahan sawah. Tabel 3 . Distribusi dan Frekuensi Luas Lahan Petani yang Mengalihkan Sebagian dan Semua Lahan
Luas lahan
Sebagian Lahan sawah
Semua Lahan Sawah
Frekuensi
Persentase (%)
Frekuensi
Persentase (%)
≤ 0,31 ha
16
43,24
11
55
≥ 0,31 ha
21
56,76
9
45
Jumlah
37
100
20
100
Luas lahan yang dimaksud dalam penelitian ini adalah luas lahan sawah yang digarap petani
selama melakukan kegiatan usahataninya. Dari hasil penelitian luas lahan yang dimiliki petani yang
mengalihkan sebagian lahan sawahnya sebanyak 43,24% ≤ 0,31 ha dan 56,76% petani yang memiliki Luas lahan yang dimaksud dalam penelitian ini adalah luas lahan sawah yang digarap petani
selama melakukan kegiatan usahataninya. Dari hasil penelitian luas lahan yang dimiliki petani yang
mengalihkan sebagian lahan sawahnya sebanyak 43,24% ≤ 0,31 ha dan 56,76% petani yang memiliki - 5 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 lahan ≥ 0,31 ha. Sementara petani yang mengalihkan semua lahan sawahnya sebesar 55% bagi petani
yang memiliki luas lahan ≤ 0,31 dan 45% petani yang lahannya ≥ 0,31 ha.Jika dijumlahkan petani yang
memiliki luas lahan sedikit akan cenderung mengalih fungsikan lahan sawahnya. 4.
Jumlah Tanggungan Dalam hal ini jumlah tanggungan bukanlah jumlah anak secara keseluruhan melainkan jumlah
anak yang masih menjadi tanggungan dikarenakan belum menikah dan masih dalam tahap menempuh
pendidikan.Dibawah ini adalah tabel jumlah tanggungan petani yang mengalihkan sebagian dan semua
lahan sawah. bel 4 . Distribusi Frekuensi Jumlah Tanggungan Petani yang Mengalihkan Sebagian dan Semua Tabel 4 . Distribusi Frekuensi Jumlah Tanggungan Petani yang Mengalihkan Sebagian dan Semua
Lahan Sawah. Jumlah tanggungan
Sebagian Lahan sawah
Semua Lahan Sawah
Frekuensi
Persentase (%)
Frekuensi
Persentase (%)
Tidak memiliki tanggungan
4
10,81
9
45
≤ 2 orang
30
81,08
11
55
≥ 2 orang
3
8,10
-
-
Jumlah
37
100
20
100 Jumlah tanggungan yang dimaksud dalam penelitian ini adalah jumlah anak yang belum menikah
dan masih dalam tahap menempuh pendidikan. Dari hasil penelitian mengenai jumlah tanggungan yang
dimiliki petani yang mengalihkan sebagian lahannya didapatkan sebesar 10,81% petani tidak memiliki
tanggungan, 81,08% petani yang memiliki jumlah tanggungan ≤ 2 orang sedangkan jumlah tanggungan ≥
2 orang sebanyak 8,10%, Hal ini berbeda dengan petani yang memutuskan mengalihkan semua lahan
sawahnya yaitu sebesar 45% untuk petani yang tidak memiliki tanggungan lagi, 55% untuk petani yang
memiliki ≤ 2 orang tanggungan sedangkan jumlah tanggungan petani yang ≥ 2 orang tidak terdapat
sama sekali. Sejatinya jumlah tanggungan yang harus ditanggung petani mempengaruhi alih fungsi lahan
dimana semakin banyak jumlah tanggungan yang harus ditanggung, maka alih fungsi lahan akan
semakin tinggi. Semakin banyak tanggungan yang dimiliki maka biaya yang dibutuhkan dalam memenuhi
kebutuhan sehari-hari semakin banyak sehingga petani akan cenderung untuk mengalih fungsikan
lahannya. Pada penelitian ini jumlah tanggungan petani tidak berpengaruh secara nyata terhadap
tingkat alih fungsi lahan sawah, hal ini dikarenakan jumlah tanggungan kurang dari 3 orang maka
dikategorikan sedikit (Winoto, 2005). 3.
Luas Lahan Semakin luas
kepemilikan lahan maka peluang petani untuk mengalih fungsikan lahannya lebih kecil dibandingkan
petani yang melakukan alih fungsi lahan.Dalam tingkat luas pemilikan lahan, petani yang memiliki lahan
cukup luas cenderung untuk tetap mempertahankan lahannya sehingga peluang terjadinya alih fungsi
lahan kecil. Sedangkan bagi petani yang memiliki lahan kecil cenderung untuk mengalih fungsikan lahan
sawahnya ke sektor lain. Hal ini diduga disebabkan karena luas lahan sangat berhubungan dengan penerimaan. Petani
yang memiliki lahan lebih luas memiliki perolehan hasil produksi lebih besar sehingga penerimaan yang
dihasilkan lebih besar dibandingkan dengan petani yang memiliki luas lahan lebih sempit. Hasil panen
dari pengolahan lahan yang lebih sempit tidak sebanding dengan modal yang dikeluarkan petani
sehingga secara tidak langsung akan mempengaruhi penerimaan yang diperoleh dalam mencukupi
kehidupan sehari-hari. 5.
Pengalaman Berusahatani Pengalaman berusahatani merupakan lama petani dalam mengusahakan kegiatan berusahatani
dihitung dalam tahun. Petani dalam mengambil keputusan dan bijaksana mengenai usahataninya selalu
mempertimbangkan resiko yang akan diterimanya. Kemampuan petani dalam menerima resiko akan
berbeda antara petani satu dengan yang lainnya. Perbedaan Kemampuan menerima resiko ini
dipengaruhi dari beberapa faktor antara lain adalah pengalaman petani. Pengalaman petani dalam
mengusahan usahatani padi sawah dapat berpengaruh terhadap keputusan petani dalam mengalih
fungsikan lahan sawahnya. Berikut Tabel 5 pengalaman berusahatani petani yang mengalihkan sebagian
dan semua lahan sawah. Tabel 5. Pengalaman Berusahatani Petani yang Mengalihkan Sebagian dan Semua Lahan Sawah. Tabel 5. Pengalaman Berusahatani Petani yang Mengalihkan Sebagian dan Semua Lahan Sawah. - 6 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS Rentang Usia
(Pengalaman
berusahatani)
Frekuensi Petani yang
Mengalihkan Sebagian
Lahan Sawah
Persentase
Frekuensi Petani yang
Mengalihkan Sebagian
Semua Lahan Sawah
Persentase
1- 6 tahun
4 jiwa
10,81 %
4 jiwa
20 %
7 - 13 tahun
21 jiwa
56,75 %
3 jiwa
15 %
14 - 20 tahun
4 jiwa
10,81 %
2 jiwa
10 %
21 - 27 tahun
2 jiwa
5,40 %
2 jiwa
10 %
28 - 34 tahun
4 jiwa
10,81 %
1 jiwa
5 %
35 – 41 tahun
2 jiwa
5,40 %
6 jiwa
30 %
42 – 49 tahun
-
-
2 jiwa
10 %
Jumlah
37 jiwa
100 %
20 jiwa
100 % Pada petani yang mengalihkan sebagian lahan terlihat jelas bahwa rentang tahun pengalaman
berusahatani yaitu dari 1- 6 tahun terdapat 4 orang petani de3ngan persentase 10,81%, 7- 13 tahun
terdapat 21 orang petani dengan persentase 56,75%, 14-20 tahun terdapat 4 orang petani dengan
persentase 10,81%, 21-27 tahun terdapat 2 orang petani dengan persentase 5,40%, 28-34 tahun
sebanyak 4 orang petani dengan persentase 10,81% dan 35-41 tahun ada 2 orang petani yang
mengalihkan lahan dengan persentase 5,40%. Sedangkan pada petani yang mengalihkan semua lahan
dapat dilihat bahwa rentang tahun pengalaman berusahatani yaitu dari 1-6 tahun ada 4 orang petani
dengan persentase 20%, 7-13 tahun 3 orang dengan persentase 15%, 14-20 tahun terdapat 2 orang
dengan persentase 10%, 21-27 tahun 2 orang petani dengan persentase 10%, 28-34 tahun Cuma ada 1
orang petani dengan persentase 5%, 35-41 tahun terdapat 6 orang petani dengan persentase 30% dan
42-49 tahun terdapat 2 orang dengan jumlah perssentase 10%. Seorang petani dikatakan berpengalaman jika lama berusahatani > 20 tahun, sementara petani
dianggap pemula apabila lama berusahatani < 20 tahun (Winoto, 2005). 5.
Pengalaman Berusahatani Pada tabel lama berusahatani
dapat dilihat bahwa jumlah petani yang memiliki pengalaman < 20 tahun sebanyak 42 petani dengan
total persentase 73,68%. Sedangkan untuk petani yang memiliki pengalaman > 20 tahun sebanyak 19
petani dengan jumlah persentase 33,33%.Semakin lama pengalaman dalam berusahatani, maka akan
semakin berat dalam pengambilan keputusan untuk alih fungsi lahan. Hal ini disebabkan karena semakin
lama pengalaman bertani, maka keahlian dalam bertani akan semakin tinggi sehingga petani akan
cenderung untuk terus mempertahankan lahannya. Hal ini menyebabkan mereka tidak akan mengalih
fungsikan lahannya. Memang pada saat peneliti terjun ke lapangan rata-rata petani yang mengalih
fungsi lahan sawahnya adalah petani yang dikategorikan petani pemula. Jumlah Ada juga nenerapa responden memilih untuk menjadi TKI di luar negeri dimana ini menjadi
fenomena yang tidak bias dipungkiri lagi menjadi sumber penerimaan yang dianggap mampu memenuhi
kebetuhan hidup serta mampu meningkatkan taraf hidup masyarakat Kerinci khususnya di Kecamatan
Keliling Danau.Selain dari hanya bersumber padi gaji PNS dan TKI ada juga beberape petani memilih
bekerja disektor perdagangan, sebgai tukang, supir, pegawai honorer bahkan ada 3 responden yang
tidak bekerja sama sekali karena faktor usia sehingga kebutuhan hidupnya ditanggung oleh anaknya. Kejadian-kejadian tersebut menunjukkan gejala akan terjadinya transformasi kegiatan dari sektor
pertanian ke sektor non pertanian. Hal ini dapat dilihat dari adanya perubahan mata pencaharian utama
dari petani.Namun, akibat keterbatasan keterampilan yang dimiliki serta pendidikan yang rendah, hanya
pekerjaan dengan upah rendah yang bisa mereka peroleh. Perubahan mata pencaharian utama yang
terjadi, secara otomatis akan berpengaruh terhadap penerimaan yang diperoleh saat ini. B
t k P
lih
L h
S
h Pengalihan sumber penerimaanbagi petani yang mengalihkan semua lahan sawahnya memang
masih ada yang tetap memilih berusahatani namun dalam hal ini bukan sektor usahatani padi sawah
melain sebagai petani gurem dan berternak yang hanya 30% saja.Sementara dari sumber penerimaan
diluar usahatani ada yang hanya menjadi PNS sekitar 3 responden dengan persentase 15% dari total 20
sampel. Ada juga nenerapa responden memilih untuk menjadi TKI di luar negeri dimana ini menjadi
fenomena yang tidak bias dipungkiri lagi menjadi sumber penerimaan yang dianggap mampu memenuhi
kebetuhan hidup serta mampu meningkatkan taraf hidup masyarakat Kerinci khususnya di Kecamatan
Keliling Danau.Selain dari hanya bersumber padi gaji PNS dan TKI ada juga beberape petani memilih
bekerja disektor perdagangan, sebgai tukang, supir, pegawai honorer bahkan ada 3 responden yang
tidak bekerja sama sekali karena faktor usia sehingga kebutuhan hidupnya ditanggung oleh anaknya. Kejadian-kejadian tersebut menunjukkan gejala akan terjadinya transformasi kegiatan dari sektor
pertanian ke sektor non pertanian. Hal ini dapat dilihat dari adanya perubahan mata pencaharian utama
dari petani.Namun, akibat keterbatasan keterampilan yang dimiliki serta pendidikan yang rendah, hanya
pekerjaan dengan upah rendah yang bisa mereka peroleh. Perubahan mata pencaharian utama yang
terjadi, secara otomatis akan berpengaruh terhadap penerimaan yang diperoleh saat ini. B
t k P
lih
L h
S
h Alih fungsi lahan sawah mulai terjadi pada awal tahun 2000-an dan mengalami peningkatan tiap
tahunnya dikarenakan tingginya permintaan lahan untuk pemukiman, sementara lahan di Kabupaten
Kerinci merupakan lahan yang perbukitan sehinnga lahan sawahlah yang menjadi pilihan para
masyarakat untuk membangun pemukiman. Sumber Penerimaan Petani Setelah Alih Fungsi Lahan Usahatani padi sawah di Kecamatan Keliling Danau Kabupaten Kerinci masih menggunakan sistem
jaringan irigasi sederhana dan setengah teknis. Sebenarnya pada awal tahun 2000 kelompok tani mulai
dibentuk di daerah penelitian, namun karena kurang adanya perhatian dari Dinas Pertanian Kabupaten
Kerinci maupun PPL kelompok tani yang telah ada tidak aktif atau tidak dijalankan. Akibat dari kurang
adanya perhatian pemerintah lahan sawah yang awalnya membentang sepanjang jalan raya mulai
berkurang karena tingginya permintaan lahan untuk pemukiman. Berusahatani padi sawahpun
dianggap kurang menjinjikan mengingat penerimaan yang diperoleh dari sektor tersebut lebih kecil
disbanding sektor lain. Untuk mengetahui sumber penerimaan petani yang mengalihkan sebagian lahan
sawahnya dapat dilihat pada Tabel 6 dibawah ini : Sumber Penerimaan Petani yang Mengalihkan Sebagian Lahan Sawahnya Sebelum dan
Sesudah Alih fungsi Lahan. Sesudah Alih fungsi Lahan. Sumber Penerimaan
Jumlah Sampel (orang)
Persentase
Usahatani Padi dan PNS
7
18,91%
Usahatani Padi dan Berdagang
4
10,81%
Usahatani Padi dan Petani Gurem
5
13,51%
Usahatani Padi dan Nelayan
4
10,81%
Usahatani Padi dan TKI
7
18,91%
Usahatani Padi dan Tukang
4
10,81%
Usahatani Padi dan Supir
2
5,40%
Hanya Usahatani Padi Sawah
4
10,81% - 7 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 Jumlah 100% Pada tabel dapat dilihat sumber penerimaan yang diperoleh oleh petani sampel sesudah alih
fungsi lahan terdiri dari beberapa sumber penerimaan. Petani yang mengalihkan sebagian lahan di
lapangan tempat penelitian berlangsung tentunya masih mengusahakan padi bsawah dan memiliki
sumber penerimaan dari sektor lain diantaranya, sebagai PNS sebanyak 7 responden dengan persentase
18,91 % dari jumlah sampel, disaektor dagang ada 4 responden dengan persentase 10,81%, disektor
petani gurem 5 responden dengan persentase 13,51%, sebagai nelayan ada 4 responden dengan
persentase 10,81%, sumber penerimaan sebagai TKI sebanyak 7 responden dengan persentase 18,91%,
sebagai tukang ada 4 responden dengan jumlah persentase 10,81%, sementara responden yang menjadi
supir hanya ada 2 responden dengan persentase 5,40% dan yang hanya berusahatani padi tanpa sumber
penerimaan smpingan ada 4 responden dengan jumlah persentase 10,91%. Sumber penerimaan dari
sektor lain ini tentunya dapat membantu petani dalam memenuhi kebutuhan keluarga sehari-hari. Dengan adanya sumber penerimaan dari sektor lain petani kiranya dapat terus berusahatni padi tanpa
harus mengalihkan semua luas lahan sawah yang dimiliki. Sementara itu mengetahui sumber
penerimaan bagi petani yang mengalihkan semua lahan sawahnya dapat dilihat pada Tabel 7 berikut ini:
Tabel 7. Sumber Penerimaan Petani yang Mengalihkan Semua Lahan Sawahnya Sesudah Alih
f
i L h Sumber Penerimaan Petani yang Mengalihkan Semua Lahan Sawahnya Sesudah Alih
fungsi Lahan. fungsi Lahan. Sumber Penerimaan
Jumlah Sampel (orang)
Persentase
PNS
3
15%
Berdagang
2
10%
Petani Gurem
3
15%
Peternak
3
15%
TKI
2
10%
Tukang
1
5%
Supir
1
5%
Honorer
2
10%
Tidak bekerja
3
15%
Jumlah
20
100% g
Sumber Penerimaan
Jumlah Sampel (orang)
Persentase
PNS
3
15%
Berdagang
2
10%
Petani Gurem
3
15%
Peternak
3
15%
TKI
2
10%
Tukang
1
5%
Supir
1
5%
Honorer
2
10%
Tidak bekerja
3
15%
Jumlah
20
100% Pengalihan sumber penerimaanbagi petani yang mengalihkan semua lahan sawahnya memang
masih ada yang tetap memilih berusahatani namun dalam hal ini bukan sektor usahatani padi sawah
melain sebagai petani gurem dan berternak yang hanya 30% saja.Sementara dari sumber penerimaan
diluar usahatani ada yang hanya menjadi PNS sekitar 3 responden dengan persentase 15% dari total 20
sampel. Jumlah Untuk mengetahui bentuk peralihan lahan sawah dapat di
lihat pada Tabel 19 dibawah ini. Bentuk Peralihan Lahan Sawah Bagi Petani yang Mengalihkan Sebagian dan Semua Lahan Bentuk Peralihan Lahan Sawah Bagi Petani yang Mengalihkan Sebagian dan Semua Lahan
Sawah. Bentuk Peralihan Lahan Sawah Bagi Petani yang Mengalihkan Sebagian dan Semua Laha
Sawah. Petani yang Mengalihkan
Sebagian Lahan Bentuk Alih fungsi
lahan
Petani yang Mengalihkan
Semua Lahan
Petani yang Mengalihkan
Sebagian Lahan - 8 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS Responden
Persentase
Responden
Persentase
Lahan Petani Gurem
3
15%
5
13,51%
Pemukiman
3
15%
32
86,48%
Lahan Ternak
14
70%
-
-
Jumlah
20
100%
37
100%
Lahan yang dijadikan pemukiman sekitar 86,48% petani yang mengalihkan sebagian lahan
sawahnya dengan jumlah responden sebanyak 32 orang hal ini tentu saja menunjukkan tinggi alih fungsi
lahan ke pemukiman. Dijadikan lahan selain untuk pemukiman ada juga beberapa responden petani
mengalihkan lahan sawahnya ke sektor usahatani lain yakni sebagai lahan ternak sebanyak 70% bagi
petani yang mengalihkan sebagian lahan dan untuk yang mengalihkan semua lahan sawah tidak ada
yang menjadikan lahan ternak melainkan 13,51% responden petani yang mengalihkan ke lahan petani
gurem, sedangkan 15% reponden dari petani yang mengalihkan semua lahan menjadi lahan pertanian
gurem dan hanya ada 15% petani yang mengalihkan semua lahan ke pemukiman. Lahan-lahan yang
memiliki lokasi dekat dengan jalan raya maka akan memiliki nilai jual lebih tinggi dibandingkan dengan
lahan yang letaknya jauh dari jalan raya. Keadaan ini sesuai dengan teori yang diungkapkan oleh Von
Thunendimana lokasi merupakan faktor yang menentukan pengunaan lahan.Hal ini pula yang
mengindikasikan mengapa petani cenderung mengalih fungsikan lahan sawahnya untuk pemukiman
terutama yang berada di pinggir jalan raya. Responden
Persentase
Responden
Persentase
Lahan Petani Gurem
3
15%
5
13,51%
Pemukiman
3
15%
32
86,48%
Lahan Ternak
14
70%
-
-
Jumlah
20
100%
37
100%
Lahan yang dijadikan pemukiman sekitar 86 48% petani yang mengalihkan sebagian lahan Lahan yang dijadikan pemukiman sekitar 86,48% petani yang mengalihkan sebagian lahan
sawahnya dengan jumlah responden sebanyak 32 orang hal ini tentu saja menunjukkan tinggi alih fungsi
lahan ke pemukiman. Jumlah Dijadikan lahan selain untuk pemukiman ada juga beberapa responden petani
mengalihkan lahan sawahnya ke sektor usahatani lain yakni sebagai lahan ternak sebanyak 70% bagi
petani yang mengalihkan sebagian lahan dan untuk yang mengalihkan semua lahan sawah tidak ada
yang menjadikan lahan ternak melainkan 13,51% responden petani yang mengalihkan ke lahan petani
gurem, sedangkan 15% reponden dari petani yang mengalihkan semua lahan menjadi lahan pertanian
gurem dan hanya ada 15% petani yang mengalihkan semua lahan ke pemukiman. Lahan-lahan yang
memiliki lokasi dekat dengan jalan raya maka akan memiliki nilai jual lebih tinggi dibandingkan dengan
lahan yang letaknya jauh dari jalan raya. Keadaan ini sesuai dengan teori yang diungkapkan oleh Von
Thunendimana lokasi merupakan faktor yang menentukan pengunaan lahan.Hal ini pula yang
mengindikasikan mengapa petani cenderung mengalih fungsikan lahan sawahnya untuk pemukiman
terutama yang berada di pinggir jalan raya. Lahan yang dijadikan pemukiman sekitar 86,48% petani yang mengalihkan sebagian lahan
sawahnya dengan jumlah responden sebanyak 32 orang hal ini tentu saja menunjukkan tinggi alih fungsi
lahan ke pemukiman. Dijadikan lahan selain untuk pemukiman ada juga beberapa responden petani
mengalihkan lahan sawahnya ke sektor usahatani lain yakni sebagai lahan ternak sebanyak 70% bagi
petani yang mengalihkan sebagian lahan dan untuk yang mengalihkan semua lahan sawah tidak ada
yang menjadikan lahan ternak melainkan 13,51% responden petani yang mengalihkan ke lahan petani
gurem, sedangkan 15% reponden dari petani yang mengalihkan semua lahan menjadi lahan pertanian
gurem dan hanya ada 15% petani yang mengalihkan semua lahan ke pemukiman. Lahan-lahan yang
memiliki lokasi dekat dengan jalan raya maka akan memiliki nilai jual lebih tinggi dibandingkan dengan
lahan yang letaknya jauh dari jalan raya. Keadaan ini sesuai dengan teori yang diungkapkan oleh Von
Thunendimana lokasi merupakan faktor yang menentukan pengunaan lahan.Hal ini pula yang
mengindikasikan mengapa petani cenderung mengalih fungsikan lahan sawahnya untuk pemukiman
terutama yang berada di pinggir jalan raya. Penerimaan Usahatani Luas Lahan
Rata-rata
(Ha)
Produksi
Rata-rata
(Ton)
Produktivitas
Rata-rata
(Ton/Ha)
Penerimaan
Rata-
ratausahatani
(Rp)
Sebelum Alih Funsi Lahan
0,33
1,08
3,22
1.500.000
Sesudah Alih Fungsi Lahan
0,28
0,34
2,21
720.541
Jumlah
0,6
1,42
5,43
2.220.541
Dari tabel sudah terlihat dengan jelas perbedaaan penerimaantan usahatani petani yang Penerimaan Usahatani Penerimaan usahatani dikatakan sebagai hasil dari suatu kegitan usahatani yang dapat
meningkatkan pendapatan rumah tangga petani. Namun pada hakekatnya para petani yang dulunya
sangat bergantung pada sektor pertanian terutama menggarap sawah sekarang mulai meninggalkan dan
beralih pada usaha lain. Beberapa dari petani sampai harus mengalihkan sawahnya untuk dijadikan
pemukiman. Para petani menilai penerimaan yang dihasilkan dari kegiatan berusahatani tak mampu lagi
menghasilkan atau dalam kata lain penerrimaan yang didapatkan tak sesuai dengan usaha yang dirasa
oleh para petani. Beberapa petani ada yang memilih membangun usaha lain, seperti menjadi pedagang,
bertani ladang, menjadi TKI di Negara tetangga dan memilih bidang pekerjaan lainnya. Berikut ini Tabel
9 dapat dilihat perbedaan penerimaan usahatani petani yang mengalihkan sebagian lahan sawah dan
yang mengalihkan semua lahan sawah di daerah penelian pada tahun 2015. Tabel 9. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya
Pada Tahun 2015. Luas Lahan
Rata-rata
(Ha)
Produksi
Rata-rata
(Ton)
Produktivitas
Rata-rata
(Ton/Ha)
Penerimaan
Rata-
ratausahatani
(Rp)
Sebelum Alih Funsi Lahan
0,33
1,08
3,22
1.500.000
Sesudah Alih Fungsi Lahan
0,28
0,34
2,21
720.541
Jumlah
0,6
1,42
5,43
2.220.541
Dari tabel sudah terlihat dengan jelas perbedaaan penerimaantan usahatani petani yang
mengalihkan sebagian lahan sawah sebelum mengalih fungsikan lahan dan sesudah alih fungsi lahan
sawanya. Jika sebelum alih fungsi lahan luas lahan sawah rata-rata 0,33 ha dapat menghasilkan 1,08 ton
dan produktivitasnya sekitar 3,22 dengan jumlah penerimaan Rp 1.500.000,- setiap satu kali masa
panen. Sementara yang terjadi sesudah alih fungsi lahan sawah terjadi pengurangan jumlah lahan rata-
rata yang ada hanya 0,27 ha dengan produksi hanya 0,34 ton dan produktivitas hanya mampu
menyentuh angka 2,20 ton/ha serta penerimaan Rp 720.541,- saja. Sedangkan pada Tabel 10 dibawah
dapat dilihat perbedaan penerimaan usahatani petani yang mengalihkan semua lahan sawahnya sebagai
berikut.Pada saat Peneliti ke lapangan penerimaan usahatani yang sedemikian tidak menjadi sumber
utama bagi pendapatan mereka, petani yang mengalihkan sebagian lahan sawah beralasan hanya
berusahatani seadanya saja demi tetap menjaga produksi padi lokal walau hanya bisa menggarap sedikit
lahan sawahnya sementara sebagian dijadikan pemukiman. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya
Pada Tahun 2015. Tabel 9. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya Tabel 9. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Sebagian Lahan Sawahnya
Pada Tahun 2015. Jumlah Dari tabel sudah terlihat dengan jelas perbedaaan penerimaantan usahatani petani yang
mengalihkan sebagian lahan sawah sebelum mengalih fungsikan lahan dan sesudah alih fungsi lahan
sawanya. Jika sebelum alih fungsi lahan luas lahan sawah rata-rata 0,33 ha dapat menghasilkan 1,08 ton
dan produktivitasnya sekitar 3,22 dengan jumlah penerimaan Rp 1.500.000,- setiap satu kali masa
panen. Sementara yang terjadi sesudah alih fungsi lahan sawah terjadi pengurangan jumlah lahan rata-
rata yang ada hanya 0,27 ha dengan produksi hanya 0,34 ton dan produktivitas hanya mampu
menyentuh angka 2,20 ton/ha serta penerimaan Rp 720.541,- saja. Sedangkan pada Tabel 10 dibawah
dapat dilihat perbedaan penerimaan usahatani petani yang mengalihkan semua lahan sawahnya sebagai
berikut.Pada saat Peneliti ke lapangan penerimaan usahatani yang sedemikian tidak menjadi sumber
utama bagi pendapatan mereka, petani yang mengalihkan sebagian lahan sawah beralasan hanya
berusahatani seadanya saja demi tetap menjaga produksi padi lokal walau hanya bisa menggarap sedikit
lahan sawahnya sementara sebagian dijadikan pemukiman. - 9 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no
DOI: 10.22437/jiseb.v21i1 JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS Tabel 10. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Semua Lahan Sawahnya
Pada Tahun 2015. Luas Lahan
Rata-rata
(Ha)
Produksi
Rata-rata
(Ton)
Produktivitas
Rata-rata
(Ton/Ha)
Penerimaan
usahatani
Rata-rata
(Rp)
Sebelum Alih Funsi Lahan
0,30
1,075
3,15
1.460.000
Sesudah Alih Fungsi Lahan
0,30 yang
dialih
fungsikan
Produksi di
sektor
usahatani lain
Produktivitas
disektor
usahatani lain
595.000
(selain
usahatani
padi sawah) bel 10. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Semua Lahan Sawahnya
Pada Tahun 2015. Perbedaan Penerimaan Usahatani Petani yang Mengalihkan Semua Lahan Sawahnya
Pada Tahun 2015 Sangat jelas bahwa sebelum alih fungsi lahan sawah petani dapat menghasilkan rata-rata Rp
1.460.000,- dengan luas lahan 0,30 ha dan produksi yang mencapai 1,075 ton serta Produktivitas yang
berada pada angka 3,15 ton/ha. Sedangkan sesudah petani mengalihkan semua lahan sawahnya luas
lahan, produksi, produktivitas maupun penerimaan usahataninya berada pada sektor usahatani lain
dengan rata-rata penerimaan Rp. 595.000,- .Semakin rendah penerimaan yang diperoleh dari hasil
usahatani, maka akan semakin tinggi peluang petani dalam melakukan alih fungsi lahan. Jika
penerimaan yang diperoleh dari hasil usahatani rendah maka ada kecenderungan untuk memilih
penerimaan diluar sektor pertanian dan lahan yang dimiliki dialih fungsikan karena penerimaan
usahatani tidak dapat memenuhi kebutuhan sehari-hari. Jumlah p
Berdasarkan hasil penelitian di lapangan faktor-faktor alih fungsi lahan sawah sangat
berhubungan dengan perbedaan tingkat penerimaan usahatani dikedua desa tersebut, untuk petani
yang mengalihkan sebagian luas lahan sawahnya sebelum alih fungsi penerimaan rata-rata usahataninya
dari Rp 1.500.000,- dan sesudah alih fungsi sebagian lahan sawah menjadi Rp 720.541,- sementara
untuk petani yang mengalihkan semua luas lahan sawahnya penerimaan usahatani sebelum alih fungsi
lahan dengan rata-rata Rp 1.460.000,- dan sesudah alih fungsi semua luas lahan sawhnya menjadi Rp
595.000,-. Kemudian untuk melihat lebih jelas perbedaan pendapatan usahatani yang dihasilkan
tersebut dilakukan uji dengan alat uji beda dua rata-rata maka diperoleh 𝑡"#$>𝑡$%& yaitu 3,35 > 1,665
terima 𝐻( tolak 𝐻) untuk petani yang mengalihkan sebagian luas lahan sawahnya, sementara untuk
petani yang mengalihkan semua lahan sawahnya 𝑡"#$>𝑡$%& yaitu 4,02 > 3,086 terima 𝐻( tolak 𝐻) artinya
terdapat derajad perbedaan yang nyata antara penerimaan usahatani sebelum dan sesudah alih fungsi
lahan sawah, dimana penerimaan usahatani sebelum alih fungsi lahan lebih besar daripada penerimaan
usahatani sesudah alih fungsi lahan. KESIMPULAN Dari hasil penelitian yang telah dilaksanakan, maka dapat disimpulkan bahwa: 1). Faktor-faktor
yang mempengaruhi alih fungsi lahan sawah terdiri dari lama pendidikan, umur petani, luas lahan,
pengalaman bertani, jumlah tanggungan. 2). Hasil analisis uji beda dua rata-rata, maka diperoleh thitung >
ttabel yaitu 3,35 > 1,665 terima H1 tolak H0 untuk petani yang mengalihkan sebagian luas lahan sawahnya,
sementara untuk petani yang mengalihkan semua lahan sawahnya thitung > ttabel yaitu 4,02 > 1,665 terima
H1 tolak H0 artinya terdapat derajat perbedaan yang nyata antara penerimaan usahatani sebelum dan
sesudah alih fungsi lahan sawah, dimana penerimaan usahatani sebelum alih fungsi lahan lebih besar
daripada penerimaan usahatani sesudah alih fungsi lahan. Terdapat perbedaan penerimaan usahatani
petani sebelum alih fungsi lahan dan sesudah alih fungsi lahan yaitu dari Rata-rata penerimaan
usahatani yang awalnya Rp. 1.500.000,-/ rata- rata 0,33 ha luas lahan turun menjadi Rp. 720.541,-/ rata-
rata 0,27 ha atau ± Rp. 4.500.000,- / 1 ha luas lahan pada satu kali musim tanam, ini terjadi pada petani
yang mengalihkan sebagian lahan sementara yang mengalihkan semua lahan penerimaan usahatani
sebelum alih fungsi lahan yakni Rp. 1.460.000,-/ rata-rata 0,30 ha lahan atau ± Rp. 5.600.000,-/ 1 lahan
menjadi Rp. 595.000/ 0,30 ha atau ± Rp. 1.800.000 / 1 ha lahan yang telah dialih fungsi ke sektor
usahatani lain pada satu kali musim tanam. UCAPAN TERIMAKASIH - 10 - JURNAL ILMIAH SOSIO-EKONOMIKA BISNIS
ISSN: 1412-8241 (p); 2621-1246 (e), Volume 21. no (1) 2018
DOI: 10.22437/jiseb.v21i1 Ucapan terimakasih disampaikan kepada Dekan Fakultas Pertanian dan ketua Program Studi
Agribisnis Fakultas Pertanian Universitas Jambi yang telah memfasilitasi pelaksanaan penelitian ini. Terimakasih kepada dosen pembimbing akademik ibu Ir. Adlaida Malik, Ms. Selain itu saya ucapkan
terimakasih kepada Bapak Camat Keliling Danau yang telah sudi memberikan segala informasi dalam
membantu penelitian ini. Kemudian ucapan terimakasih kepada Bapak Mukhsin sebagai Kepala Desa
Koto Dian dan Bapak Erizal selaku Kepala Desa Koto tuo yang telah member izin untuk melakukan
penelitian di Desa Koto Dian dan Koto Tuo dengan judul Hubungan Faktor-faktor Alih fungsi Lahan Padi
Sawah dan Perbedaan Tingkat Penerimaan Usahatani Petani di Kecamatan Keliling Danau Kabupaten
Kerinci. DAFTAR PUSTAKA Badan Pusat Statistik Provinsi Jambi. 2013. Jambi Dalam Angka. Jambi
Badan Pusat Statistik Provinsi Jambi. 2013. Keliling Danau Dalam angka.Jambi. Barlowe R. 1978. Land Resource economics. Third edition.Prentice. Hall inc, New jersey. Hasan, Iqbal. Analisi Data Penelitian dengan Statistik. PT Bumi Aksara. Jakarta
Kacabdis Pertanian Dan Perkebunan Tanaman Pangan Kecamatan Keliling Danau. Kerinci.Jambi. Mosher, A.T. 1983.Menggerakkan dan Membangun Pertanian. CV Yasaguna. Jakarta
Nazir M. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta. Utomo. 1992. Konversi Lahan Sawah di Indonesia.ITB. Bandung. Winoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bogor. Winoto.2005. Konversi Lahan Sawah Di Indonesia. Penebar Swadaya Badan Pusat Statistik Provinsi Jambi. 2013. Jambi Dalam Angka. Jambi
Badan Pusat Statistik Provinsi Jambi. 2013. Keliling Danau Dalam angka.Jambi. Barlowe R. 1978. Land Resource economics. Third edition.Prentice. Hall inc, New jersey. Hasan, Iqbal. Analisi Data Penelitian dengan Statistik. PT Bumi Aksara. Jakarta
Kacabdis Pertanian Dan Perkebunan Tanaman Pangan Kecamatan Keliling Danau. Kerinci.Jambi. Mosher, A.T. 1983.Menggerakkan dan Membangun Pertanian. CV Yasaguna. Jakarta
Nazir M. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta. Utomo. 1992. Konversi Lahan Sawah di Indonesia.ITB. Bandung. Winoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bogor. Winoto.2005. Konversi Lahan Sawah Di Indonesia. Penebar Swadaya Badan Pusat Statistik Provinsi Jambi. 2013. Jambi Dalam Angka. Jambi
Badan Pusat Statistik Provinsi Jambi. 2013. Keliling Danau Dalam angka.Jambi. Barlowe R. 1978. Land Resource economics. Third edition.Prentice. Hall inc, New jersey. Hasan, Iqbal. Analisi Data Penelitian dengan Statistik. PT Bumi Aksara. Jakarta
Kacabdis Pertanian Dan Perkebunan Tanaman Pangan Kecamatan Keliling Danau. Kerinci.Jambi. Mosher, A.T. 1983.Menggerakkan dan Membangun Pertanian. CV Yasaguna. Jakarta
Nazir M. 1988. Metode Penelitian. Ghalia Indonesia, Jakarta. Utomo. 1992. Konversi Lahan Sawah di Indonesia.ITB. Bandung. Winoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bog
Winoto.2005. Konversi Lahan Sawah Di Indonesia. Penebar Swadaya inoto J. 1995. Alih Guna Lahan Pertanian: Permasalahan dan Implikasi. Institut Pertanian Bogor,Bogor. - 11 -
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Population Connectivity Measures of Fishery-Targeted Coral Reef Species to Inform Marine Reserve Network Design in Fiji
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Scientific reports
| 2,016
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cc-by
| 9,156
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Population Connectivity Measures
of Fishery-Targeted Coral Reef
Species to Inform Marine Reserve
Network Design in Fiji received: 28 July 2015
accepted: 07 December 2015
Published: 25 January 2016 Erin K. Eastwood1,†,*, Elora H. López1,‡,* & Joshua A. Drew1,2 Erin K. Eastwood1,†,*, Elora H. López1,‡,* & Joshua A. Drew1,2 Coral reef fish serve as food sources to coastal communities worldwide, yet are vulnerable to mounting
anthropogenic pressures like overfishing and climate change. Marine reserve networks have become
important tools for mitigating these pressures, and one of the most critical factors in determining
their spatial design is the degree of connectivity among different populations of species prioritized for
protection. To help inform the spatial design of an expanded reserve network in Fiji, we used rapidly
evolving mitochondrial genes to investigate connectivity patterns of three coral reef species targeted by
fisheries in Fiji: Epinephelus merra (Serranidae), Halichoeres trimaculatus (Labridae), and Holothuria atra
(Holothuriidae). The two fish species, E. merra and Ha. trimaculatus, exhibited low genetic structuring
and high amounts of gene flow, whereas the sea cucumber Ho. atra displayed high genetic partitioning
and predominantly westward gene flow. The idiosyncratic patterns observed among these species
indicate that patterns of connectivity in Fiji are likely determined by a combination of oceanographic
and ecological characteristics. Our data indicate that in the cases of species with high connectivity,
other factors such as representation or political availability may dictate where reserves are placed. In
low connectivity species, ensuring upstream and downstream connections is critical. Coral reefs are some of the planet’s most complex and diverse ecosystems, providing nearly US$ 30 billion in
goods and services to global economies annually, through tourism, fisheries, and coastal protection1. However,
the vivid splendor of these ecosystems is subject to numerous anthropogenic stressors including overfishing, pol-
lution, sedimentation, and climate change, and nearly every coral reef worldwide is currently affected by human
activities2–4. One tool commonly used to mitigate global reef degradation is the creation of marine protected areas (MPAs) –
spatially explicit areas of ocean where human activities are regulated or prohibited. One subset of MPAs are
no-take marine reserves, wherein direct harvesting of marine resources is prohibited both spatially and tempo-
rally. When effectively implemented, no-take marine reserves are important tools for conservation and fisheries
management. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 28 July 2015
accepted: 07 December 2015
Published: 25 January 2016 Population Connectivity Measures
of Fishery-Targeted Coral Reef
Species to Inform Marine Reserve
Network Design in Fiji They provide a refuge for exploited species, allow for fish and invertebrate stocks to recover, repro-
duce, and reseed adjacent unprotected areas through larval export and adult movement, while they protecting
existing habitat from further degradation5–8.i g
g
Networks of reserves confer additional benefits, protecting a greater diversity of habitats and promoting the
stability of meta-populations9,10. The optimal orientation for reserve networks requires information concerning
the degree of demographic or larval exchange occurring between populations11,12. Reserve networks that explic-
itly incorporate connectivity in their design are more resilient to threats like overfishing, disease, and climate
change because neighboring reserves can help reseed reefs in the event of disturbances, thereby boosting the
stability of the system as a whole11,13,14. 1Department of Ecology, Evolution, and Environmental Biology, Columbia University, NY. 2Department of
Vertebrate Zoology, American Museum of Natural History, NY. †Present address: National Oceanic and Atmospheric
Administration, Climate Program Office, 1315 East-West Highway, Silver Spring MD 20910. ‡Present address:
Department of Biology, Stanford University, Hopkins Marine Station, Pacific Grove, CA. *These authors contributed
equally to this work. Correspondence and requests for materials should be addressed to J.A.D. (email: j.drew@
columbia.edu) Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 1 www.nature.com/scientificreports/ Figure 1. Sampling locations within Fiji, with island names in bold and village names adjacent to points. Division boundaries and path of the Bligh Water Current (denoted by red arrows) are approximate. Map ©
d-maps.com available at http://dmaps.com/carte.php?num_car= 28136&lang= en Figure 1. Sampling locations within Fiji, with island names in bold and village names adjacent to points. Division boundaries and path of the Bligh Water Current (denoted by red arrows) are approximate. Map ©
d-maps.com available at http://dmaps.com/carte.php?num_car= 28136&lang= en Species’ behaviour, locomotion, life history, and habitat all play into how organisms interact with their phys-
ical environment, and thus influence connectivity. The larvae of many reef fishes possess an array of sensory
modalities and the physiological capacity for directional swimming that allows for non-random settlement15–18,
and some species with prolonged pelagic larval stages display natal reef preference and genetic structuring across
populations18,19. Despite the capabilities of some larvae, oceanic currents can limit genetic connectivity between
populations if individuals are unable to traverse those currents, as this reduces gene flow20–23. Conversely, ocean
currents can promote connectivity if they transport larvae from upstream to downstream sites, thus generating
asymmetrical gene flow24. Population Connectivity Measures
of Fishery-Targeted Coral Reef
Species to Inform Marine Reserve
Network Design in Fiji y
gl
In 2005, the Fiji government set the goal of protecting 30% of its marine waters by 202025. As of 2010, local
management had effectively protected a considerable proportion of Fiji’s priority ecosystems for management26. However, the amount of area that can be protected by ad-hoc community-based management is nearing satura-
tion, and these efforts need to be scaled up significantly if the national goal is to be reached26. Because of this, a
systematic conservation planning approach that includes consideration of the connectivity and complementarity
of potential reserve sites across the entirety of Fiji has been recommended27. Additional scientific input, particu-
larly information concerning population connectivity, will be necessary if an expanded network of MPAs is to
effectively utilize limited conservation resources in the country.l f
y
y
Previous work on connectivity among reefs in Fiji revealed asymmetrical gene flow along an east-west gradi-
ent for three of five coral reef fish species studied, potentially indicating that the Bligh Waters – a fast-moving cur-
rent that bisects the main islands – could be facilitating larval transport for multiple taxa within Fiji28. However,
this study was based on a suite of species that were not fisheries targets. A more relevant framework for conser-
vation planning might instead be crafted based on the connectivity dynamics of economically and nutrition-
ally significant species. Small-scale inshore fisheries provide food for about 50% of rural households in Fiji and
contribute over $48 million to the national economy every year29,30, so the sustainable management of species
contributing to these fisheries is paramount for ensuring food security. Here, we expand upon previous work and
investigate the role of the Bligh Waters in shaping the connectivity patterns of three coral reef species commonly
targeted by inshore fisheries across the Fijian archipelago (Fig. 1) – the honeycomb grouper Epinephelus merra
(Serranidae), the three-spot wrasse Halichoeres trimaculatus (Labridae), and the black sea cucumber Holothuria
atra (Holothuriidae). Results
G
ti atra populations at the island-
assemblage level in Fiji. Significant p-values are in bold. Vanua Levu
Taveuni
Naviti
Vanuabalavu
(A) E. merra Vanua Levu
–
Taveuni
0.00000
–
Naviti
0.05327
0.01336
–
Vanuabalavu
0.00702
0.00000
0.00000
–
Viti Levu
0.00602
0.00000
0.00602
0.00000
(B) Ha. trimaculatus Vanua Levu
–
Taveuni
0.00547
–
Naviti
0.00000
0.00000
–
Vanuabalavu
0.00000
0.00000
0.00000
–
Viti Levu
0.07232
0.00196
0.02021
0.01350
(C) Ho. atra Vanua Levu
–
Taveuni
0.3287
–
Naviti
0.0865
0.00000
–
Vanuabalavu
0.5042
0.00000
0.1395
–
Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. atra populations at the island-
assemblage level in Fiji. Significant p-values are in bold. Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. atra populations at the island-
assemblage level in Fiji. Significant p-values are in bold. Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. atra populations at the island-
assemblage level in Fiji. Significant p-values are in bold. Population Structure and Power Analyses. E. merra was characterized by very low and non-significant
measures of genetic differentiation at the island level (pairwise ΦST values in Table 3A), after an AMOVA
grouping the two Vanuabalavu sites together (Naracivo and Donicali) resulted in non-significant F-statistics
for among-group (FCT), within-group (FSC), and within-population (FST) genetic differentiation (Table 4A). Sampling locations were also assembled into 4 groups that reflect the political units of Fiji (e.g. grouping Nagigi
and Naselesele together, as they both belong to the Northern Division), and this second AMOVA also revealed
non-significant F-statistics for each hierarchical level (Table 4B). gi
Ha. trimaculatus displayed slightly different patterns of genetic partitioning. This species’ pairwise ΦST
values were similarly low but exhibited significant north-south genetic partitioning between the Vanua Levu
village of Nagigi and the Viti Levu village of Nabukavese (ΦST = 0.07 p = 0.032, Table 3B). An AMOVA clustering
Ha. trimaculatus populations into 4 groups reflecting political units within Fiji also revealed non-significant
F-statistics for each hierarchical level (Table 4C). Analysis of Ho. atra populations returned a wide range of pairwise ΦST values (Table 3C). Very low values
were observed between Taveuni and both the eastern island of Vanuabalavu and the western island and Naviti,
and relatively high values between Vanua Levu and all other island sites. The highest pairwise ΦST value occurred
between Vanua Levu and Vanuabalavu. Results
G
ti Genetic Diversity. We sequenced 475 bp of the mtDNA control region from 82 individuals of E. merra,
376 bp of the same locus from 91 individuals of Ha. trimaculatus, and 376 bp of the COI gene from 40 individuals
of Ho. atra (Table 1). Both fish species displayed high haplotype diversity and had highly polymorphic control
regions (87 and 71 polymorphic loci within E. merra and Ha. trimaculatus sequences, respectively), while Ho. atra
COI sequences were considerably less genetically diverse (Table 2) and had only 15 polymorphic loci. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 2 www.nature.com/scientificreports/ Division
Island (Village)
Latitude,
Longitude
Epinephelus merra
(N = 82)
Halichoeres trimaculatus
(N = 91)
Holothuria atra (N = 40)
Northern
Vanua Levu (Nagigi)
S 16° 80’ 44.18”
W 179° 48'05.53”
20
23
10
Northern
Taveuni (Naselesele)
S 16° 41’ 41.19”
W 179° 52’ 1.52”
12
21
5
Western
Naviti (Malevu)
S 17° 6’ 54.12”
E 177° 16’ 51.18”
11
16
7
Central
Viti Levu (Nabukavesi)
S 18° 13’ 20.42”
E 178° 15’ 58.08”
16
15
–
Eastern
Vanuabalavu (Naracivo, Donicali)
S 17° 13’ 15.84”
W 178° 57’ 53.57”
23
16
18
Table 1. Geographic locations of sampling sites, and sample sizes for each species. Table 1. Geographic locations of sampling sites, and sample sizes for each species. Table 1. Geographic locations of sampling sites, and sample sizes for each species. Table 1. Geographic locations of sampling sites, and sample sizes for each species. Species
Individuals
Haplotypes
π
Θs (SD)
Sequence
Length
Larval
Duration
Epinephelus merra
82
66
0.03
16.67 (4.51)
475
39 days
Halichoeres trimaculatus
91
75
0.03
13.97 (3.77)
376
27 days
Holothuria atra
40
8
0.01
3.18 (1.61)
376
18–25 days
Table 2. Results of sequencing the mitochondrial control region, and larval durations for each study
species64,73,74. Table 2. Results of sequencing the mitochondrial control region, and larval durations for each study
species64,73,74. Vanua Levu
Taveuni
Naviti
Vanuabalavu
(A) E. merra Vanua Levu
–
Taveuni
0.00000
–
Naviti
0.05327
0.01336
–
Vanuabalavu
0.00702
0.00000
0.00000
–
Viti Levu
0.00602
0.00000
0.00602
0.00000
(B) Ha. trimaculatus Vanua Levu
–
Taveuni
0.00547
–
Naviti
0.00000
0.00000
–
Vanuabalavu
0.00000
0.00000
0.00000
–
Viti Levu
0.07232
0.00196
0.02021
0.01350
(C) Ho. atra Vanua Levu
–
Taveuni
0.3287
–
Naviti
0.0865
0.00000
–
Vanuabalavu
0.5042
0.00000
0.1395
–
Table 3. Pairwise ΦST values for (A) E. merra (B) Ha. trimaculatus and (C) Ho. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 Results
G
ti Analysis of molecular variance using the mitochondrial control region between (A) five groups of
E. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages,
(B) four groups of E. merra (Northern Division, Eastern Division, Western Division, Central Division)
reflecting political boundaries, and (C) four groups of Ha. trimaculatus (Northern Division, Eastern Division,
Western Division, Central Division) reflecting political boundaries. Negative values are presented, but are
effectively equal to zero. Figure 2. Haplotype network of Ho. atra samples. Each circle represents a haplotype. Size of the circle is
scaled to the number of individuals that share that haplotype, Hash marks on connecting lines indicate the
number of base pair differences between haplotypes. Shading represents the island where individuals were
sampled. Figure 2. Haplotype network of Ho. atra samples. Each circle represents a haplotype. Size of the circle is
scaled to the number of individuals that share that haplotype, Hash marks on connecting lines indicate the
number of base pair differences between haplotypes. Shading represents the island where individuals were
sampled. island of Naviti had the highest number of individuals with private alleles, and the eastern island of Vanuabalavu
had the lowest measures of intra-population genetic diversity.i Results of the statistical power analyses revealed that in both fish species, the control region markers had
sufficient power to detect genetic differentiation above a ΦST value of 0.01, which corresponds with low levels of
genetic differentiation and high amounts of connectivity (Fig. 3)31. Migration Estimates. MIGRATE analyses resolved high levels of migration but with complex patterns of
directionality among populations. For E. merra, the individuals from Nabukavesi, located on the southern large
island of Viti Levu appear to be a major source population, providing high numbers of migrants per generation
to other localities – in some cases, up to 10 times the amount migrants received (Table 5). These patterns are the
opposite of those observed in Ha. trimaculatus – with the site on northern large island of Vanua Levu serving
as a source and Viti Levu as a sink (Table 5). Additionally, in both species the western island of Naviti provided
more migrants per generation to the far eastern island of Vanuabalavu than vice versa (3.5 times more in E. merra,
2.5 times more in Ha. trimaculatus).i MIGRATE analyses of Ho. Results
G
ti Because genetic partitioning was detected between the same-Division
islands of Vanua Levu and Taveuni at this initial step, an AMOVA further grouping locations together was not
performed. Further, the majority of Ho. atra samples (n = 35) fit into one of three haplotypes (Fig. 2). The most
highly represented haplotype (n = 29) was found in individuals from all four sampling locations. The western Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 3 www.nature.com/scientificreports/ Source of variation
d.f. Sum of
squares
Percentage of
variation
Statistics
P
(A) Among groups
4
22.069
− 2.02
FCT = − 0.020
0.785
Among populations within groups
1
6.865
1.98
FSC = 0.019
0.218
Within populations
76
437.262
100.04
FST = 0.000
0.424
(B) Among groups
3
17.781
1.59
FCT = 0.016
0.298
Among populations within groups
1
4.288
− 1.71
FSC = − 0.017
0.617
Within populations
77
444.127
100.13
FST = − 0.001
0.490
(C) Among groups
3
14.387
− 0.10
FCT = − 0.001
0.723
Among populations within groups
1
5.034
0.61
FSC = 0.006
0.295
Within populations
85
377.046
99.49
FST = 0.005
0.281
Table 4. Analysis of molecular variance using the mitochondrial control region between (A) five groups of
E. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages,
(B) four groups of E. merra (Northern Division, Eastern Division, Western Division, Central Division)
reflecting political boundaries, and (C) four groups of Ha. trimaculatus (Northern Division, Eastern Division,
Western Division, Central Division) reflecting political boundaries. Negative values are presented, but are
effectively equal to zero. Source of variation
d.f. Sum of
squares
Percentage of
variation
Statistics
P
(A) Among groups
4
22.069
− 2.02
FCT = − 0.020
0.785
Among populations within groups
1
6.865
1.98
FSC = 0.019
0.218
Within populations
76
437.262
100.04
FST = 0.000
0.424
(B) Among groups
3
17.781
1.59
FCT = 0.016
0.298
Among populations within groups
1
4.288
− 1.71
FSC = − 0.017
0.617
Within populations
77
444.127
100.13
FST = − 0.001
0.490
(C) Among groups
3
14.387
− 0.10
FCT = − 0.001
0.723
Among populations within groups
1
5.034
0.61
FSC = 0.006
0.295
Within populations
85
377.046
99.49
FST = 0.005
0.281 Table 4. Analysis of molecular variance using the mitochondrial control region between (A) five groups of
. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages, Table 4. Table 4. Analysis of molecular variance using the mitochondrial control region between (A) five groups of
E. merra (Naviti, Vanua Levu, Taveuni, Vanuabalavu, Viti Levu) reflecting island-level assemblages,
(B) four groups of E. merra (Northern Division, Eastern Division, Western Division, Central Division)
reflecting political boundaries, and (C) four groups of Ha. trimaculatus (Northern Division, Eastern Division,
Western Division, Central Division) reflecting political boundaries. Negative values are presented, but are
effectively equal to zero. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. POWSIM analysis results showing power to detect structure among populations of Epinephelus
merra and Halichoeres trimaculatus at different FST levels. Power is expressed as the proportion of significant
outcomes after 1,000 replicates. Dashed line is at 0.80, the minimum acceptable power level put forth by72. Figure 3. POWSIM analysis results showing power to detect structure among populations of Epinephelus
merra and Halichoeres trimaculatus at different FST levels. Power is expressed as the proportion of significant
outcomes after 1,000 replicates. Dashed line is at 0.80, the minimum acceptable power level put forth by72. Species
Vanua Levu
Taveuni
Naviti
Vanuabalavu
Viti Levu
E. merra
Vanua Levu
–
5850.0
543.3
896.7
576.7
Taveuni
3183.3
–
276.7
1516.7
1256.7
Naviti
7490.0
2676.7
–
656.7
690.0
Vanuabalavu
6536.7
2436.7
190.0
–
2023.3
Viti Levu
5530.0
1403.3
116.7
1790.0
–
Ha. trimaculatus
Vanua Levu
–
296.7
916.7
1683.3
1050.0
Taveuni
523.3
–
683.3
450.0
1330.0
Naviti
736.7
450.0
–
1636.7
3930.0
Vanuabalavu
316.7
430.0
623.3
–
1936.7
Viti Levu
550.0
516.7
1123.3
1250.0
–
Ho. atra
Vanua Levu
–
522.3
479.7
555
–
Taveuni
455
–
514.3
411.7
–
Naviti
438.3
487
–
388.3
–
Vanuabalavu
533
672.3
582.3
–
–
Table 5. Results of MIGRATE analysis for each species. Median number of recruits per generation is shown,
with migration occurring from row to column Table 5. Results of MIGRATE analysis for each species. Median number of recruits per generation is shown,
with migration occurring from row to column. Table 5. Results of MIGRATE analysis for each species. Median number of recruits per generation is shown,
with migration occurring from row to column. Finally, the Mantel test revealed negative or flat levels of correlation between pairwise measures genetic
differentiation and geographic distance in all three species (E. merra R2 = 0.001, Ha. trimaculatus R2 = 0.005,
Ho. atra R2 = 0.010), indicating that none of these species’ patterns of genetic differentiation can be explained by
geographic distance between populations. Results
G
ti atra sequences showed that three of the five highest migration values were from
the eastern island of Vanuabalavu out to more western sites. The highest estimated median number of migrants
per generation was from Vanuabalavu to Taveuni (672.3), while the lowest estimated number of migrants per
generation was from Malevu to Vanuabalavu (388.3-Table 5). There were more westward moving migrants than
eastward moving migrants for four of the six pairs of sites. (Table 3). Tajima’s D values were negative for both E. merra and Ha. trimaculatus (−1.15228 and −1.20336, respec-
tively), and positive for Ho. atra (0.71194). All values were non-significant (p = 0.109, p = 0.094, p = 0.712,
respectively). Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 4 Discussionf More oceanographic investigation is necessary to understand if larvae spawned at
Taveuni are brought out to sea by a current that bypasses the reefs on the southern edge of Vanua Levu. Skillings
et al. 2011 found Ho. atra to be genetically differentiated across distances comparable to that between Vanua Levu
and Taveuni (74.8 km), suggesting that there are many other factors besides this species’ larval potential to drift
great distances that determine larval dispersal and consequent connectivity.h g
p
q
y
The most frequent Ho. atra haplotype, accounting for almost 58% of samples, is found in all four sampled loca-
tions, suggesting an overall trend of connectivity across the region despite observed differences in the degree of
connectivity in each pair of sites. The boom-bust demographic cycle of Ho. atra38 may explain how all populations
came to share the dominant haplotype, but still display levels of differentiation between populations. This shared
haplotype may be the vestige of a time when there were large population sizes and high rates of migration, while
the varying levels of genetic differentiation may be the result of subsequent population contraction and lesser
gene flow across the archipelago. This is supported by Ho. atra’s positive and non-significant Tajima’s D, which
indicates that the sea cucumber populations studied may have undergone recent demographic contraction39. p p
y
g
g p
Results of the MIGRATE analyses were complex and idiosyncratic among all three species studied (Table 5). Gene flow patterns support a scenario of high connectivity for both fish species, with the magnitude of migrants
exchanged among populations estimated at hundreds to thousands per generation. Patterns of directionality in
E. merra and Ha. trimaculatus were complex and occasionally contradictory – for instance, Vanua Levu exported
more migrants than it received from other locations in E. merra, whereas in Ha. trimaculatus the opposite was
true. Both fish species also exhibited higher numbers of migrants coming from the western locality of Naviti and
settling in the eastern island of Vanuabalavu than vice versa, indicating that western populations could be seeding
eastern localities in spite of the prevailing westward-moving oceanic currents. Ho. atra shows a different pattern,
in which the larvae seem to follow the oceanic currents resulting in the eastern island of Vanuabalavu exporting
higher numbers of larval migrants to the western localities than vice versa. Discussionf This concurs with previous studies
showing unidirectional patterns of east-to-west gene flow in several taxa28. Ho. atra’s east to west pattern of gene
flow is also supported by the haplotype diversities of each location, as the western population of Naviti, in the
Yasawa islands, has the highest intra-population genetic diversity while Vanuabalavu, which lies farthest to the
east in the Lau group, has the lowest. This suggests that larvae spawned in Nagigi, Taveuni, Vanuabalavu and other
unsampled populations may potentially recruit more often to reefs in the western Yasawa Islands, promoting high
genetic diversity. These contrasting patterns of gene flow observed between the fishes and the sea cucumber could
be due to demographic stochasticity, differences in reproductive mode or timing, or seasonal variation in the flow
of the Bligh Waters, which is common in other intra-pelagic currents40.h g
p
g
The dearth of literature on the inter- and intra-annual physical oceanography of the Fijian archipelago makes
it difficult to assess the role of variation in currents on the data presented here. Genetic connectivity studies
conducted in regions with better-studied oceanography have shown both inter-annual and intra-annual ocean-
ographic features to be responsible for population connectivity or structure. Galarza et al.41 found oceanic fronts
in the Mediterranean Sea to be associated with genetic structuring for several fish species, presumably due to
the inability of larvae spawned on one side of the front to disperse across the front and admix with populations
on the other side. Similarly, the Kuroshio Current in the East China Sea limits dispersal and therefore generates
population differentiation between mudskippers in China and those in Japan42. Mesoscale eddies, which occur on
a length scale of less than 100 km and last less than one month, have been implicated in keeping newly spawned
larvae close to their natal site, as opposed to dispersing out into the open ocean43. Interpretations of genetic
structuring and connectivity patterns found across species studied in the Fijian archipelago, as well as the idiosyn-
crasies in the data, almost certainly require a better understanding of the underlying oceanography. This clearly
presents a priority for future research.hi p
p
y
The Tajima’s D values for both E. merra and Ha. trimaculatus were strongly negative and while non-significant,
they may indicate that both species may have undergone rapid population expansion in recent evolutionary his-
tory. Discussionf Different patterns of connectivity were observed for each of the three species in this study, highlighting the com-
plexity of larval transport and population demography in the marine environment. High amounts of gene flow
and very low amounts of genetic structure were observed for both fish species, indicating that on the relatively
small spatial scale of the Fijian archipelago, populations of E. merra and Ha. trimaculatus are interconnected. High gene flow is a common phenomenon in marine species, as the marine environment has relatively few phys-
ical barriers and long pelagic larval durations often promote long-distance dispersal32–34.h t
The high level of connectivity we observed among populations of E. merra is not surprising, and mirrors con-
nectivity levels found in the Philippines35 and the Western Indian Ocean36. Ha. trimaculatus generally exhibited
high levels of connectivity among populations as well, but this species did show a subtle yet significant amount of
genetic partitioning between the northern island of Vanua Levu and southern island of Viti Levu. These findings
support the hypothesis that the Bligh Waters may be a major oceanographic feature affecting connectivity in Fiji – a
|pattern observed in a previous comparative phylogeographic study28.l Fast-flowing currents like the Bligh Waters can act as barriers to dispersal by advecting larvae out of the system
before they are able to settle on reefs opposite of the current, thus preventing genetic exchange between popu-
lations20–23. Ha. trimaculatus’ low amount of genetic partitioning may indicate that the Bligh Waters has been
acting as an oceanographic barrier for a relatively short evolutionary time scale, or that historical factors could Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 5 www.nature.com/scientificreports/ also have contributed to this species’ subtle phylogeographic structure – for instance, sea level shifts influencing
habitat availability37. y
In contrast, the sea cucumber Ho. atra exhibited complex patterns of connectivity among the four populations
sampled. There was little to no genetic differentiation between Taveuni and the islands of the Lau group, but high
amounts of partitioning between Taveuni and Vanua Levu despite these two locations’ geographic proximity. The populations in the Lau group were also genetically divergent from those in Vanua Levu. This may be due
to oceanographic characteristics of the Somosomo Strait, the narrow channel that passes between the islands
of Taveuni and Vanua Levu. www.nature.com/scientificreports/ www.nature.com/scientificreports/ study did miss populations exhibiting partitioning at these levels, the conclusions drawn would be effectively the
same – that both fish species exhibit high amounts of connectivity between their populations31. study did miss populations exhibiting partitioning at these levels, the conclusions drawn would be effectively the
same – that both fish species exhibit high amounts of connectivity between their populations31. i
p
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In Ho. atra, on the other hand, this study observed very low haplotype diversities across all locations, even in
Vanuabalavu where sample sizes were high (n = 18). While low sample sizes understandably lead to low observed
genetic diversity, it is also likely that this species’ reproductive ecology (e.g. fission, or budding) has contributed to
higher local densities of genetically identical individuals than in exclusively sexually reproducing species. Thus,
while results for this species should certainly be taken with caution, it is possible that low sample sizes in this
study may be capturing an adequate amount of the gene pool at each location.t y
y
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q
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Critiques for mtDNA-based phylogeographic studies often concern the variable rate of mutation observed
between these markers and others – mtDNA often reach higher FST values than nuclear DNA45 – and the lack of
power caused by high levels of diversity46. However, rapid mutation rates of mtDNA have been shown to provide
high sensitivity for detecting genetic differentiation on short evolutionary time scales, rendering these markers
highly useful for studies like this one investigating population-level connectivity47–49. In addition, a recent anal-
ysis has shown that mtDNA markers consistently display higher power to detect population divergence than any
single nuclear marker when sample sizes are sufficient50. As a result, mtDNA sequences have been the markers
of choice for marine connectivity studies in the Indo-Pacific for the past 20 years51. While multi-locus analyses
certainly allow for a more nuanced evaluation of connectivity, it is not necessarily the case that adding nuclear loci
would reveal significantly more information52. gi
y
Additionally, use of markers that have been used for previous phylogeography studies allows for easier com-
parison of results across taxa and locations51. The control region was used to analyse genetic connectivity in the
only other study of fish biogeography in Fiji28, and thus we chose this ‘legacy marker’ for our analyses of the fish
data. www.nature.com/scientificreports/ Similarly, the COI was used in a Hawai’ian biogeography study of Ho. atra53, so we chose to use the same
marker in this study for the sake of comparison. Using relatively inexpensive markers and technologies for analy-
ses in biogeography studies makes such studies more doable across underrepresented taxa and regions, and thus
broadens the scope of known genetic connectivity patterns across the Pacific. This is especially critical from a
management perspective. Conclusions and Management Implicationsh The high connectivity observed among populations of the fish species suggests that prioritizing one region over
another for the placement of marine reserves in Fiji is not necessary for the conservation and persistence of
E. merra and Ha. trimaculatus populations. Instead, each region sampled should be considered equally important
for protection, as each node in these meta-populations contributes ecologically and economically significant
amounts of migrants to the others. This finding provides managers with greater flexibility in conservation pri-
oritization, as they can focus on areas that have strong local support for conservation, or areas like the Vatu-i-Ra
seascape (between Viti Levu and Vanua Levu), which have been identified as having exceptional habitat quality54. Placing some portion of habitat within these regions under protection will promote the persistence and resilience
of the entire system, while enabling fine-scale locations of protected areas to be determined by habitat representa-
tion, social and economic factors, or other aspects of reserve network design. This may help alleviate the tensions
that often exist during marine spatial planning, between scientific information and social or economic priorities55. C
l
i h H
l i
ll
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h
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hi h ti
Conversely, with Ho. atra, some populations are well connected to each other and others are isolated, which
necessitates a more nuanced spatial management scenario. For instance, since the Vanua Levu and Taveuni pop-
ulations are genetically differentiated, it is recommended that they be treated as separate management areas
to preserve their genetic diversity, despite the fact that both lie within Fiji’s Northern Division. Additionally,
since the Vanuabalavu population in the Lau archipelago to the east likely provides larval recruits and thereby
genetic diversity to western reefs, this region should be a conservation priority for the management of the species. Upstream areas, such as Vanuabalavu for Ho. atra and Naviti for the fish, should be considered priorities for mon-
itoring, as the health of downstream populations is largely reliant on the well-being of upstream areas. Ultimately, our data suggest management at the larger Division or Provincial scale may be appropriate for
species like E. merra and Ha. trimaculatus that exhibit high amounts of connectivity within the country, but for
those like Ho. atra with low connectivity and high genetic partitioning among populations, management at the
provincial or local scale may be required to ensure the persistence of isolated populations. Discussionf Because the program MIGRATE assumes stable population dynamics, migration estimates can be strongly
influenced by population expansion or contraction. Thus, the results of gene flow in the fish species should be
taken with caution, particularly in the case of directionality. While the Tajima’s D value for Ho. atra was slightly
positive, potentially indicating that this species has undergone a recent population bottleneck, the p-value for
this estimate was much larger than those of the fishes and therefore likely does not have a significant effect on
MIGRATE analyses39. y
Another caveat of this study is its low sample sizes for E. merra at two locations (n = 10 and 12), and for
Ho. atra at three locations (n = 10, 7, and 5). High amounts of nucleotide and haplotype diversity, like those found
in E. merra, can sometimes be a relic of low sample sizes - however, high genetic diversity is a very common find-
ing in marine fishes, and studies with very large sample sizes often observe high haplotype diversities as well33,44. Additionally, Ha. trimaculatus displayed almost identical measures of haplotype and nucleotide diversity, while
having larger sample sizes than E. merra at nearly every location. Our power analyses show that with the sample
sizes used for E. merra and Ha. trimaculatus, the diverse mtDNA control region confers low statistical power in
detecting very weak levels of differentiation (ΦST of 0.01 or below), thus limiting our study’s ability to rule out
a type II error within this range. However, the magnitude of differentiation below 0.01 is so low that even if our Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 6 Methods
d Study species. Epinephelus merra and Halichoeres trimaculatus are both widespread Indo-Pacific coral reef
fishes, and are commonly targeted by fishermen in Fiji56,57. E. merra is typically caught by spear or by hook and
line, and is most commonly used for subsistence and occasionally sold in fish markets. Ha. trimaculatus is caught
primarily via net, and is more often used for subsistence or fed to livestock, depending on their size at harvest. Both species are found in protected seaward reefs and lagoonal areas, and both have small home ranges, rarely
moving more than 1 km away from their adult home reefs58,59.ii g
y
Holothuria atra, known as the black sea cucumber or the lollyfish, is a major commercial fisheries target
throughout the Indo-Pacific60,61. Ho. atra can reproduce both sexually via broadcast spawning, and asexually by
fissioning into anterior and posterior parts and then regenerating a full body from each part38,62,63. Ho. atra’s larval
duration lasts approximately 18–25 days53,64, and its range extends from the Western Indian Ocean to the Eastern
Pacific Ocean. Like the fishes, they tend to be relatively sedentary as adults with congeners traveling < 20 m per
day65. Ethics Statement. The study protocol was approved by the Columbia University Animal Care Committee
(protocol no. AC-AAAF6300) and followed the laws of the Republic of Fiji and with permission of the traditional
marine resource owners. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 7 www.nature.com/scientificreports/ Sample collection. We sampled at six locations across Fiji (Fig. 1). Study sites were chosen to maximize
regional coverage within the country, and sites were spread across the four Divisions of Fiji (Northern, Western,
Central, and Eastern). Sample collection. We sampled at six locations across Fiji (Fig. 1). Study sites were chosen to maximize
regional coverage within the country, and sites were spread across the four Divisions of Fiji (Northern, Western,
Central, and Eastern). ,
)
Fish were collected by net, spear or through purchase from local fishermen, while Ho. atra were collected by
hand (Table 1). After samples were collected, gill clips from fish, or dermal clips from Ho. atra were taken and
stored in ethanol or frozen in liquid nitrogen to preserve DNA. The whole samples were then fixed in formalin for
accession into the ichthyology collections at the American Museum of Natural History. Genetic Analyses. Genomic DNA was extracted from gill clips and dermal samples using a DNeasy Tissue
Kit (Qiagen, Hilden, Germany). Methods
d For fish samples, we amplified the mitochondrial control region by PCR using
the primers CR-A and CR-E66. For Ho. atra we amplified part of the cytochrome c oxidase 1 (COI) gene using
primers designed by53. PCR parameters can be found in Supplementary Note S1 online. Sequences were gen-
erated on an ABI 3730 sequencer (Applied Biosystems, Inc., Foster City, CA, USA) and sequence quality was
checked and sequences were aligned using Geneious 8.067. Sequences were deposited in GENBANK with the
following accession numbers: E. merra KT353114-KT353195, Ha. trimaculatus KT329098-KT329188, Ho. atra
KT378456-KT378495. Data Analysis. To investigate the amount of genetic differentiation occurring between sampled populations,
pairwise ΦST measures (a modified F-statistic specific to mitochondrial DNA) were calculated for each species in
ARLEQUIN 3.568, using 1000 replicates to estimate significance. Because of high haplotype diversities observed
in the fish species and relatively low sample sizes, the statistical power of the control region marker was evaluated
with POWSIM69, using a Ne of 5,000 and adjusting generation time (t) to assess power at multiple FST values
(0.001, 0.0025, 0.005, 0.01, and 0.02). Power was expressed as the proportion of significant outcomes for 1,000
replicates. p
To test whether regional-level genetic structuring was present, analyses of molecular variance (AMOVAs)
were also performed in ARLEQUIN. For E. merra, each of the 6 sampling localities was initially treated as a
separate entity, and when no significant differences were detected between the villages of Donicali and Naracivo
(13 km apart on the island of Vanuabalavu), the data were pooled into island-level assemblages and reanalysed. Extremely low sample sizes for Ha. trimaculatus and Ho. atra in Donicali (n = 3 and n = 4, respectively) would
have made individual locality results unreliable, so these two species were grouped at the island-level at the outset. Further, if no differentiation was detected between the island-level assemblages for each species, analyses were
repeated with populations grouped by Division to investigate whether Fiji’s large-scale political boundaries are
appropriate for the management of biological populations.l pp
p
g
g
p p
We calculated magnitude and direction of gene flow at the island-level using MIGRATE-n70. Methods
d Two runs of
1,000,000 generations were conducted for each species with an initial 25% burn-in, Because of this program’s
underlying assumptions that population size and migration rates have not changed over time, Tajima’s D statistics
were determined in ARLEQUIN, to assess the likelihood of recent demographic expansion or contraction for
each species. p
To test whether any observed genetic differences could be due to geographic distance versus phylogeogra
barriers, a Mantel test was also performed for each species using Isolation By Distance 3.2371. References 1. Cesar, H., Burke, L. & Pet-Soede, L. The Economics of Worldwide Coral Reef Degradation. (2003). Available at: http://eprints.eriub
org/48/. Date of access: 01/09/2013.h y
f
p
fh
f
3. Roberts, C. et al. Marine biodiversity hotspots and conservation priorities for tropical reefs. Science 295, 1280–84 (i y
p
p
p
,
4 Myers R & Worm B Rapid worldwide depletion of predatory fish communities Nature 423, 280–83 (2003) y
p
p
p
4. Myers, R. & Worm, B. Rapid worldwide depletion of predatory fish communities. Nature 423, 280–83 (2003).f i
5. Selig, E. & Bruno, J. A global analysis of the effectiveness of marine protected areas in preventing coral loss. PLoS One 5,
(2010).h (
)
6. Halpern, B. The impact of marine reserves: do reserves work and does reserve size matter? Ecol. Appl. 13, 117–137 (2003).fi h
pp
7. Roberts, C., Bohnsack, J., Gell, F., Hawkins, J. & Goodridge, R. Effects of marine reserves on adjacent fisheries. Science 294, 1920–2
(2001).i 8. Gaines, S., White, C., Carr, M. & Palumbi, S. Designing marine reserve networks for both conservation and fisheries management. Proc. Natl. Acad. Sciences 107, 18286–93 (2010).i ,
(
)
9. Kritzer, J. & Sale, P. Metapopulation ecology in the sea: from Levins’ Model to marine ecology and fisheries science. Fish Fish 5,
131–40 (2004). (
)
10. Kininmonth, S. et al. Dispersal connectivity and reserve selection for marine conservation. Ecol. Modelling 222, 1272–82 (201 10. Kininmonth, S. et al. Dispersal connectivity and reserve selection for marine conservation. Ecol. Modelling 222, 1272–82 (2011). 11. Almany, G. et al. Connectivity, biodiversity conservation and the design of marine reserve networks for coral reefs. Coral Reefs 28,
339–51 (2009). 2. Roberts, C. et al. Application of ecological criteria in selecting marine reserves and developing reserve networks. Ecol. Appl. 13
215–28 (2003). 13. McLeod, E., Salm, R., Green, A. & Almany, J. Designing marine protected area networks to address the impacts of climate change. Front. Ecol. Environ. 7, 362–70 (2008).i 14. Green, A. et al. Designing marine reserves for fisheries management, biodiversity conservation, and climate change adaptation
Coast. Manage. 42, 143–59 (2014).ii g
(
)
15. Dixson, D. et al. Coral reef fish smell leaves to find island homes. Proc. Royal Soc. B: Biol. Sci. 275, 2831–39 (2008).fi ii
16. Simpson, S., Meekan, M., Jeffs, A., Montgomery, J. & McCauley, R. Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 20. Barber, P., Palumbi, S., Erdmann, M. & Moosa, M. Sharp genetic breaks among populations of Haptosquilla pulchella (Stomatop
indicate limits to larval transport: patterns, causes, and consequences. Mol. Ecol. 11, 659–74 (2002).i 21. Rocha, L. Patterns of distribution and processes of speciation in Brazilian reef fishes. Journ. Biogeog. 30, 1161–71 (2003).h p
pi
g
g
22. Thornhill, D., Mahon, A., Norenburg, J. & Halanych, K. Open-ocean barriers to dispersal: a test case with the Antarctic Polar
and the ribbon worm Parborlasia corrugatus (Nemertea: Lineidae). Mol. Ecol. 17, 5104–5117 (2008). g
(
)
,
(
)
23. Treml, E., Halpin, P., Urban, D. & Pratson, L. Modeling population connectivity by ocean currents, a graph-theoretic approach for
marine conservation Landscape Ecol 23 19–36 (2008) 23. Treml, E., Halpin, P., Urban, D. & Pratson, L. Modeling population connectivity by ocean currents, a graph-theoretic approac
marine conservation. Landscape Ecol. 23, 19–36 (2008). p
g p p
marine conservation. Landscape Ecol. 23, 19–36 (2008). p
,
(
)
24. Vuilleumier, S. & Possingham, H. Does colonization asymmetry matter in metapopulations? Proc. Royal Soc. London B: Biol. Sci. 273, 1637–42 (2006). 25. Jupiter, S., Mills, M., Compley, J., Batibasaga, A. & Jenkins, A. Fiji marine ecological gap assessment: interim progress report. (2010)
Wildlife Conservation Society, Suva, Fiji. 26 pp. Available at: http://www.wcsfiji.com/Resources/Reports.aspx Date of access:
01/04/2015.f 26. Mills, M., Jupiter, S., Pressey, R., Ban, N. & Comley, J. Incorporating effectiveness of community-based management in a nat
marine gap analysis for Fiji. Cons. Biol. 25, 1155–64 (2011). 27. Mills, M., Adams, V., Pressey, R., Ban, N. & Jupiter, S. Where do national and local conservation actions meet? Simulating the
expansion of ad hoc and systematic approaches to conservation into the future in Fiji: marginal benefits of conservation planning. Cons. Letters 5, 387–98 (2012).i 28. Drew, J. & Barber, P. Comparative phylogeography in Fijian coral reef fishes: a multi-taxa approach towards marine reserve design. PLoS ONE 7, e47710 (2012). 29. Asian Development Bank. Fisheries Sector Review: Republic of the Fiji Islands. Technical Assistance Report TA4403, 1 (2005). Available at: http://www.adb.org/projects/documents/fisheries-sector-review-0. Date of access: 01/09/2013.i 29. Asian Development Bank. Fisheries Sector Review: Republic of the Fiji Islands. Technical Assistance Report TA4403, 1 (2005). Available at: http://www.adb.org/projects/documents/fisheries-sector-review-0. Date of access: 01/09/2013.i g
ji
0. Food and Agriculture Organization of the United Nations (FAO). Fiji: Fishery and Aquaculture Country Profile. (2005). Available
at: http://www.fao.org/fishery/countrysector/FI-CP_FJ/1 Date of access: 01/09/2013. www.nature.com/scientificreports/ Strong genetic but not spatial subdivision of two reef fish species targeted by
fishers on the Great Barrier Reef. Fish. Res. 102, 16–25 (2010).hf fishers on the Great Barrier Reef. Fish. Res. 102, 16–25 (2010).h i
5. Nabholz, B., Glémin, S. & Galtier, N. The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA
diversity across birds and mammals. BMC Evol. Biol. 9, 54 (2009). i
45. Nabholz, B., Glémin, S. & Galtier, N. The erratic mitochondrial h
diversity across birds and mammals. BMC Evol. Biol. 9, 54 (200 y
6. Galtier, N., Nabholz, B., Glémin, S. & Hurst, G. Mitochondrial DNA as a marker of molecular diversity: a reappraisal. Mol. Ecol. 18
4541–4550 (2009).i 47. Avise, J. et al. Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Syst. 18, 489–522 (1987).ffl 48. Waples, R. Separating the wheat from the chaff: patterns of genetic differentiation in high gene flow species. Journ. Hered
438–450 (1998). (
)
49. Hedgecock, D., Barber, P. & Edmands, S. Genetic approaches to measuring connectivity. Oceanography 20, 70–79 (2007). 49. Hedgecock, D., Barber, P. & Edmands, S. Genetic approaches g
g
y
g
p y
50. Larsson, L., Charlier, J., Laikre, L. & Ryman, N. Statistical power for detecting genetic divergence – organelle versus nuclear markers. Cons. Gen. 10, 1255–1264 (2009).hi 1. Keyse, J. et al. The scope of published population genetic data for Indo-Pacific marine fauna and future research opportunities in the
region. Bull. Marine Sci. 90, 47–78 (2014). g
52. Karl, S., Toonen, R., Grant, W. & Bowen, B. Common misconceptions in molecular ecology: echoes of the modern synthesis. Mol. Ecol. 21, 4171–4189 (2012). 53. Skillings, D., Bird, C. & Toonen, R. Gateways to Hawai’i: genetic population structure of the tropical sea cucumber Holothuria atra. Journ. Marine Biol. 2011, 1–16 (2011). 4. Jupiter, S., Mcclennen, C. & Matthews, E. “Vatu-i-Ra Seascape, Fiji”. In Climate and Conservation, pp. 155–169 (Island Press/Center
for Resource Economics, 2012).fii 55. Gurney, G. et al. Efficient and equitable design of marine protected areas in Fiji through inclusion of stakeholder-specific objectives
in conservation planning. Cons. Biol. 29, 1378–1389 (2015).fif 56. Jennings, S. & Polunin, N. Relationships between catch and effort in Fijian multispecies reef fisheries subject to different levels of
exploitation. Fish. Manage. Ecol. 2, 89–101 (1995).i p
g
(
)
7. Golden, A., Naisilsisili, W., Ligairi, I. & Drew, J. www.nature.com/scientificreports/ p
gi
y
y
1. Wright, S. Evolution and the Genetics of Populations, Vol. 4: Variability within and among populations. Univ. Chicago Press, Chicago
(1978).ii 32. Craig, M., Eble, T. & Bowen, B. High genetic connectivity across the Indian and Pacific Oceans in the reef fish Myripristis berndti
(Holocentridae). Mar. Ecol. Prog. Ser 334, 245–54 (2007).i g
3. Horne, J., van Herwerden, L., Choat, J. & Robertson, D. High population connectivity across the Indo-Pacific: congruent lack o
phylogeographic structure in three reef fish Congeners. Mol. Phylogenet. Evol. 49, 629–38 (2008). i
34. Teske, P., Forget, F., Cowley, P., von der Heyden, S. & Beheregaray, L. Connectivity between marine reserves and exploited areas in
the philopatric reef fish Chrysoblephus laticeps (Teleostei: Sparidae). Mar. Biol. 157, 2029–42 (2010).li i
35. Matias, A., Anticamara, J. & Quilang, J. High gene flow in reef fishes and its implications for ad-hoc no-take marine reserves. MDN
24, 584–95 (2013). 6. Muths, D., Tessier, E. & Bourjea, J. Genetic structure of the reef grouper Epinephelus merra in the West Indian Ocean appear
congruent with biogeographic and oceanographic boundaries. Mar. Ecol. 36, 447–461 (2014).i 37. Ludt, W., Bernal, M., Bowen, B. & Rocha, L. Living in the past: phylogeography and population histories of Indo-Pacific wrasses
(genus Halichoeres) in shallow lagoons versus outer reef slopes. PLoS ONE 9, e38042 (2012). 8. Uthicke, S., Schaffelke, B. & Byrne, M. A boom-bust phylum? Ecological and evolutionary consequences of density variations in
echinoderms. Ecol. Monographs 79, 3–24 (2009).i g
p
39. Tajima, F. Evolutionary relationship of DNA sequences in finite populations. Genetics 105, 437–460 (1983). j
y
p
qi
p p
40. Hu, J., Kawamura, H., Hong, H. & Qi, Y. A review on the currents in the South China Sea: seasonal circulation, South China Sea
Warm Current and Kuroshio Intrusion. Journ. Oceanog. 56, 607–24 (2000).hli 1. Galarza, J. et al. The influence of oceanographic fronts and early-life-history traits on connectivity among littoral fish species. PNAS
106, 1473–1478 (2009). 42. He, L., Mukai, T., Chu, K., Ma, Q. & Zhang, J. Biogeographical role of the Kuroshio Current in the amphibious mudskipper
Periophthalmus modestus indicated by mitochondrial DNA data. Sci. Rep. 5, 15645 (2015).ii 3. Karnauskas, M., Chérubin, L. & Paris, C. Adaptive significance of the formation of multi-species fish spawning aggregations near
submerged capes. PLoS ONE 6, e22067 (2011).i g
4. Evans, R., van Herwerden, L., Russ, G. & Frisch, A. References Settlement-stage coral reef fish prefer the higher-frequency
invertebrate-generated audible component of reef noise. Animal Behav. 75, 1861–68 (2008).hi 17. Igulu, M. et al. The potential role of visual cues for microhabitat selection during the early life phase of a coral reef fish (Lu
fulviflamma). Journ. Exper. Marine Biol. Ecol. 401, 118–25 (2011).hi 7. gu u,
. et al.h
e pote t a
o e o v sua cues o
c o ab tat se ect o du
g t e ea y
e p ase o a co a
e
fulviflamma). Journ. Exper. Marine Biol. Ecol. 401, 118–25 (2011). Thi f
fl
p
18. Jones, G., Planes, S. & Thorrold, S. Coral reef fish larvae settle close to home. Current Biol. 15, 1314–18 (2005).i l
. Jones, G., Planes, S. & Thorrold, S. Coral reef fish larvae settle cloi J
,
,
,h
,i
,
(
)
9. Robertson, D. Population maintenance among tropical reef fishes: inferences from small-island endemics. Proc. Natl. Acad. Sci. 98
5667–5670 (2001). Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 8 (
)
0. Bruckner, A., Johnson, K. & Field, J. Conservation strategies for sea cucumbers: can a CITES Appendix II listing promote sustainable
international trade? SPC Beche-de-mer Info. Bull. 18, 24–33 (2003). www.nature.com/scientificreports/ 72. Cohen, J. Statistical power analysis for the behavioral sciences. Second edition, Acad. Press (1988).l 3. Planes, S., Parroni, M. & Chauvet, C. Evidence of limited gene flow in three species of coral reef fishes in the lagoon of New
Caledonia. Marine Biol. 130, 361–368 (1998).i 73. Planes, S., Parroni, M. & Chauvet, C. Evidence of limited gene flow in three species of coral reef fishes in the lagoon of New
Caledonia. Marine Biol. 130, 361–368 (1998).i 4. Victor, B. Duration of the planktonic larval stage of on hundred species of Pacific and Atlantic wrasses (family Labridae). Marine
Biol. 90, 317–326 (1986). 74. Victor, B. Duration of the planktonic larval stage of on hundred species of Pacific and Atlantic wrasses (family Labridae). Marine
Biol. 90, 317–326 (1986). Author Contributions Designed Experiment J.A.D. Conducted Experiment J.A.D., E.K.E. and E.H.L. Wrote Manuscript J.A.D., E.K.E. and E.H.L. Prepared Figures E.K.E. and E.H.L. All authors reviewed the Manuscript. Acknowledgements g
We would like to thank the Fijian Locally Managed Marine Area (FLMMA) Network for supporting this research,
as well as Waisea Naisilisili, Apete Tamani, Liliana Rabuku, and the people of Nagigi, Naselesele, Malevu,
Donicali, Naracivo, and Nabukavesi for welcoming us into their homes and communities. We would also like
to thank the Sackler Institute for Comparative Genomics for sequencing facilities and assistance. Funding for
EHL was provided by the Columbia University Earth Institute Student Travel Grant, The Explorers Club’s Youth
Activity Fund, the Columbia College Class of 1939 Summer Research Fellowship, the Department of Ecology,
Evolution and Environmental Biology Department Thesis Research Award, and the Columbia University Scholars
Program (CUSP) Summer Enhancement Fellowship. Funding for EKE was provided by the Columbia University
Department of Ecology, Evolution and Environmental Biology and 45 SciFund challenge contributors. Funding
to JAD was provided by the US State Department Embassy in Suva, Fiji and the Mindlin Foundation. No funders
had any input into the development or execution of this work. www.nature.com/scientificreports/ 61. Purcell, S. et al. Sea cucumber fisheries: global analysis of stocks, management measures and drivers of overfishing. Fish Fish
34–59 (2013).f 61. Purcell, S. et al. Sea cucumber fisheries: global analysis of stocks, management measures and drivers of overfishing. Fish Fish. 14,
34–59 (2013). 62 Ch
S Ch
C & Al
d
S Fi i
d it
ff
t
l ti
t
t
f H l th
i
t
(E hi
d
t
H l th
id
) 34–59 (2013). 62. Chao, S., Chen, C. & Alexander, S. Fission and its effect on population structure of Holothuria atra (Echinodermata: Holothuroidea) 2. Chao, S., Chen, C. & Alexander, S. Fission and its effect on population structure of Holothuria atra (Echinodermata: Holothuroidea
in Taiwan. Marine Biol. 116, 109–115 (1993).i (
)
63. Conand, C. Asexual reproduction by fission in Holothuria atra: variability of some parameters in populations from the tropical
Indo-Pacific. Oceanologica Acta 19, 209–216 (1996). i
Indo-Pacific. Oceanologica Acta 19, 209–216 (1996). i
g
4. Laxminarayana, A. Induced spawning and larval rearing of the sea cucumbers, Bohadschia mamorata and Holothuria atra in
Mauritius. SPC Beche-de-mer Info. Bull. 22, 48–52 (2005). f
65. Navarro, P., García-Sanz, S., Barrio, J. & Tuya, F. Feeding and movement patterns of the sea cucumber Holothuria sanctori. Marine
Biol. 160, 2957–2966 (2013). (
)
6. Lee, W., Conroy, J., Howell, W. & Kocher, T. Structure and evolution of teleost mitochondrial control regions. Journ. Mol. Evol. 41
54–66 (1995).t (
)
7. Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis o
sequence data. Bioinformatics, 28, 1647–1649 (2012).fi q
f
68. Excoffier, L. & Lischer, H. Arlequin Suite Ver 3.5: A new series of programs to perform population genetics analyses under Linux and
Windows. Mol. Ecol. Res. 10, 564–67 (2010).f 9. Ryman, N. & Palm, S. POWSIM: A computer program for assessing statistical power when testing for genetic differentiation. Mol
Ecol. 6, 600–602 (2006).i 0. Beerli, P. & Palczewski, M. Unified framework to evaluate panmixia and migration direction among multiple sampling locations
Genetics 185, 313–26 (2010). 71. Jensen, J., Bohonak, A. & Kelley, S. Isolation by distance, web service. BMC Genetics 6: 13. v.3.23 (2005). Available at: http://ibdws. sdsu.edu/Date of access: 01/04/2015. 1. Jensen, J., Bohonak, A. & Kelley, S. Isolation by distance, web service. BMC Genetics 6: 13. v.3.23 (2005). Available at: http://ibdws
sdsu.edu/Date of access: 01/04/2015. www.nature.com/scientificreports/ Combining natural history collections with fisher knowledge for community-based
conservation in Fiji. PLoS ONE 9, e98036 (2014).hh 58. Liu, M. & Yeeting, B. 2008. Epinephelus merra. The IUCN Red List of Threatened Species. Version 2014.3. < www.iucnredlist.org> . Date of access: 12/04/2015.i 59. Green, A. et al. Larval dispersal and movement patterns of coral reef fishes, and implications for marine reserve network design. Biol. Reviews 90, 1215–1247 (2014). Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 9 www.nature.com/scientificreports/ Scientific Reports | 6:19318 | DOI: 10.1038/srep19318 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare How to cite this article: Eastwood, E. K. et al. Population Connectivity Measures of Fishery-Targeted Coral
Reef Species to Inform Marine Reserve Network Design in Fiji. Sci. Rep. 6, 19318; doi: 10.1038/srep19318
(2016) How to cite this article: Eastwood, E. K. et al. Population Connectivity Measures of Fishery-Targeted Coral
Reef Species to Inform Marine Reserve Network Design in Fiji. Sci. Rep. 6, 19318; doi: 10.1038/srep19318
(2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
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Cosmic CARNage II: the evolution of the galaxy stellar mass function in observations and galaxy formation models
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Accepted XXX. Received YYY; in original form ZZZ Cosmic CARNage II
1 Cosmic CARNage II
1 Department of Physics & Astronomy, University of Sussex, Brighton, BN1 9QH, UK Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 Department of Astronomy and Yonsei University Observatory, Yonsei University, 03722 Seoul, Republic of Kore 23Centro de Astro-Ingenieria, Universidad Catolica de Chile, Santiago, Chile
24 24International Centre for Radio Astronomy Research, University of Western Australia, 35 Stirling Highway, C
Western Australia 6009, Australia by U
e s ty o
on 17 July 2018 Cosmic CARNage II: the evolution of the galaxy stellar
mass function in observations and galaxy formation models Rachel Asquith,1⋆Frazer R. Pearce,1 Omar Almaini,1 Alexander Knebe,2,3
Violeta Gonzalez-Perez,4,5 Andrew Benson,6 Jeremy Blaizot,7,8,9 Jorge Carretero,10,11
Francisco J. Castander,10 Andrea Cattaneo,12 Sof´ıa A. Cora,13,14 Darren J. Croton,15
Julien E. Devriendt,16 Fabio Fontanot,17 Ignacio D. Gargiulo,13,14 Will Hartley,18
Bruno Henriques,19 Jaehyun Lee,20 Gary A. Mamon,21 Julian Onions,1
Nelson D. Padilla,22,23 Chris Power,24 Chaichalit Srisawat,25 Adam R. H. Stevens,15,24
Peter A. Thomas,25 Cristian A. Vega-Mart´ınez,13 Sukyoung K. Yi26 1School of Physics & Astronomy, University of Nottingham, Nottingham NG7 2RD, UK f
y
y,
y
f
g
,
g
,
2Departamento de F´ısica Te´orica, M´odulo 15, Facultad de Ciencias, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain
3Centro de Investigaci´on Avanzada en F´ısica Fundamental (CIAFF), Facultad de Ciencias, Universidad Aut´onoma de Madrid,
28049 Madrid, Spain 2Departamento de F´ısica Te´orica, M´odulo 15, Facultad de Ciencias, Universidad Aut´onoma de Madrid, 28049 Madrid, Spain
3Centro de Investigaci´on Avanzada en F´ısica Fundamental (CIAFF), Facultad de Ciencias, Universidad Aut´onoma de Madrid,
28049 Madrid, Spain 4Institute for Computational Cosmology, Department of Physics, University of Durham, South Road, Durham, DH1 3LE, UK
5Institute of Cosmology & Gravitation, University of Portsmouth, Dennis Sciama Building, Portsmouth, PO1 3FX, UK
6Carnegie Observatories, 813 Santa Barbara Street, Pasadena, CA 91101, USA 4Institute for Computational Cosmology, Department of Physics, University of Durham, South Road, Durham, DH1 3LE, UK
5Institute of Cosmology & Gravitation, University of Portsmouth, Dennis Sciama Building, Portsmouth, PO1 3FX, UK
6Carnegie Observatories, 813 Santa Barbara Street, Pasadena, CA 91101, USA 7Universit`e de Lyon, Lyon, F-69003, France y
,
y
,
,
8Universit`e Lyon 1, Observatoire de Lyon, 9 avenue Charles Andr`e, Saint-Genis Laval, F-69230, France
9 8Universit`e Lyon 1, Observatoire de Lyon, 9 avenue Charles Andr`e, Saint-Genis Laval, F-69230, France
9C
S
C
A
S 11Port d’Informaci´o Cient´ıfica (PIC) Edifici D, Universitat Aut`onoma de Barcelona (UAB), E-08193 Bellaterra (Barcelona), Spain. 12GEPI, Observatoire de Paris, CNRS, 61, Avenue de l’Observatoire 75014, Paris France Instituto de Astrofısica de La Plata (CCT La Plata, CONICET, UNLP), Paseo del Bosque s/n, B1900FWA, La Plata, Argentina. 1
INTRODUCTION galaxies throughout their lives, one approach is to link a
population of galaxies at high redshift to a population at
low redshift that could be their descendants. This can be
done by selecting galaxies at a constant comoving number
density when ranked by mass or luminosity (Mundy et al. 2015). This method was partly motivated by the need to
overcome ‘progenitor bias’, where new young star-forming
galaxies enter the sample at low redshift that are not present
at high redshift (Shankar et al. 2015). Not accounting for
this bias correctly can lead to a poor selection of the set
of galaxies being connected as progenitor and descendent
and therefore incorrect conclusions being drawn about their
evolution. Locally, low-mass galaxies tend to be disky, blue and
star-forming, whereas high-mass galaxies are more likely
to be spheroidal, red and passive (e.g. Kennicutt 1998;
Strateva et al. 2001; Kauffmann et al. 2003; Baldry et al. 2004). At high redshift (z
>
1) we also observe this
bimodality in the galaxy population (Kovaˇc et al. 2014;
Cirasuolo et al. 2007), but do not definitively know the
mechanisms by which these galaxies evolve into the popula-
tions we observe locally. Various mechanisms have been sug-
gested to move galaxies from the ‘blue cloud’ to the ‘red se-
quence’ and shut offtheir star formation in a process known
as ‘quenching’. Potential quenching mechanisms include en-
vironmental effects and feedback from active galactic nuclei
(AGN) at high masses (for a review see Benson 2010), but
these processes are still not fully understood. A powerful method to trace galaxies through redshift is
to use semi-analytic models (SAMs) (for a review see Benson
2010; Somerville & Dav´e 2015), a type of galaxy formation
model in which simple analytic prescriptions (in connection
with merger trees from either cosmological simulations or
extended Press-Schechter formalisms) are used to model the
physical processes occurring during galaxy formation and
evolution. These models are able to evolve the same popula-
tion of galaxies through redshift and connect them without
the limitations of observational methods. These models are
also computationally inexpensive, so can be used to simu-
late large volumes and produce large catalogues of galaxies
with which to compare observational data. By comparing
the models to key observables, e.g. the evolution of the stel-
lar mass function (SMF), we can learn about the physics of
galaxy formation. ⋆E-mail: rachel.asquith@nottingham.ac.uk ABSTRACT We present a comparison of the observed evolving galaxy stellar mass functions with the predictions
of eight semi-analytic models and one halo occupation distribution model. While most models are
able to fit the data at low redshift, some of them struggle to simultaneously fit observations at high
redshift. We separate the galaxies into ‘passive’ and ‘star-forming’ classes and find that several of the
models produce too many low-mass star-forming galaxies at high redshift compared to observations,
in some cases by nearly a factor of 10 in the redshift range 2.5 < z < 3.0. We also find important
differences in the implied mass of the dark matter haloes the galaxies inhabit, by comparing with
halo masses inferred from observations. Galaxies at high redshift in the models are in lower mass
haloes than suggested by observations, and the star formation efficiency in low-mass haloes is higher
than observed. We conclude that many of the models require a physical prescription that acts to
dissociate the growth of low-mass galaxies from the growth of their dark matter haloes at high
redshift. Key words:
methods:numerical – galaxies:haloes – galaxies: evolution – cosmol-
ogy:theory – dark matter Cosmic CARNage II: the evolution of the galaxy stellar
mass function in observations and galaxy formation models 14Facultad de Ciencias Astron´omicas y Geof´ısicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, B1900FWA, La Plata, Argentina
15C
t
f
A t
h
i
d S
ti
S i b
U i
it
f T
h
l
H
th
Vi t
i
3122 A
t
li s y Geofısicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, B1900FWA, La Plata, Argentina
rcomputing, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia Facultad de Ciencias Astronomicas y Geofısicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, B190
Centre for Astrophysics and Supercomputing, Swinburne University of Technology, Hawthorn, Victoria 3122, Au Astrophysics, University of Oxford, Denys Wilkinson Building, Keble Road, Oxford, OX1 3RH, UK 17INAF - Astronomical Observatory of Trieste, via Tiepolo 11, I-34143 Trieste, Italy
18 servatory of Trieste, via Tiepolo 11, I-34143 Trieste, Ita 17INAF - Astronomical Observatory of Trieste, via Tiepolo 11, I-34143 Trieste, Italy 18Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT
19Max-Planck-Institut f¨ur Astrophysik, Karl-Schwarzschild-Str. 1, 85741 Garching b. M¨unchen, Germany 20Korea Institute for Advanced Study, 85 Hoegiro Dongdaemun-gu, Seoul 02455 Korea 22Instituto de Astrofisica, Universidad Catolica de Chile, Santiago, Chile 23Centro de Astro-Ingenieria, Universidad Catolica de Chile, Santiago, Chile
24 24International Centre for Radio Astronomy Research, University of Western Australia, 35 Stirling Highway, Crawley,
Western Australia 6009, Australia 25Department of Physics & Astronomy, University of Sussex, Brighton, BN1 9QH, UK
26 2 R. Asquith et al. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 1
INTRODUCTION (2013) had on their own model,
but found that it did not make much difference to the ob-
served number density of low-mass galaxies. They conclude
that this is due to the sensitivity of the results to how the
gas reservoirs are tracked and treated in the different codes. Croton et al. (2016) also had similar problems with this ap-
proach and found that it did not solve the problems with
fitting the stellar mass function. This presents difficulties to
the modelling community, as it means that different mod-
els may require different changes to get them to match the
observed evolution. This excess of low-mass galaxies at high redshift was
investigated by Fontanot et al. (2009), who found that in
three different SAMs, galaxies in the mass range 9
<
log(M∗/M⊙) < 11 form too early and have little ongoing star
formation at late times. They concluded that the physical
processes operating on these mass scales, such as supernova
feedback, needed a re-think. Weinmann et al. (2012) later
used two SAMs and two cosmological hydrodynamical sim-
ulations and examined the evolution of the observed number
density of galaxies. They found that although the models fit
well at z = 0, the low-mass galaxies were formed at early
times. They conclude that as the current form of feedback
is mainly dependent on host halo mass and time, it is un-
likely to be able to separate the growth of galaxies from the
growth of their dark matter haloes. It is also possible to try and match the galaxy stellar
mass function at all redshifts without changing the physics
involved in the model. For example, Rodrigues et al. (2017)
used Galform to identify a small region of parameter space
where the model matched the observational data out to z =
1.5, without needing to adapt any of the physics involved. They found that the parameters controlling the feedback
processes were most strongly constrained, suggesting that
these processes are important when fitting the evolution of
the galaxy stellar mass function. Halo occupation distribution (HOD) models, rather
than modelling the physical processes that we think go
into galaxy formation, use statistical methods to match
galaxies to their corresponding dark matter haloes (e.g. Berlind & Weinberg 2002; Zheng et al. 2005). 1
INTRODUCTION However, they allow these functions to
scale with halo mass and redshift in an arbitrary way which
may not be physically motivated. Hirschmann et al. (2016)
also investigated this problem with their model and found
that they improved their agreement with observations by ei-
ther reducing the gas ejection rate with cosmic time or vary-
ing the reincorporation timescale with halo mass, classed as
‘ejective’ and ‘preventative’ feedback schemes respectively. Although their results improve from their fiducial model,
they still find too many low-mass, red, old galaxies between
0.5 < z < 2.0. galaxies form earlier with their abundance changing little
from z ∼1 to the present day, whereas there is a rapid evo-
lution in the number of low-mass galaxies at late times (e.g. Fontana et al. 2004, 2006; Faber et al. 2007; Pozzetti et al. 2007; Marchesini et al. 2009, 2010; Pozzetti et al. 2010;
Ilbert et al. 2010, 2013; Muzzin et al. 2013). This is some-
times referred to as ‘mass assembly downsizing’ (Cowie et al. 1996; Cimatti et al. 2006; Lee & Yi 2013). )
After
much
work
understanding
both
AGN
feed-
back and the mass assembly of high-mass galaxies (e.g. Benson et al. 2003;
Di Matteo et al. 2005;
Bower et al. 2006; Croton et al. 2006), models are now able to reproduce
the high-mass end of the galaxy stellar mass function over
a range of redshifts. However, models still typically overpro-
duce the number of low-mass galaxies at high redshift. The
main reason for this discrepancy appears to be that galaxies
in the models follow the growth of their dark matter haloes
too closely (Weinmann et al. 2012; Somerville & Dav´e 2015;
Guo et al. 2016). Halo mass growth is the main driver of gas
accretion rate in galaxies, which then in turn drives the star
formation rate. The star formation history then traces the
dark matter mass accretion history, which, in the favoured
ΛCDM structure formation scenario, is approximately self-
similar for haloes of different masses. However, in the real
Universe it appears that there is not such a tight correlation
(White et al. 2015; Guo et al. 2016). However,
the
effect
of
adjusting
certain
physical
prescriptions
can
be
vastly
different
between
models. White et al. (2015) investigated what effect replicating the
changes in Henriques et al. 1
INTRODUCTION If models are not able to reproduce ob-
servational results it may mean that they are missing key
physics which is important in galaxy formation and evolu-
tion. Model galaxies can also be separated into ‘star-forming’
and ‘passive’ types, to test for the quenching processes which
transform galaxies from star-forming to passive. One way to study this problem is to directly observe
galaxies forming and evolving in the distant Universe. At
high redshift (z > 1), deep near-infrared observations are
vital to select galaxies by rest-frame optical light. Selecting
high-redshift galaxies using optical imaging will introduce
strong biases against dusty galaxies or those with evolved
(i.e. passive) stellar populations (e.g. Cowie et al. 1996). It
is only recently that deep near-infrared surveys have been
conducted with the required depth and area to produce large
galaxy samples at high redshift, sufficient to allow accurate
determinations of the galaxy stellar mass function while min-
imising the influence of cosmic variance. In particular, the
UKIDSS Ultra Deep Survey (UDS) (Lawrence et al. 2007,
Almaini et al. in prep.) and UltraVISTA (McCracken et al. 2012) are now deep enough to detect typical (i.e. M∗) galax-
ies to z ∼3, over large volumes of the distant Universe
(∼100 × 100 projected comoving Mpc at z = 3). Using these
surveys, we can directly test model predictions for the build-
up of the galaxy populations, rather than inferring their evo-
lution by extrapolating back in time. However, each galaxy
is only being seen at one point in its life and we cannot infer
the full evolutionary history. While it has been shown that SAMs are able to re-
produce the SMF at z = 0, they struggle to simultane-
ously match observations at both low and high redshift
(e.g. Fontanot et al. 2009; Weinmann et al. 2012; Guo et al. 2011; Knebe et al. 2015). This has only become clearer in re-
cent years as observational surveys have been able to probe
down to lower masses as well as probing to higher redshifts. Observational evidence appears to point towards a seem-
ingly ‘anti-hierarchical’ formation scenario where high-mass In order to get a cohesive picture of what happens to Cosmic CARNage II 3 times and increasing the dependence of stellar feedback on
halo mass at high redshift were the most successful at qual-
itatively matching the evolution of the number density of
low-mass galaxies. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 2
SIMULATION DATA We also note that we compare to different observational
data than the combined dataset used to calibrate the models. The data from Davidzon et al. (2017) is more recent than
the calibration dataset and also allows us to split our sam-
ple into passive and star-forming galaxies. When comparing
Davidzon et al. (2017) to the stellar mass function calibra-
tion data at z = 0 and z = 2, the two largely agree, although
the former has smaller error bars. This is encouraging as it
shows good agreement between different observations. The
eight
SAMs
we
will
be
using
are
DLB07
(De Lucia & Blaizot
2007),
Galform
(Gonzalez-Perez et al. 2014), GalICS-2.0 (Cattaneo et al. 2017, although the exact version used for this comparison
is the one described in the appendix of Knebe et al. (2015)),
Lgalaxies
(Henriques et al. 2013),
Morgana
(Monaco et al. 2007),
Sag
(Cora et al. 2018),
Sage
(Croton et al. 2016) and ySAM (Lee & Yi 2013). The
single HOD model is Mice (Carretero et al. 2015). A
brief description of the physical prescriptions used in each
model is given in the Appendix of Knebe et al. (2015). Any
changes to any of the models since then are included in
Appendix A. The
eight
SAMs
we
will
be
using
are
DLB07
(De Lucia & Blaizot
2007),
Galform
(Gonzalez-Perez et al. 2014), GalICS-2.0 (Cattaneo et al. 2017, although the exact version used for this comparison
is the one described in the appendix of Knebe et al. (2015)),
Lgalaxies
(Henriques et al. 2013),
Morgana
(Monaco et al. 2007),
Sag
(Cora et al. 2018),
Sage
(Croton et al. 2016) and ySAM (Lee & Yi 2013). The
single HOD model is Mice (Carretero et al. 2015). A
brief description of the physical prescriptions used in each
model is given in the Appendix of Knebe et al. (2015). Any
changes to any of the models since then are included in
Appendix A. Inspecting the top panels, what is clear is that the ob-
servational number counts are evolving, with the high-mass
end largely in place by z = 3, while the low-mass end rises at
late times. Whilst the models match the observations well at
low redshift, the strong evolution at the low-mass end is not
seen for most of the galaxy formation models. 1
INTRODUCTION We also note
that the stellar masses from the models have been convolved
with a 0.08(1+z) dex scatter to account for the observational
errors when measuring stellar mass. This value comes from
Conroy et al. (2009), who estimate an error of ∼0.2 dex at
z = 2 when fixing the stellar population synthesis model. of the required physics or that the underlying observational
datasets are incomplete or are physically incompatible with
each other. In the Cosmic CARNage mock galaxy comparison
project (Knebe et al. 2018, hereafter referred to as Paper
I) we sought to address some of these issues by requiring
the participants to calibrate their models to the same set of
observational data. These data included the galaxy stellar
mass function at z = 0 and z = 2, the star formation rate
function at z = 0.15, the black-hole bulge-mass relation at
z = 0, and the cold gas mass fraction at z = 0. Participants
were free to weight these five calibrations as they saw fit,
and were asked to generate their “best-fit” model that took
all of them into account, i.e. calibration set ‘-c02’ in Paper
I. The underlying cosmological dark-matter-only simula-
tion was run using the Gadget-3 N-body code (Springel
2005) with parameters given by the Planck cosmology
(Planck Collaboration et al. 2014, Ωm = 0.307, ΩΛ = 0.693,
Ωb
=
0.048, σ8
=
0.829,
h
=
0.677, ns
=
0.96). We use 5123 particles of mass 1.24 × 109h−1M⊙in a
box of comoving width 125h−1Mpc. The halo catalogues
were extracted from 125 snapshots and identified using
Rockstar (Behroozi et al. 2013a). The halo merger trees
were then generated using the ConsistentTrees code
(Behroozi et al. 2013b). We will build on previous work by investigating the evo-
lution of the SMF for the eight SAMs and one HOD model
that were used in Paper I. These models are all calibrated
to the same observational data and are all run on the same
background dark matter only simulation, which means that
we can discount the differences due to the underlying cosmo-
logical framework when considering the differences between
the models. Our aim is then to see if the current physical
prescriptions used in any of the galaxy formation models can
produce a realistic population of galaxies at both low and
high redshift. 1
INTRODUCTION As these mod-
els are applied independently at each redshift, the evolution
of each galaxy is not tracked, although they can be con-
nected to their progenitors and descendants via dark matter
merger trees. HOD models by design are able to reproduce
the SMF at each redshift and are therefore able to repro-
duce the population of galaxies at any given time. This type
of model is a very useful tool for learning about the rela-
tionship between galaxies and their host dark matter haloes
and how this changes as a function of redshift. For example,
Berlind et al. (2003) found that low-mass haloes are mainly
populated by young galaxies and high-mass haloes by older
galaxies. Monte Carlo Markov Chain (MCMC) methods were
used by Henriques et al. (2013) in an attempt to fit the
stellar mass function at all redshifts, but they could not
find a single set of parameters that allowed this. They then
changed the reincorporation timescale for ejected gas to be
inversely proportional to halo mass and independent of red-
shift and found that they were able to fit observed numbers
of low-mass galaxies from 0 < z < 3. However, the passive
fraction of low-mass galaxies was still too high. Their model
was later updated further in Henriques et al. (2015) where
they also reduce ram-pressure stripping in low-mass haloes,
make radio-mode AGN feedback more efficient at low red-
shift, and reduce the gas surface density threshold for star
formation. They then find that their model reproduces the
observed abundance and passive fraction of low-mass galax-
ies, both at high and low redshift. Galaxy formation models such as HODs and SAMs
must be calibrated using observational datasets. Varying the
calibration dataset, even for the same model may produce
significantly different catalogues. Essentially, the calibration
datasets introduce tension, and it may not be possible for
a single model to fit all the required observational datasets
simultaneously. This could be because the model lacks some Another
attempt
to
solve
this
problem
was
by
White et al. (2015), who tried three different physically mo-
tivated methods to decouple the accretion rate in galaxies
from their star formation rate. They found that changing the
gas accretion to be less efficient in low-mass haloes at early R. Asquith et al. 4 A description of how the models were calibrated to the
same observational data is given in Paper I. 3.1
Evolution of the Galaxy Stellar Mass Function We start by examining the evolution of the stellar mass func-
tion in Figure 1, shown for the whole sample in the top row. The coloured lines are the stellar mass functions for each of
the models, computed for each redshift bin using single snap-
shots at z = 0.8,2.0 and 3.0 for each redshift bin respectively. We note that the precise choice of snapshot does not af-
fect our conclusions. The observations from Davidzon et al. (2017) are based on the UltraVISTA near-infrared survey of
the COSMOS field and are shown as a black line and dark
shaded region. When finding the best-fit Schechter param-
eters to their stellar mass functions, they take into account
the errors in measuring stellar mass, known as Eddington
bias. As they have applied this correction, when plotting
the stellar mass function we do not apply the 0.08(1+ z) dex
scatter to the stellar mass values. In Appendix B we have in-
cluded a version of Figure 1 where the model stellar masses
do have this scatter applied, to show the differences to the
SMF. The rest of this paper is structured as follows: in Section
2 we will briefly explain the underlying dark matter simula-
tion and the parameters used. In Section 3 we will present
the results for the evolution of the SMF and the passive frac-
tion. We will then show how the specific star formation rate
of star-forming galaxies in the models evolves. We will also
examine the average halo mass as a function of stellar mass
and the stellar mass - halo mass relation for all the models. In Section 4 we will present our discussion and in Section 5
we will present our conclusions. 1
INTRODUCTION We will investigate the evolution of the SMF in
the redshift range 0.5 < z < 3.0 for all nine galaxy formation
models and determine if models still struggle to simultane-
ously match observations both at low and high redshift. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
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RESULTS 3.1
Evolution of the Galaxy Stellar Mass Function 3.1
Evolution of the Galaxy Stellar Mass Function Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 2
SIMULATION DATA The excep-
tions to this are Mice, Lgalaxies and Sag, which all show
an increasing number density of low-mass galaxies towards
low redshift. As Mice is an HOD model it has been designed
to match the evolution of the SMF. Lgalaxies and Sag
likely do a better job of matching the SMF at high redshift The models have all been run on the same underlying
dark matter simulation which may be different to the one
used in the above reference papers. This can lead to changes
in the predictions of each model, as can varying the initial
mass function, yield, stellar population synthesis model and
calibration data set used. Cosmic CARNage II
5 5 Cosmic CARNage II
5
−5
−4
−3
−2
log(φ[Mpc−3dex−1])
0.5 < z < 0.8
All
1.5 < z < 2.0
Davidzon+17
DLB07
Galform
GalICS-2.0
LGALAXIES
2.5 < z < 3.0
MICE
MORGANA
SAG
SAGE
ySAM
−5
−4
−3
−2
log(φ[Mpc−3dex−1])
Passive
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−5
−4
−3
−2
log(φ[Mpc−3dex−1])
Star-forming
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
-5
-4
-3
-2
log(φ[Mpc−3dex−1])
-5
-4
-3
-2
log(φ[Mpc−3dex−1])
-5
-4
-3
-2
log(φ[Mpc−3dex−1])
Figure 1. The evolution of the stellar mass function for all the models over the range 0.5 < z < 3.0. The stellar mass function for the
whole, passive and star-forming samples are shown in the top, middle and bottom panels, respectively, as coloured lines. The black line
is the observational best-fit mass functions from Davidzon et al. (2017), with the dark grey shaded region showing the 1σ errors. For
the models, each redshift bin contains one snapshot, at redshifts z = 0.8, 2.0 and 3.0 respectively. We can see that the models match
well at low redshift (by construction), but deviate further from the observations at high redshift. The number density of the lowest mass
objects is nearly constant in the models but changes by more than 0.5 dex in the observations. Most of the low-mass galaxies that are
not present in the observations at high redshift seem to be star-forming. 2
SIMULATION DATA 2.5 < z < 3.0
MICE
MORGANA
SAG
SAGE
ySAM
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
-5
-4
-3
-2
log(φ[Mpc−3dex−1]) log(M∗[M⊙]) 4
3
2
log(φ[Mpc−3dex−1]) 0.0
10.5
11
log(M∗[M⊙] 0.0
10.5
11. log(M∗[M⊙]) 0.0
10.5
11. log(M∗[M⊙]) Figure 1. The evolution of the stellar mass function for all the models over the range 0.5 < z < 3.0. The stellar mass function for the
whole, passive and star-forming samples are shown in the top, middle and bottom panels, respectively, as coloured lines. The black line
is the observational best-fit mass functions from Davidzon et al. (2017), with the dark grey shaded region showing the 1σ errors. For
the models, each redshift bin contains one snapshot, at redshifts z = 0.8, 2.0 and 3.0 respectively. We can see that the models match
well at low redshift (by construction), but deviate further from the observations at high redshift. The number density of the lowest mass
objects is nearly constant in the models but changes by more than 0.5 dex in the observations. Most of the low-mass galaxies that are
not present in the observations at high redshift seem to be star-forming. ejected mass with redshift to make supernova feedback more
efficient at high redshift. due to the physics involved in the treatment of gas. Both
follow the prescription suggested in Henriques et al. (2013)
of scaling the reincorporation timescale of ejected gas with
the inverse of the halo mass. This means the process of gas
being reincorpoated back into the halo takes longer for low-
mass haloes, shifting the growth of galaxies in these haloes
from early to late times. Sag also scales the reheated and At the high-mass end, the models underestimate the
number density compared to observations, with Mice and
GalICS-2.0 as the exceptions. One alternative reason
for this tension at the high-mass end may be due to
Davidzon et al. (2017) underestimating their uncertainties R. Asquith et al. 6 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
0.0
0.2
0.4
0.6
0.8
1.0
Passive fraction
0.5 < z < 0.8
Davidzon+17
DLB07
Galform
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
1.5 < z < 2.0
GalICS-2.0
LGALAXIES
MICE
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
2.5 < z < 3.0
MORGANA
SAG
SAGE
ySAM
0.0
0.2
0.4
0.6
0.8
1.0
Passive fraction
Figure 2. 2
SIMULATION DATA The sSFR of star-forming galaxies in the models
matches observations well but there is less of a trend with mass
in some models. 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−2.5
−2.0
−1.5
−1.0
−0.5
log(sSFR[Gyr−1])
Elbaz+07
DLB07
Galform
GalICS-2.0
LGALAXIES
MICE
MORGANA
SAG
SAGE
ySAM
z = 0.0
Figure 4. As for Figure 3, but for z = 2.0 and observations from
Daddi et al. (2007). The model data is taken from one snapshot
at z = 2.0. Here all of the models lie almost completely below the
observational best-fit range. 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−2.5
−2.0
−1.5
−1.0
−0.5
log(sSFR[Gyr−1])
Elbaz+07
DLB07
Galform
GalICS-2.0
LGALAXIES
MICE
MORGANA
SAG
SAGE
ySAM
z = 0.0
Figure 3. The relationship between mass and sSFR at z = 0.0 for
star-forming galaxies in the nine models. The model data is taken
from one snapshot at z = 0.0. The grey shaded region is taken
from Elbaz et al. (2007) and shows the observational best-fit to
this relation. The sSFR of star-forming galaxies in the models
matches observations well but there is less of a trend with mass
in some models. 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−2.5
−2.0
−1.5
−1.0
−0.5
log(sSFR[Gyr−1])
Elbaz+07
DLB07
Galform
GalICS-2.0
LGALAXIES
MICE
MORGANA
SAG
SAGE
ySAM
z = 0.0
Figure 4. As for Figure 3, but for z = 2.0 and observations from
Daddi et al. (2007). The model data is taken from one snapshot
at z = 2.0. Here all of the models lie almost completely below the
observational best-fit range. 3.2
Star-forming and Passive Galaxy Stellar Mass
Functions
We explore the mass growth further in the bottom two rows
of Figure 1, splitting the population into passive (middle
row) and star-forming (bottom row) galaxies We separate 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−2.5
−2.0
−1.5
−1.0
−0.5
log(sSFR[Gyr−1])
Elbaz+07
DLB07
Galform
GalICS-2.0
LGALAXIES
MICE
MORGANA
SAG
SAGE
ySAM
z = 0.0 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−2.5
−2.0
−1.5
−1.0
−0.5
log(sSFR[Gyr−1])
Elbaz+07
DLB07
Galform
GalICS-2.0
LGALAXIES
MICE
MORGANA
SAG
SAGE
ySAM
z = 0.0 Figure 3. The relationship between mass and sSFR at z = 0.0 for
star-forming galaxies in the nine models. The model data is taken
from one snapshot at z = 0.0. The grey shaded region is taken
from Elbaz et al. 2
SIMULATION DATA (2007) and shows the observational best-fit to
this relation. The sSFR of star-forming galaxies in the models
matches observations well but there is less of a trend with mass
in some models. Figure 4. As for Figure 3, but for z = 2.0 and observations from
Daddi et al. (2007). The model data is taken from one snapshot
at z = 2.0. Here all of the models lie almost completely below the
observational best-fit range. 2
SIMULATION DATA The evolution of the passive fraction over the range 0.5 < z < 3.0. The coloured lines, black solid lines and grey shaded regions
are the same as in Figure 1, as are the snapshots used in each redshift bin for the models. For a few models the passive fraction is too
high at low masses, particularly at low redshift. The models match well at high masses at low redshift, but generally underpredict the
passive fraction for high-mass galaxies at high redshift. 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
0.0
0.2
0.4
0.6
0.8
1.0
Passive fraction
0.5 < z < 0.8
Davidzon+17
DLB07
Galform
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
1.5 < z < 2.0
GalICS-2.0
LGALAXIES
MICE
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
2.5 < z < 3.0
MORGANA
SAG
SAGE
ySAM
0.0
0.2
0.4
0.6
0.8
1.0
Passive fraction
Figure 2. The evolution of the passive fraction over the range 0.5 < z < 3.0. The coloured lines, black solid lines and grey shaded regions
are the same as in Figure 1, as are the snapshots used in each redshift bin for the models. For a few models the passive fraction is too
high at low masses, particularly at low redshift. The models match well at high masses at low redshift, but generally underpredict the Passive fraction 10.0
10.5
11.0
log(M∗[M⊙]) 10.0
10.5
11.0
log(M∗[M⊙]) Figure 2. The evolution of the passive fraction over the range 0.5 < z < 3.0. The coloured lines, black solid lines and grey shaded regions
are the same as in Figure 1, as are the snapshots used in each redshift bin for the models. For a few models the passive fraction is too
high at low masses, particularly at low redshift. The models match well at high masses at low redshift, but generally underpredict the
passive fraction for high-mass galaxies at high redshift. 9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−2.5
−2.0
−1.5
−1.0
−0.5
log(sSFR[Gyr−1])
Elbaz+07
DLB07
Galform
GalICS-2.0
LGALAXIES
MICE
MORGANA
SAG
SAGE
ySAM
z = 0.0
Figure 3. The relationship between mass and sSFR at z = 0.0 for
star-forming galaxies in the nine models. The model data is taken
from one snapshot at z = 0.0. The grey shaded region is taken
from Elbaz et al. (2007) and shows the observational best-fit to
this relation. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 3.2
Star-forming and Passive Galaxy Stellar Mass
Functions We explore the mass growth further in the bottom two rows
of Figure 1, splitting the population into passive (middle
row) and star-forming (bottom row) galaxies. We separate
passive and star-forming galaxies using a redshift-dependent
specific star formation rate (sSFR) cut of sSFR(z)
=
1/(3tH(z)) where tH(z) is the Hubble time at that red-
shift. We test the robustness of this cut by examining the
change in our results when using slightly different cuts of
sSFR(z) = 1/(2tH(z)) and sSFR(z) = 1/(4tH(z)). We find that
the shape of the stellar mass function changes very little and when accounting for Eddington bias, as it is very difficult
to accurately measure all of the sources of error. Due to the
steep slope of the SMF at high masses this would have a
greater impact at the high-mass end of the SMF. The im-
pact of Eddington bias on the SMF are discussed further in
Appendix B. Cosmic CARNage II
7 Cosmic CARNage II
7 Cosmic CARNage II
7
−1.5
−1.0
−0.5
0.0
log(φ/φ0)
9 < log(M∗[M⊙]) < 10
All
Davidzon+17
DLB07
Galform
GalICS-2.0
−2.5
−2.0
−1.5
−1.0
−0.5
0.0
0.5
log(φ/φ0)
Passive
0.5
1.0
1.5
2.0
2.5
3.0
z
−1.0
−0.5
0.0
0.5
log(φ/φ0)
Star-forming
10 < log(M∗[M⊙]) < 11
LGALAXIES
MICE
MORGANA
0.5
1.0
1.5
2.0
2.5
3.0
z
11 < log(M∗[M⊙]) < 12
SAG
SAGE
ySAM
0.5
1.0
1.5
2.0
2.5
3.0
z
2 4
6
8
10
11
tlb[Gyr]
2 4
6
8
10
11
tlb[Gyr]
2 4
6
8
10
11
tlb[Gyr]
-1.5
-1.0
-0.5
0.0
log(φ/φ0)
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
log(φ/φ0)
-1.0
-0.5
0.0
0.5
log(φ/φ0)
Figure 5. The evolution of the number density φ, in bins of stellar mass. This is normalised by the number density at 0.2 < z < 0.5,
which we call φ0. We show three mass bins as indicated (left to right panels) for all galaxies (top panels), passive galaxies (middle
panels) and star-forming galaxies (bottom panels). The dark grey shaded regions and black lines with circular points show data from
Davidzon et al. (2017). The coloured points and lines are for the nine models. In the lowest mass bin, most of the models assemble the
galaxies before the observations. The models match the observations well at intermediate masses, but the observational number density
increases before many of the models at high mass. 3.2
Star-forming and Passive Galaxy Stellar Mass
Functions 11 < log(M∗[M⊙]) < 12
SAG
SAGE
ySAM
2 4
6
8
10
11
tlb[Gyr]
-1.5
-1.0
-0.5
0.0
log(φ/φ0) 0.5
1.0
1.5
2.0
2.5
3.0
z
-1.0
-0.5
0.0
0.5
log(φ/φ0) Star-forming Figure 5. The evolution of the number density φ, in bins of stellar mass. This is normalised by the number density at 0.2 < z < 0.5,
which we call φ0. We show three mass bins as indicated (left to right panels) for all galaxies (top panels), passive galaxies (middle
panels) and star-forming galaxies (bottom panels). The dark grey shaded regions and black lines with circular points show data from
Davidzon et al. (2017). The coloured points and lines are for the nine models. In the lowest mass bin, most of the models assemble the
galaxies before the observations. The models match the observations well at intermediate masses, but the observational number density
increases before many of the models at high mass. SMF, as these galaxies will have very blue and red colours
respectively. makes no difference to any of the conclusions that we draw. In the observations the passive and star-forming galaxies
are seperated using the (NUV - r) vs (r - J) colour-colour
diagram as described in Ilbert et al. (2013), which is best
suited to differentiate fully quiescent galaxies from those
with residual star formation. In practice, the exact location
of the split makes little difference to the low-mass end of
the star-forming SMF and the high-mass end of the passive Splitting the galaxy population in this way reveals that
the main source of the difference between the observations
and the models comes from the star-forming population:
low-mass star-forming galaxies appear to be far too com-
mon at high redshift in the models and the star-forming
SMF evolves little from z = 3 to z = 0.5. The exceptions to
this are Mice and Lgalaxies, which appear consistent with R. Asquith et al. 8 8
R. Asquith et al. 3.2
Star-forming and Passive Galaxy Stellar Mass
Functions 11.5
12.0
12.5
13.0
13.5
14.0
14.5
log(M200[M⊙])
0.5 < z < 1.0
All
1.0 < z < 2.0
Baryon fraction
Hartley+13
DLB07
Galform
GalICS-2.0
LGALAXIES
2.0 < z < 3.6
MICE
MORGANA
SAG
SAGE
ySAM
11.5
12.0
12.5
13.0
13.5
14.0
14.5
log(M200[M⊙])
Passive
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
11.5
12.0
12.5
13.0
13.5
14.0
14.5
log(M200[M⊙])
Star-forming
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
11.5
12.0
12.5
13.0
13.5
14.0
14.5
log(M200[M⊙])
11.5
12.0
12.5
13.0
13.5
14.0
14.5
log(M200[M⊙])
11.5
12.0
12.5
13.0
13.5
14.0
14.5
log(M200[M⊙])
Figure 6. The average halo mass in the models compared to measurements from observations between z = 0.5 and z = 3.6. Observational
measurements of the average halo mass from Hartley et al. (2013) are derived from clustering and are shown as stars. The values for
each model are shown as coloured lines. For the models, the mass of the main host halo was used rather than the subhalo, to better
compare with observational halo mass measurements from clustering. The top panels cover the full galaxy sample, the middle panels
are for passive galaxies and the bottom panels are for star-forming galaxies. The black dashed line shows the universal baryon fraction. For the models, we use snapshots at z = 1.0, 2.0 and 3.5 for each redshift bin respectively. For the passive sample, the halo masses from
observations are approximately constant, but decrease by up to a factor of 10 in the models with increasing redshift. For the star-forming
sample, the observations show halo mass increasing with increasing redshift, whereas in the models there is no real trend with redshift. th
b
ti
t l
t
3 F
th
i
t
fid
h
i
ll
ti
t d
t
d
th
t
f log(M∗[M⊙]) Figure 6. The average halo mass in the models compared to measurements from observations between z = 0.5 and z = 3.6. Observational
measurements of the average halo mass from Hartley et al. (2013) are derived from clustering and are shown as stars. The values for
each model are shown as coloured lines. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 3.3
Evolution of the Passive Fraction rates of the subset of star-forming galaxies. Figure 3 shows
the average sSFR as a function of mass at z = 0.0 for each of
the models as a solid coloured line. The grey shaded region is
taken from Elbaz et al. (2007), who used SDSS data to find
a fit to the correlation between SFR and mass at z = 0. Their
sample is made up of 19590 galaxies with redshifts z = 0.04−
0.1 and is complete to MB ≤−20. Brinchmann et al. (2004)
used Hα emission to derive the SFR of these galaxies and
the stellar masses were derived by Kauffmann et al. (2003),
who fit using a library of star formation histories to find the
most likely stellar mass. Another way of looking at this result is to examine the pas-
sive fraction, which is shown in Figure 2. Again, the shaded
regions indicate the observations taken from the same source
as used for Figure 1. The passive fraction indicates the ratio
of passive to star-forming galaxies. At low masses, some of
the models, such as DLB07, Galform and Morgana, tend
to overestimate the passive fraction compared to observa-
tions. This has been seen previously and appears to be linked
to how environmental processes are taken into account in
the models (Lagos et al. 2014; Gonzalez-Perez et al. 2018). At low redshift the number of star-forming galaxies matches
observations well, so this difference is due to the lack of a
turnover or flattening of the passive SMF. At higher red-
shifts, the overproduction of low-mass star-forming galaxies
would act to decrease the passive fractions. However, this is
still too high in some models, again due to the rising number
density towards low masses in the passive SMF. Most of the models match the observations well here,
with Lgalaxies and Sag lying in the observational region
at all masses. Some models appear to evolve less with mass
than the observations suggest, with some showing almost
no trend, whereas the sSFR implied by the observations de-
creases by over 0.5 dex between 109M⊙and 1012M⊙. This
means that some of the models, such as Galform, match at
low masses but not high masses, and others such as DLB07
and ySAM match at high masses but not low masses. This
was also discussed in Guo et al. 3.2
Star-forming and Passive Galaxy Stellar Mass
Functions For the models, the mass of the main host halo was used rather than the subhalo, to better
compare with observational halo mass measurements from clustering. The top panels cover the full galaxy sample, the middle panels
are for passive galaxies and the bottom panels are for star-forming galaxies. The black dashed line shows the universal baryon fraction. For the models, we use snapshots at z = 1.0, 2.0 and 3.5 for each redshift bin respectively. For the passive sample, the halo masses from
observations are approximately constant, but decrease by up to a factor of 10 in the models with increasing redshift. For the star-forming
sample, the observations show halo mass increasing with increasing redshift, whereas in the models there is no real trend with redshift. to find a physically motivated way to reduce the star forma-
tion rates of low-mass galaxies at high redshift. The same
galaxies at later times would then have lower stellar masses
and star formation rates. This would then act to redistribute
the passive SMF in the models to better match the observa-
tions. the observations at low masses up to z = 3. For the passive
galaxies, the number density at low masses does evolve with
redshift in the models, as seen in the observations. However,
most of the SAMs show rising number density towards lower
masses, in contrast with the observations which appear to
show a turnover or flattening of the passive SMF towards
lower masses. In order to solve these problems, models need Cosmic CARNage II
9 9 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
log(M200[M⊙])
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
z = 0.1
Behroozi+13
Baryon fraction
DLB07
Galform
11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
log(M200[M⊙])
z = 1.0
GalICS-2.0
LGALAXIES
MICE
MORGANA
11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
log(M200[M⊙])
z = 2.0
SAG
SAGE
ySAM
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙]) 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
log(M200[M⊙])
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
z = 0.1
Behroozi+13
Baryon fraction
DLB07
Galform
11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
log(M200[M⊙])
z = 1.0
GalICS-2.0
LGALAXIES
MICE
MORGANA
11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5
log(M200[M⊙])
z = 2.0
SAG
SAGE
ySAM
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
Figure 7. 3.3
Evolution of the Passive Fraction (2016), who used data from
two SAMs, Galform and Lgalaxies, and one hydrody-
namical simulation, Eagle. They found that the median
sSFR remained almost constant with mass, in contrast with
observations. At low redshift, the models tend to match the observa-
tions well at high masses, but one model, Sage, underpre-
dicts the passive fraction. This is mainly due to an under-
prediction for the number of high-mass passive galaxies. As
shown by Stevens & Brown (2017), detailing the structural
evolution of galaxy discs with the Dark Sage variant of
the model (Stevens et al. 2016) leads to more sensible pas-
sive fractions. In the redshift range 1.5 < z < 2.0 the mod-
els tend to underpredict the fraction of high-mass galaxies,
mainly due to the lack of high-mass passive galaxies above
z ∼1. The model which best matches the observed passive
fraction for high-mass galaxies is DLB07, which slightly un-
derpredicts the number density of both high-mass passive
and star-forming galaxies in this redshift range. The relationship between sSFR and stellar mass at z =
2.0 is then shown in Figure 4. Here the observations are taken
from Daddi et al. (2007), who use galaxies in the GOODS-S
field to find the correlation between SFR and mass at z = 2. They are complete to K < 22 and use only 24µm selected
galaxies in order to exclude passive galaxies. The SFRs were
estimated using the UV and the stellar masses were derived
by Fontana et al. (2004) using SED fitting. This comparison highlights large differences between
the observations and models at this redshift, with the mod-
els almost completely outside the observational range. The
sSFR of star-forming galaxies in the models is on average
around 0.5 dex lower than measured in the observations. 3.2
Star-forming and Passive Galaxy Stellar Mass
Functions Comparison of the average stellar mass for each halo mass bin in the models to the abundance matching model of Behroozi et al. (2013c), considering only central galaxies. The results from the models are shown as coloured lines and the stellar mass - halo mass
relation from Behroozi et al. (2013c) is shown as a dark grey shaded region and black line. The black dashed line shows the universal
baryon fraction. The panels are for each redshift, increasing from left to right, using snapshots at z = 0.0, 1.0 and 2.0 respectively. Looking
at the data from Behroozi et al. (2013c), we can see that the average stellar mass stays fairly constant with redshift, but increases in the
models towards low redshift, particularly at high masses. log(M200[M⊙]) log(M200[M⊙]) log(M200[M⊙]) Figure 7. Comparison of the average stellar mass for each halo mass bin in the models to the abundance matching model of Behroozi et al. (2013c), considering only central galaxies. The results from the models are shown as coloured lines and the stellar mass - halo mass
relation from Behroozi et al. (2013c) is shown as a dark grey shaded region and black line. The black dashed line shows the universal
baryon fraction. The panels are for each redshift, increasing from left to right, using snapshots at z = 0.0, 1.0 and 2.0 respectively. Looking
at the data from Behroozi et al. (2013c), we can see that the average stellar mass stays fairly constant with redshift, but increases in the
models towards low redshift, particularly at high masses. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
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on 17 July 2018 3.5
Growth of the Galaxy Stellar Mass Function In Figure 5 we examine the growth of the stellar mass func-
tion as a function of mass and redshift. This is found by
taking the value of the number density φ at fixed stellar
mass for a certain redshift bin and normalising it by the
value of φ in the lowest redshift bin 0.2 < z < 0.5, which
we call φ0. This allows for easier comparison between the
models and observations and will highlight when the num-
ber density of different populations increases. The dark grey
region and black line with circular points shows data from
Davidzon et al. (2017). The coloured lines then show the
number density evolution for the nine models. The black
dotted line shows where the number density is equal to the
number density in the lowest redshift bin. For the models, here we use the mass of the main host
halo for each galaxy rather than the mass of its subhalo. Host haloes do not reside within another halo, whereas
subhaloes are contained within a host halo. Although us-
ing the host halo is not necessarily the usual choice when
analysing simulation data, it allows us to compare to obser-
vational measurements of halo mass from galaxy clustering,
which effectively measure the mass of the main host haloes
(Mo & White 2002). For this reason we also include both
centrals and satellites, in order to best mimic the observa-
tional measurements. Assuming galaxy clustering measure-
ments can correctly recover the host halo mass, we can then
directly compare the observations and models. Looking at the passive galaxies, we can see that the
models struggle to match the observed growth of the mass
function at low masses, as the number density of low-mass
galaxies increases in the models at higher redshift than the
observations. The only exception is Mice, which has very
few galaxies with mass below 1010M⊙above z ∼1. At in-
termediate masses the models match the observations well,
but at high masses the growth of the mass function occurs
in observations before many of the models. y
For the star-forming galaxies, at low masses there is a
similar problem with several of the models; the mass func-
tion grows too much at high redshift. Just under half of
the models have more low-mass star-forming objects in the
highest redshift bin than the lowest redshift one. 3.4
Relationship between Mass and Specific Star
Formation Rate In order to better compare star formation in the observations
and the models, we also look at the specific star formation 10 R. Asquith et al. of the stellar mass function. Looking at the lower panel of
Figure 1, we can see that DLB07 overpredicts the num-
ber of low-mass star-forming galaxies at both low and high
redshift. Sage agrees well with observations at low redshift
but overproduces low-mass star-forming galaxies at high red-
shift. However, looking at the lower left panel of Figure 5,
we can see that DLB07 matches observations of the growth
of the mass function better than Sage. These models there-
fore have slightly different problems; DLB07 has too many
low-mass galaxies at all redshifts, but the number density in-
creases at the correct rate. Conversely, Sage has the correct
number at low redshift, but the number density increases
too early. The models therefore predict a slower evolution of the sSFR
with redshift than observations. This has been previously
seen by Mitchell et al. (2014), who find that when they scale
the reincorporation time of gas with redshift they are able
to better match the evolution of the stellar mass function,
but still underestimate the sSFR of high-mass galaxies at
z ∼2. Hirschmann et al. (2016) also found that their ejec-
tive models predicted lower than observed sSFRs at high
redshift, even when they could reproduce the growth of the
stellar mass function. The models therefore predict a slower evolution of the sSFR
with redshift than observations. This has been previously
seen by Mitchell et al. (2014), who find that when they scale
the reincorporation time of gas with redshift they are able
to better match the evolution of the stellar mass function,
but still underestimate the sSFR of high-mass galaxies at
z ∼2. Hirschmann et al. (2016) also found that their ejec-
tive models predicted lower than observed sSFRs at high
redshift, even when they could reproduce the growth of the
stellar mass function. Reducing the star formation rates of galaxies above z ∼
2, as suggested in Section 3.2, may help to solve this problem. If galaxies have a lower star formation rate at higher redshift,
their resulting mass at lower redshift will be lower. A galaxy
with the same star-formation rate at z = 2 will then have a
higher sSFR as it will have a lower stellar mass. 3.6
Average Halo Mass In this section we study the average halo mass the galax-
ies reside within, shown in Figure 6. For the models we use
single snapshots at z = 1.0,2.0 and 3.5 for each redshift bin
respectively. The dashed black line indicates the universal
baryon fraction, i.e. where all the baryonic material within
the halo has been converted into stars. Each of the coloured
lines indicates the average halo mass values for a different
model, while the black points with errorbars are average
halo mass values taken from Hartley et al. (2013), who use
the UDS DR8 data to estimate the halo masses from mea-
surements of galaxy clustering (e.g. Mo & White 2002, and
references therein). Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
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on 17 July 2018 3.5
Growth of the Galaxy Stellar Mass Function However,
several of the models are more in line with the growth of the
observed mass function, namely Sag and Mice. At interme-
diate masses, the number density of star-forming galaxies
increases at higher redshift in the models than in the ob-
servations. The model that is most discrepant, Morgana,
has more intermediate mass star-forming galaxies between
1.0 < z < 1.5 than in the lowest redshift bin. For high-mass
galaxies, the number density evolves little since high red-
shift in the observations. Mice reproduces this trend well
but in other models the number density increases at lower
redshift. This may be in part due to the fact that there are
low numbers of the highest mass galaxies which will natu-
rally introduce more scatter in the proportional change in
number density. Splitting the sample into passive and star-forming
galaxies in Figure 6 we see that there are marked differ-
ences between the observations and the models. For passive
galaxies, the average halo mass in observations stays con-
stant over redshift in the observations, but rises towards
low redshift in the models. For the star-forming population,
while the observations indicate a general downsizing trend
in halo mass of about an order of magnitude between high
and low redshift, all the models show virtually no change. It
is clear that the models start significantly below the observa-
tions at 2.0 < z < 3.5 and only agree with the observations
by 0.5 < z < 1.0. Both passive and star-forming low-mass
galaxies are therefore in lower mass haloes on average in the
models than in the observations at high redshift. One thing that can affect the average halo mass values
in the models is the halo mass definition used, as this can
lead to differences of up to 20 percent (Jiang et al. 2014). Al-
though this may account for some of the scatter between the
models, the differences between the observations and models
cannot be explained by this alone. Another factor that could
affect the observational measurments of halo mass from clus- We can also see interesting differences between mod-
els when comparing the stellar mass function to the growth Cosmic CARNage II
11 Cosmic CARNage II
11 Cosmic CARNage II
11 changes the least with redshift is Mice; as this is an HOD
model it naturally matches the SMHM relation better than
the SAMs. 3.7
Stellar Mass - Halo Mass Relation In Figure 7 we display measurements of the average stel-
lar mass of central galaxies in bins of halo mass, com-
paring the models with the abundance matching model of
Behroozi et al. (2013c). The dashed black line indicates the
universal baryon fraction and the dark grey region and
black solid line show the fit to the stellar mass - halo mass
(SMHM) relation from Behroozi et al. (2013c). The coloured
lines show the average stellar mass values for each different
model. In the absence of a new population of low-mass, star-
forming galaxies being observed at z ∼2, many of the mod-
els would need improvements in order to reproduce obser-
vations. They would need to produce far fewer low-mass
star-forming galaxies at essentially all but the latest times. Shifting star formation from high-mass haloes at high red-
shift to low-mass haloes at low redshift would also produce
better agreement with observations of galaxy clustering. Re-
ducing the number of low-mass star-forming objects would
also have to be achieved without reducing the number of
high-mass objects significantly. At low redshift, the results from the models and SMHM
relation agree well at low and intermediate halo masses. However, above halo masses of ∼1013.5M⊙the average stel-
lar mass of centrals in the models is higher than suggested
by the SMHM relation. This means that at low redshift, star
formation in high-mass haloes is more efficient in the mod-
els. The exceptions to this are Lgalaxies and Mice, which
agree with the SMHM relation at nearly all halo masses. For most of the models, the slope of the relation at high
halo masses does flatten, but not to the extent seen from
the SMHM relation. Some of the models, such as Lgalaxies and Sag, do
fit the low-mass end of both the star-forming and passive
stellar mass function at high redshift. This is likely due to
their implementation of the physics involved in the treat-
ment of gas, in particular the reincorporation timescales. Mice also matches observations at high redshift, but as this
is a HOD model it matches by construction. However, there
are still some observables that even these models struggle to
match, such as the relation between stellar mass and specific
star formation rate and the average halo mass that galax-
ies occupy. 4
DISCUSSION Comparing several galaxy formation models allows us to dis-
tinguish areas that are challenging for the current generation
of models and therefore provide direction for the future de-
velopment of the field as a whole. The main issue highlighted
in this paper is the fact that most of the models produce too
many low-mass, star-forming galaxies at early times. Obser-
vationally these appear either to not exist or to be missed
by the surveys. This is a difficult area observationally with
the answer to this question only becoming evident when the
stellar mass functions are reliably pushed to lower masses. At present they are tantalisingly close to indicating a clear
turnover in the space density of passive galaxies at low-mass,
which would significantly challenge many of the models fea-
tured here. 3.5
Growth of the Galaxy Stellar Mass Function tering is ‘halo assembly bias’, which refers to the fact that
halo clustering can depend on other properties besides halo
mass. For example, Gao et al. (2005) found that at fixed
halo mass, haloes that assembled earlier are more clustered
than those that assembled later. Therefore, galaxies in older
haloes will be more strongly clustered than they should be
for their halo mass, which means that their halo masses will
be measured as higher than they actually are. This could
alleviate some of the discrepancy between the observations
and models. For example, if the passive galaxies observed at
low redshift are associated to older haloes, then their halo
masses could have been overestimated. changes the least with redshift is Mice; as this is an HOD
model it naturally matches the SMHM relation better than
the SAMs. 3.7
Stellar Mass - Halo Mass Relation Whilst this could be due to problems with the
observational measurements of these quantities, this could
point towards areas where the models still need to improve. As we move to higher redshift the SMHM relation
changes little. The peak of the relation moves to slightly
higher halo masses and the average stellar mass for low-mass
haloes decreases by ∼0.4 dex at 1011.5M⊙. In the mod-
els the average stellar mass for low-mass haloes decreases
slightly with increasing redshift, but is above the SMHM
relation by z = 1.0 for most models. This discrepancy can
be partially explained by the cut in stellar mass applied
at M∗= 109M⊙h−1, which may have skewed the distribu-
tion towards higher stellar masses. This might be enough
to explain the difference for models such as Lgalaxies or
Galform, but the discrepancy is too large for Morgana,
DLB07 and ySAM. In these models, the average stellar
mass for low-mass haloes at high redshift is too high. This
means that star formation in these objects is very efficient,
leading to an increase in the number of low-mass galaxies at
z ∼2. This is likely due to the way that the physics involved
in the gas cycle is implemented in these models. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
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on 17 July 2018 ACKNOWLEDGEMENTS We thank Carnegie Observatories for their support and hos-
pitality during the workshop ‘Cosmic CARNage’ where all
the calibration issues were discussed and the roadmap laid
out for the work presented here. • Whilst most of the models are able to match the ob-
served stellar mass function at low redshift, they tend to
overproduce the number density of low-mass galaxies at high
redshift. The authors would further like to express special thanks
to the Instituto de Fisica Teorica (IFT-UAM/CSIC in
Madrid) for its hospitality and support, via the Centro de
Excelencia Severo Ochoa Program under Grant No. SEV-
2012-0249, during the three week workshop ‘nIFTy Cos-
mology’ where this work developed. We further acknowl-
edge the financial support of the 2014 University of West-
ern Australia Research Collaboration Award for ‘Fast Ap-
proximate Synthetic Universes for the SKA’, the ARC Cen-
tre of Excellence for All Sky Astrophysics (CAASTRO)
grant number CE110001020, and the ARC Discovery Project
DP140100198. We also recognise support from the Universi-
dad Autonoma de Madrid (UAM) for the workshop infras-
tructure. • In most of the models the low-mass end of the star-
forming stellar mass function is already largely in place at
high redshift (z > 1), in contrast to observations. This is be-
cause the models appear to produce too many star-forming
galaxies below the knee of the stellar-mass function at early
times. • In most of the models the low-mass end of the star-
forming stellar mass function is already largely in place at
high redshift (z > 1), in contrast to observations. This is be-
cause the models appear to produce too many star-forming
galaxies below the knee of the stellar-mass function at early
times. • The passive stellar mass function from the models
evolves with redshift as in the observations, but does not
have the same turnover or flattening in the number density
at the low-mass end. • Whilst most of the models match the passive fraction
well at high masses, for some of the models the passive frac-
tion is too high at low masses. This is despite the overpro-
duction of low-mass star-forming galaxies. We would like to thank Rachel Somerville, Gabriella
De Lucia and Pierluigi Monaco for kindly providing useful
discussion and comments. 5
CONCLUSIONS In this paper we have contrasted nine different galaxy forma-
tion models and compared them to the latest high-redshift
observations. In doing so we have highlighted the areas in
which the models find particular difficulty in matching the
observations. We can see from this project that some of the
models still have trouble simultaneously matching the stellar
mass function at both low and high redshift. The galaxies
look roughly correct at z = 0, but for many models there are
too many low-mass galaxies at z ∼2, as has also been seen
previously (e.g. Fontana et al. 2006; Fontanot et al. 2009;
Weinmann et al. 2012; Henriques et al. 2012; Guo et al. 2016). For intermediate- and high-mass haloes, the average
stellar mass generally decreases with increasing redshift in
the models and the slope of the relation decreases. This sug-
gests that star formation was less efficient in the models at
high redshift. At z = 0.1 the models overpredict the stellar
mass in high-mass haloes, but slightly underpredict it by
z = 2.0. For intermediate-mass haloes, the average stellar
mass is too low in the models at z = 2.0 by up to 0.5 dex,
as is the case for Galform at 1012.5M⊙. The model that 12 R. Asquith et al. high redshift will help shed light on these issues and identify
further areas of improvement for the models. To explore this further, we split galaxies into pas-
sive and star-forming populations. We find that there are
too many star-forming galaxies with stellar masses below
1011M⊙in many of the models at z ∼2. In summary, while some of the models are remarkably
successful at reproducing the evolution of the stellar mass
function, there remain significant issues. In particular: Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
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on 17 July 2018 ACKNOWLEDGEMENTS • Most of the models are able to reproduce the relation-
ship between sSFR and the mass of the star-forming galaxies
at low redshift, but underpredict the sSFR at high redshift. RA is funded by the Science and Technology Funding
Council (STFC) through a studentship. AK is supported
by the Ministerio de Econom´ıa y Competitividad and the
Fondo Europeo de Desarrollo Regional (MINECO/FEDER,
UE) in Spain through grant AYA2015-63810-P. He fur-
ther thanks Denison Witmer for california brown and
blue. VGP acknowledges support from a European Re-
search Council Starting Grant (DEGAS-259586). This work
used the DiRAC Data Centric system at Durham Uni-
versity, operated by the Institute for Computational Cos-
mology on behalf of the STFC DiRAC HPC Facility
(www.dirac.ac.uk). This equipment was funded by BIS Na-
tional E-infrastructure capital grant ST/K00042X/1, STFC
capital grant ST/H008519/1, and STFC DiRAC Opera-
tions grant ST/K003267/1 and Durham University. DiRAC
is part of the National E-Infrastructure. FJC acknowl-
edges support from the Spanish Ministerio de Econom´ıa y
Competitividad project AYA2012-39620. SAC acknowledges
funding from Consejo Nacional de Investigaciones Cient´ıfi-
cas y T´ecnicas (CONICET, PIP-0387), Agencia Nacional
de Promoci´on Cient´ıfica y Tecnol´ogica (ANPCyT, PICT-
2013-0317), and Universidad Nacional de La Plata (G11-
124), Argentina. DJC acknowledges receipt of a QEII Fel-
lowship from the Australian Government. FF acknowledges
financial contribution from the grants PRIN MIUR 2009
‘The Intergalactic Medium as a probe of the growth of
cosmic structures’ and PRIN INAF 2010 ‘From the dawn
of galaxy formation’. The work of BH was supported by
Advanced Grant 246797 GALFORMOD from the Euro-
pean Research Council. NDP was supported by BASAL
PFB-06 CATA, and Fondecyt 1150300. Part of the calcu-
lations presented here were run using the Geryon cluster at
the Center for Astro-Engineering at U. Catolica, which re-
ceived funding from QUIMAL 130008 and Fondequip AIC-
57. CP acknowledges support of the Australian Research • Observational measurements of halo mass, estimated
from galaxy clustering, indicate clear downsizing in the av-
erage halo mass occupied by star-forming galaxies as a func-
tion of redshift. This is not clearly indicated by any of the
models; both star-forming and passive galaxies in the mod-
els occupy haloes with lower masses than those inferred from
observations at z = 2. Cosmic CARNage II
13 Cosmic CARNage II
13 13 Council (ARC) through Future Fellowship FT130100041
and Discovery Project DP140100198. WC and CP acknowl-
edge support of ARC DP130100117. PAT acknowledges
support from the Science and Technology Facilities Coun-
cil (grant number ST/L000652/1). SKY acknowledges sup-
port from the Korean National Research Foundation (NRF-
2017R1A2A1A05001116). This study was performed under
the umbrella of the joint collaboration between Yonsei Uni-
versity Observatory and the Korean Astronomy and Space
Science Institute. The supercomputing time for the numer-
ical simulations was kindly provided by KISTI (KSC-2014-
G2-003). Faber S. M., et al., 2007, ApJ, 665, 265 Fontana A., et al., 2004, A&A, 424, 23 Fontana A., et al., 2006, A&A, 459, 745 Fontanot F., De Lucia G., Monaco P., Somerville R. S., Santini
P., 2009, MNRAS, 397, 1776 Gan J., Kang X., van den Bosch F. C., Hou J., 2010, MNRAS,
408, 2201 Gao L., Springel V., White S. D. M., 2005, MNRAS, 363, L66 Gonzalez-Perez V., Lacey C. G., Baugh C. M., Lagos C. D. P.,
Helly J., Campbell D. J. R., Mitchell P. D., 2014, MNRAS,
439, 264 Gonzalez-Perez V., et al., 2018, MNRAS, 474, 4024 Gunn J. E., Gott J. R. I., 1972, ApJ, 176, 1 )
The authors contributed to this paper in the following
ways: RA analysed the data, created the plots and wrote
the paper along with FRP and OA. AK & CP formed part
of the core team and along with FRP organised the nIFTy
workshop where this work was initiated. AB organised the
follow-up workshop ‘Cosmic CARNage’ where all the discus-
sions about the common calibration took place and out of
which this paper emerged. JO supplied the simulation, halo
catalogue and merger tree for the work presented here. WH
supplied the halo mass measurements from the UDS that
were used in this work. The remaining authors performed
the SAM or HOD modelling using their codes, in particular
FJC, AC, SC, DC, FF, VGP, BH, JL, ARHS, CVM, and
SKY actively ran their models. All authors proof-read and
commented on the paper. Guo Q., et al., 2011, MNRAS, 413, 101 Guo Q., et al., 2016, MNRAS, 461, 3457 Hartley W. G., et al., 2013, MNRAS, 431, 3045 Henriques B. M. B., White S. D. M., Lemson G., Thomas P. A.,
Guo Q., Marleau G.-D., Overzier R. A., 2012, MNRAS, 421,
2904 Henriques B. M. B., White S. D. References Kovaˇc K., et al., 2014, MNRAS, 438, 717 Lagos C. D. P., Baugh C. M., Zwaan M. A., Lacey C. G., Baldry I. K., Glazebrook K., Brinkmann J., Ivezi´c ˇZ., Lupton
R. H., Nichol R. C., Szalay A. S., 2004, ApJ, 600, 681 Lagos C. D. P., Baugh C. M., Zwaan M. A., Lacey C. G.,
Gonzalez-Perez V., Power C., Swinbank A. M., van Kampen
E., 2014, MNRAS, 440, 920 Gonzalez-Perez V., Power C., Swinbank A. M., van Kampen
E., 2014, MNRAS, 440, 920 E., 2014, MNRAS, 440, 920 y
Behroozi P. S., Wechsler R. H., Wu H.-Y., 2013a, ApJ, 762, 109 Lawrence A., et al., 2007, MNRAS, 379, 1599 Behroozi P. S., Wechsler R. H., Wu H.-Y., Busha M. T., Klypin
A. A., Primack J. R., 2013b, ApJ, 763, 18 Lee J., Yi S. K., 2013, ApJ, 766, 38 Behroozi P. S., Wechsler R. H., Conroy C., 2013c, ApJ, 770, 57 Marchesini D., van Dokkum P. G., F¨orster Schreiber N. M., Franx Marchesini D., van Dokkum P. G., F¨orster Schreib
M., Labb´e I., Wuyts S., 2009, ApJ, 701, 1765 Marchesini D., van Dokkum P. G., F¨orster Schreibe Marchesini D., van Dokkum P. G., Forster Schreiber
M., Labb´e I., Wuyts S., 2009, ApJ, 701, 1765 M., Labb´e I., Wuyts S., 2009, ApJ, 701, 1765 Benson A. J., 2010, Phys. Rep., 495, 33 Marchesini D., et al., 2010, ApJ, 725, 1277 Benson A. J., Bower R. G., Frenk C. S., Lacey C. G., Baugh
C. M., Cole S., 2003, ApJ, 599, 38 McCarthy I. G., Frenk C. S., Font A. S., Lacey C. G., Bower R. G.,
Mitchell N. L., Balogh M. L., Theuns T., 2008, MNRAS, 383,
593 Berlind A. A., Weinberg D. H., 2002, ApJ, 575, 587 Berlind A. A., et al., 2003, ApJ, 593, 1 McCracken H. J., et al., 2012, A&A, 544, A156 Bower R. G., Benson A. J., Malbon R., Helly J. C., Frenk C. S.,
Baugh C. M., Cole S., Lacey C. G., 2006, MNRAS, 370, 645 Mitchell P. D., Lacey C. G., Cole S., Baugh C. M., 2014, MNRAS,
444, 2637 Brinchmann J., Charlot S., White S. D. M., Tremonti C., Kauff-
mann G., Heckman T., Brinkmann J., 2004, MNRAS, 351,
1151 Mo H. J., White S. D. M., 2002, MNRAS, 336, 112 Monaco P., Fontanot F., Taffoni G., 2007, MNRAS, 3 Mundy C. J., Conselice C. J., Ownsworth J. Cosmic CARNage II
13 M., Thomas P. A., Angulo R. E.,
Guo Q., Lemson G., Springel V., 2013, MNRAS, 431, 3373 Henriques B. M. B., White S. D. M., Thomas P. A., Angulo R.,
Guo Q., Lemson G., Springel V., Overzier R., 2015, MNRAS,
451, 2663 Hirschmann M., De Lucia G., Fontanot F., 2016, MNRAS, 461,
1760 Ilbert O., et al., 2010, ApJ, 709, 644 Ilbert O., et al., 2013, A&A, 556, A55 Jiang L., Helly J. C., Cole S., Frenk C. S., 2014, MNRAS, 440,
2115 This research has made use of NASA’s Astrophysics
Data System (ADS) and the arXiv preprint server. This research has made use of NASA’s Astrophysics
Data System (ADS) and the arXiv preprint server. Kauffmann G., et al., 2003, MNRAS, 341, 33 Kennicutt Jr. R. C., 1998, ARA&A, 36, 189 Kimm T., Yi S. K., Khochfar S., 2011, ApJ, 729, 11 Knebe A., et al., 2015, MNRAS, 451, 4029 Knebe A., et al., 2018, MNRAS, 475, 2936 ACKNOWLEDGEMENTS • Observational measurements of halo mass, estimated
from galaxy clustering, indicate clear downsizing in the av-
erage halo mass occupied by star-forming galaxies as a func-
tion of redshift. This is not clearly indicated by any of the
models; both star-forming and passive galaxies in the mod-
els occupy haloes with lower masses than those inferred from
observations at z = 2. • The average stellar mass is higher in low-mass haloes at
high redshift in the models compared to observations, mean-
ing that star formation in low-mass haloes is more efficient
in the models than in the real Universe. • The average stellar mass is higher in low-mass haloes at
high redshift in the models compared to observations, mean-
ing that star formation in low-mass haloes is more efficient
in the models than in the real Universe. Achieving consistent results at both z = 0 and z = 2 with
a population of galaxies that evolves strongly with redshift is
clearly difficult. The HOD model, Mice, obtains good results
but the galaxies present at z = 2 are not evolved directly into
the z = 0 population. Of the SAMs, the Lgalaxies and Sag
models best match the growth of the observed mass func-
tions, but they share the same trends as the other models
for the specific star formation rate and average halo mass
within which the objects reside. Both of these models found
that they needed to modify the treatment of the gas cycle
in order to match the evolution of the low-mass end of the
stellar mass function. This is very promising for the galaxy
formation modelling community, which has long struggled
with this issue. While it is clear that current galaxy formation models
can reproduce a variety of observational data, we have iden-
tified key areas of tension. Some models still overpredict the
number of low-mass galaxies at high redshift, but even the
models that can match the evolution of the galaxy stellar
mass function underpredict the specific star formation rates
of galaxies at early times. Future observational surveys at APPENDIX B: STELLAR MASS FUNCTION
INCLUDING EDDINGTON BIAS In Figure 1 we have compared the stellar mass function
from the models to observational data from Davidzon et al. (2017). We do not scatter the stellar masses in the models
with the 0.08(1 + z) dex scatter used to mimic observational
uncertainties, as Davidzon et al. (2017) have accounted for
this when finding the best-fit Schechter parameters to their
stellar mass function. Here we present an alternative version of Figure 1,
shown in Figure B1, where we do apply the scatter to the
stellar mass values in the models. We compare to obser-
vations from Muzzin et al. (2013), who do not take these
uncertainties into account when fitting to the stellar mass
function. Like Davidzon et al. (2017), the observations from
Muzzin et al. (2013) are based on the UltraVISTA near-
infrared survey of the COSMOS field. Supernova feedback and winds The mass reheated by super-
nova feedback involves an explicit redshift dependence and
an additional modulation with virial velocity, according to
a fit to results from FIRE (Feedback in Realistic Environ-
ments) hydrodynamical simulations (Muratov et al. 2015). Comparing Figure B1 to Figure 1, we can see that the
main difference to the SMF is at the high-mass end and that
the low-mass end is largely unaffected. Due to the redshift
dependence of the scatter we apply to the stellar masses, the
differences are also larger at high redshift. As an example,
the value of φ increases by over 0.5 dex at 1011M⊙in the
redshift bin 2.5 < z < 3.0 in Lgalaxies when the scatter is
applied. Gas ejection and reincorporation The energy input by mas-
sive stars eject some of the hot gas out of the halo, according
to the energy conservation argument presented by Guo et al. (2011). The energy injected by massive stars is proportional
to the mean kinetic energy of supernova ejecta per unit
mass of stars formed, and includes the same explicit red-
shift dependence and the additional modulation with virial
velocity as the reheated mass. The ejected gas mass is re-
incorporated back onto the corresponding (sub)halo within a
timescale that depends on the inverse of the (sub)halo mass
(Henriques et al. 2013). In Figure 1, it appears that most of the models under-
predict the number of high-mass galaxies at high redshift,
with only Mice and GalICS-2.0 matching observations. 14
R. Asquith et al. 14 Strateva I., et al., 2001, AJ, 122, 1861 Strateva I., et al., 2001, AJ, 122, 1861 Orphans The positions and velocities of orphan galaxies are
obtained from the integration of the orbits of subhaloes that
will not longer be identified. The orbits are integrated nu-
merically, considering the last known position, velocity and
virial mass of subhaloes as initial conditions, and taking into
account mass loss by TS and dynamical friction effects, fol-
lowing some aspects of the works by Gan et al. (2010) and
Kimm et al. (2011). A merger event occurs when the halo-
centric distance becomes smaller that 10 percent of the virial
radius of the host halo. Tecce T. E., Cora S. A., Tissera P. B., Abadi M. G., Lagos
C. D. P., 2010, MNRAS, 408, 2008 Weinmann S. M., Pasquali A., Oppenheimer B. D., Finlator K.,
Mendel J. T., Crain R. A., Macci`o A. V., 2012, MNRAS, 426,
2797 White C. E., Somerville R. S., Ferguson H. C., 2015, ApJ, 799,
201 Zheng Z., et al., 2005, ApJ, 633, 791 A1
Sag The changes implemented in SAG are described in detail in
Cora et al. (2018). We summarize them here: The changes implemented in SAG are described in detail in
Cora et al. (2018). We summarize them here: Cooling Both central and satellite galaxies experience gas
cooling processes. Satellite galaxies keep their hot gas haloes
which are gradually removed by the action of ram pressure
stripping (RPS), modelled according to McCarthy et al. (2008), and tidal stripping (TS). When the mass of the
hot gas halo becomes smaller than 10 percent of the total
baryonic mass of the galaxy, it is assumed that it no longer
shields the cold gas disc from the action of RPS, which is
modelled following the criterion from Gunn & Gott (1972);
see Tecce et al. (2010) for more details. Values of ram pres-
sure experienced by galaxies in haloes of different mass as
a function of halo-centric distance and redshift are obtained
from fitting formulae derived from the self-consistent infor-
mation provided by the hydrodynamical simulations anal-
ysed by Tecce et al. (2010), as described in Vega-Mart´ınez
et al. (in prep.). A2
Sage A description of the physical prescriptions of each model
is available in the Appendix of Knebe et al. (2015). Here
we present a brief description of the changes to any of the
models since then: The only change in Sage is to the radio mode AGN feed-
back. It is explained in detail in Croton et al. (2016) and
summarized here: AGN feedback The radio mode AGN feedback has been
modified in Sage since Croton et al. (2006). There is now
a heating radius, inside which gas is prevented from cool-
ing. This heating radius increases with subsequent heating
episodes and can not decrease. AGN feedback The radio mode AGN feedback has been
modified in Sage since Croton et al. (2006). There is now
a heating radius, inside which gas is prevented from cool-
ing. This heating radius increases with subsequent heating
episodes and can not decrease. Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 References R., 2015, MNRAS,
450, 3696 Carretero J., Castander F. J., Gazta˜naga E., Crocce M., Fosalba
P., 2015, MNRAS, 447, 646 Muratov A. L., Kereˇs D., Faucher-Gigu`ere C.-A., Hopkins P. F
Quataert E., Murray N., 2015, MNRAS, 454, 2691 Cattaneo A., et al., 2017, MNRAS, 471, 1401 Quataert E., Murray N., 2015, MNRAS, 454, 2691 Cimatti A., Daddi E., Renzini A., 2006, A&A, 453, L29 Cimatti A., Daddi E., Renzini A., 2006, A&A Muzzin A., et al., 2013, ApJ, 777, 18 Cirasuolo M., et al., 2007, MNRAS, 380, 585 Planck Collaboration et al., 2014, A&A, 571, A16 Conroy C., Gunn J. E., White M., 2009, ApJ, 699, 486 Pozzetti L., et al., 2007, A&A, 474, 443 Cora S. A., et al., 2018, preprint (arXiv:1801.03883) Pozzetti L., et al., 2010, A&A, 523, A13 Cowie L. L., Songaila A., Hu E. M., Cohen J. G., 1996, AJ, 112,
839 Rodrigues L. F. S., Vernon I., Bower R. G., 2017, MNRAS, 466,
2418 Croton D. J., et al., 2006, MNRAS, 365, 11 Shankar F., et al., 2015, ApJ, 802, 73 Croton D. J., et al., 2016, ApJS, 222, 22 Somerville R. S., Dav´e R., 2015, ARA&A, 53, 51 Daddi E., et al., 2007, ApJ, 670, 156 Springel V., 2005, MNRAS, 364, 1105 Davidzon I., et al., 2017, A&A, 605, A70 De Lucia G., Blaizot J., 2007, MNRAS, 375, 2 Stevens A. R. H., Brown T., 2017, MNRAS, 471, 447 Di Matteo T., Springel V., Hernquist L., 2005, Nature, 433, 604 Stevens A. R. H., Croton D. J., Mutch S. J., 2016, MNRAS, 461,
859 Elbaz D., et al., 2007, A&A, 468, 33 Elbaz D., et al., 2007, A&A, 468, 33 y
y
on 17 July 2018 14
R. Asquith et al. by University of
on 17 July 2018 Downloaded from https://academic.oup.com/mnras/advance-article-abstract/doi/10.1093/mnras/sty1870/5054051
by University of Nottingham user
on 17 July 2018 by U
e s ty o
on 17 July 2018 APPENDIX B: STELLAR MASS FUNCTION
INCLUDING EDDINGTON BIAS However, in Figure B1 the models and observations agree
better at high redshift for several other models, namely Gal-
form and Morgana. The data from Muzzin et al. (2013)
form part of the combined dataset used to calibrate the mod-
els, so it is natural that the models may match this data
better. AGN feedback AGN are produced from the growth of central
BHs. When this growth takes place from cold gas accretion
during gas cooling, it depends on the mass of the hot gas
atmosphere, following Henriques et al. (2015). 15 Cosmic CARNage II
1 Cosmic CARNage II
15
−5
−4
−3
−2
log(φ[Mpc−3dex−1])
0.5 < z < 1.0
All
1.5 < z < 2.0
Muzzin+13
DLB07
Galform
GalICS-2.0
LGALAXIES
2.5 < z < 3.0
MICE
MORGANA
SAG
SAGE
ySAM
−5
−4
−3
−2
log(φ[Mpc−3dex−1])
Passive
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
−5
−4
−3
−2
log(φ[Mpc−3dex−1])
Star-forming
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
-5
-4
-3
-2
log(φ[Mpc−3dex−1])
-5
-4
-3
-2
log(φ[Mpc−3dex−1])
-5
-4
-3
-2
log(φ[Mpc−3dex−1])
Figure B1. Alternative version of Figure 1, applying the 0.08(1 + z) dex scatter to the stellar mass values in the models. Here we
compare to observational data from Muzzin et al. (2013), who do not take into account Eddington bias when finding the best-fit Schechter
parameters. Here the models match the observations better at high masses and high redshift. 2.5 < z < 3.0
MICE
MORGANA
SAG
SAGE
ySAM
9.0
9.5
10.0
10.5
11.0
11.5
12.0
log(M∗[M⊙])
-5
-4
-3
-2
log(φ[Mpc−3dex−1]) log(M∗[M⊙]) 4
3
2
log(φ[Mpc−3dex−1]) log(φ[Mpc−3dex−1]) 10.0
10.5
11. log(M∗[M⊙]) 10.0
10.5
11
log(M∗[M⊙]) Figure B1. Alternative version of Figure 1, applying the 0.08(1 + z) dex scatter to the stellar mass values in the models. Here we
compare to observational data from Muzzin et al. (2013), who do not take into account Eddington bias when finding the best-fit Schechter
parameters. Here the models match the observations better at high masses and high redshift.
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Hyponatremia due to Severe Primary Hypothyroidism in an Infant
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Frontiers in pediatrics
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Abbreviations: AVP, arginine vasopressin; BUN, blood urea nitrogen; FENa, fractional excretion of sodium in the urine;
FEUrate, fractional excretion of urate; TSH, thyroid stimulating hormone. Edited by: Wassim Chemaitilly,
St. Jude Children’s Research
Hospital, USA Hyponatremia due to Severe Primary
Hypothyroidism in an Infant
Nickolas T. Agathis1, Ingrid M. Libman2 and Michael L. Moritz2*
1 Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, USA, 2 Children’s Hospital of Pittsburgh of UPMC, The
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Hyponatremia has been reported in the elderly with hypothyroidism and myxedema, but
this has not been a universal finding in clinical studies and there have been only a few
reports in children. We report a case of an infant who developed hyponatremia due to
severe primary hypothyroidism. A 4-month-old ex-preterm male, who had been euthyroid
on the newborn screen, developed unexplained hospital-acquired hyponatremia (serum
Na 124 mEq/L) while on full oral feeds. He was euvolemic, appeared well and was without
myxedema. An evaluation of hyponatremia was negative with the exception of severe
primary hypothyroidism (TSH 315.4 IU/mL, repeat 540 IU/mL). The hyponatremia resolved
with thyroxine supplementation. This case demonstrates that severe hypothyroidism can
result in hyponatremia in infants. It is critical to consider hypothyroidism in the evaluation
of an infant with unexplained hyponatremia as untreated hypothyroidism can lead to
profound developmental delays. Specialty section: Specialty section:
This article was submitted to Pediatric
Endocrinology, a section of the
journal Frontiers in Pediatrics This article was submitted to Pediatric
Endocrinology, a section of the
journal Frontiers in Pediatrics Received: 05 August 2015
Accepted: 23 October 2015
Published: 10 November 2015 CASE REPORT
published: 10 November 2015
doi: 10.3389/fped.2015.00096 CASE REPORT CASE REPORT Accepted: 23 October 2015
Published: 10 November 2015 A 4-month-old male was born at 32-week gestation as a result of premature rupture of mem-
branes with a birth weight of 1782 g and APGAR scores of 3 and 8 at 1 and 5 min postdelivery,
respectively. His newborn screen was reported as normal. His past medical history was significant Keywords: child development, hyponatremia, hypothyroidism, infant, newborn, thyroxine INTRODUCTION Reviewed by:
Mohamad Maghnie,
University of Genova, Italy
Laura Gabriela Gonzalez Briceño,
Hôpital Necker – Enfants Malades,
France Hyponatremia is the most common electrolyte abnormality encountered in hospitalized patients. It
is primarily the result of excess free water intake in conjunction with impaired free water excretion
due to arginine vasopressin (AVP) excess. One potential cause of hyponatremia is hypothyroidism. This association has been primarily reported in the elderly with myxedema (1, 2), with only a few
reports in children (3–6). The mechanisms linking the two entities are not entirely clear, but there
is evidence to support both prerenal and renal mechanisms. There is controversy in the literature
whether hypothyroidism produces hyponatremia, as this has not been a universal finding in all
studies. *Correspondence:
Michael L. Moritz
michael.moritz@chp.edu We report a 4-month-old ex-preterm male with a complicated past medical history and who was
reported to have a normal newborn screen, who developed unexplained hyponatremia. This led
to the diagnosis of severe hypothyroidism with resolution of the hyponatremia following thyroxin
supplementation. Citation: At 16 weeks of age, weight 4.57 kg, having been admitted to
the hospital since birth, he was switched from total parenteral
nutrition to enteral feeds consisting of Neocate and breast milk. At
that time, his serum sodium was 136 mEq/L. Serum chemistries
were done 4 days later, which revealed a serum sodium level of
124 mEq/L and plasma osmolality of 260 mOsm/kg confirmed on
two repeated measurements (Table 1). There was no apparent
explanation for the hyponatremia, since there was no apparent
volume depletion, vomiting, diarrhea, or gastrostomy tube loss. He was stable from a respiratory standpoint and was not requiring
oxygen. His only medications were famotidine, lansoprazole, and
methadone for fentanyl withdrawal. He was not receiving diuretics
or intravenous fluids. He was neurologically asymptomatic with-
out lethargy, irritability, vomiting, or feeding intolerance. His total
feeds in the previous 24 h were 110 mL/kg and his weight had
increased by 400 g since the previous sodium level 4 days earlier. Family history was remarkable for hypothyroidism in the
maternal grandmother and a paternal aunt. To further evaluate
the cause of hypothyroidism, a thyroid ultrasound and thyroid
scan were done, which were both normal. The scan demonstrated
homogeneous uptake at the expected location of the thyroid
gland on both sides of the neck with no heterotopic uptake seen. y
g
p
y
An evaluation of hyponatremia included serum and urine
biochemistries including liver function tests, thyroid function
tests, a cortisol level, serum and urine osmolality levels, and
a uric acid level (Table 1). Spot urine electrolytes revealed a TABLE 1 | Laboratory investigations. Citation: Front. Pediatr. 3:96. doi: 10.3389/fped.2015.00096 November 2015 | Volume 3 | Article 96 1 Frontiers in Pediatrics | www.frontiersin.org Hyponatremia due to Hypothyroidism Agathis et al. low urine sodium level of 23 mEq/L and a low fractional excre-
tion of sodium (FENa) of 0.2%; this combination of findings
was suggestive of a prerenal state. He had severe hypouricemia
with a uric acid level of 0.9 mg/dL and an elevated fractional
excretion of urate (FEUrate) of 45%. This combination of find-
ings was suggestive of a Syndrome of Inappropriate Antidi-
uretic Hormone secretion (SIADH)-like state. Thyroid function
tests revealed severe hypothyroidism (TSH 315.4 IU/mL) (normal
range 1.7–9.1 IU/mL), which was confirmed on repeat measure-
ment with a free T4 of 0.52 ng/dL (normal range 0.8–1.8 ng/dL)
and a TSH of 540 IU/mL, with a normal cortisol. The hypona-
tremia was initially corrected with a 24-h infusion of 0.9% sodium
chloride at a rate of 4 mL/kg/h and thyroid supplementation, to
a sodium level of 138 mEq/L. The hypothyroidism was treated
with 37.5 mcg daily of levothyroxine on Day 1 and 2, which
was increased to 50 mcg daily on day 3. The intravenous fluids
were discontinued on day 1 and the sodium level decreased to
133 mEq/L on day 2. The serum sodium then normalized without
further intravenous fluids and without oral sodium supplemen-
tation. On day 4, repeat urine and serum chemistries revealed a
serum sodium of 136 mEq/L, a spot urine sodium of 63 mEq/L
with FENa of 0.7% and resolving hypouricemia with an FEUrate
that decreased to 22% and eventually to 18% on day 73 (Table 1). Due to mild hyperkalemia an ACTH stimulation test was done
which was normal. for an omphalocele repaired at 2 days of life. Between 14 and
15 weeks of life, he had a pyloric stenosis treated with a pyloromy-
otomy, gastroesophageal reflux treated with a Nissen fundoplica-
tion and gastrostomy tube, and a bilateral herniorrhaphy. Other
complications included influenza-A pneumonia during the first
month of life requiring 17 days of mechanical ventilation, chronic
lung disease, and a grade I intraventricular hemorrhage noted at
6 weeks of age on head ultrasound, which had resolved at 4 months
of age. An echocardiogram revealed a resolved patent ductus
arteriosus with a clinically insignificant patent foramen ovale and
a normal renal sonogram. November 2015 | Volume 3 | Article 96 DISCUSSION We report a case of an ex-preterm infant found to have hospital-
acquired hyponatremia most likely due to hypothyroidism as
other causes of hyponatremia were excluded. While hyponatremia
from SIADH can be due to medications, methadone and lan-
soprazole are extremely infrequent causes of hyponatremia and
the hyponatremia resolved promptly following treatment of the
hypothyroidism despite continuing these medications. The exact
etiology of the hypothyroidism has not yet been identified. Con-
genital hypothyroidism can not be excluded as the newborn screen
for congenital hypothyroidism may have given a false negative
result as the child was an ex-preterm infant of 32-week gestation
and was also ill requiring an omphalocele repair at 2 days of age. Also the neonatal screening was not repeated 2 weeks after the
first screening as recommended by consensus guidelines (7). The
associated features of omphalocele, pyloric stenosis, and cardiac
anomalies are suggestive of a genetic cause, which would merit
further evaluation. Acquired hypothyroidism is unlikely; however,
thyroid autoantibodies were not checked. This case report supports the assertion that severe hypothy-
roidism is associated with hyponatremia, yet other investigators
have not been able to find an association. In a sample of 445
hypothyroid adults, the frequency of hyponatremia was no dif-
ferent than in euthyroid controls (17). Similarly, there was no
difference in sodium concentrations or the incidence of hypona-
tremia in a homogenous group of 32 congenital hypothyroid
infants compared to age matched controls (18). Warner et al. was able to demonstrate a statistically significant, but seemingly
clinically insignificant, relationship between hypothyroidism and
hyponatremia, with a fall in serum sodium concentration of
0.14 mEq/L for every 10 IU/L rise in TSH (19). Based on these
findings, our patient’s TSH of 539 IU/L is associated with an aver-
age fall in serum sodium concentration of approximately 7 mEq/L
from baseline. Therefore, this model was partially accurate in
predicting that our patient could develop clinically significant
hyponatremia. The association between hyponatremia and hypothyroidism
has been recognized primarily in adults with severe myxedema (1,
2), but is an extremely unusual finding in an infant. There have
been four previous reports in children (3–6), three of whom were
infants with congenital hypothyroidism, of which one was water
intoxicated (5), and another an 8-year-old child with hypothy-
roidism as a result of brain injury from status epilepticus (4). Citation: Day -4
Day 0
Day 1
Day 2
Day 4
Day 7
Day 73
SERUM (REFERENCE RANGES)
Na (mEq/L)
136
124
138
133
136
134
137
K (mEq/L)
5.2
4.9
5.3
5.4
5.4
6.3
6.3
Cl (mEq/L)
106
88
103
100
107
97
96
CO2 (mEq/L)
23.0
31.0
26.0
28.0
26.0
29.0
34
BUN (mEq/L)
25
15
12
13
19
21
16
Cr (mg/dL)
0.3
0.2
0.2
0.2
0.2
0.3
0.2
Glucose (mg/dL)
89
69
94
100
62
Uric acid (mg/dL)
0.9
1.4
Osmolality (mOsm/kg)
260
285
Cortisol (ug/dL) (3–23)
11.5
2.2
TSH (IU/mL) (1.7–9.1)
315.4
539.86
92.9
0.27
T3 (ng/dL) (1.03–2.29)
0.85
1.50
2.50
T4 (ug/dL) (7–15)
2.3
5.8
12.3
Free T4 (ng/dL) (0.8–1.8)
0.52
2.09
URINE
Specific gravity
1.006
pH
8.0
Osm (mOsm/kg)
389
349
172
Na (mEq/L)
23
63
21
K (mEq/L)
57.2
39.2
15.8
Cl (mEq/L)
32
69
<15
Cr (mg/dL)
15
14
6
Uric acid (mg/dL)
30.3
21.4
9.4
FENa (%)
0.2
0.7
0.5
FEUrate (%)
45
22
18 2 Hyponatremia due to Hypothyroidism Agathis et al. at 0.2% with an elevated BUN-to-creatinine ratio of 80, reflective
of appropriate renal compensation secondary to decreased effec-
tive circulating volume. The decreased urinary sodium excretion
might also partially reflect decreased sodium intake as the patient
was on a low sodium enteral diet. Patients with SIADH can have
a low urine sodium concentration and FENa, if they are sodium
restricted (13). Our patient also had significant hypouricemia and
an elevated fractional excretion of urate of 45% (normal 13–26%)
(14), which is consistent with proximal tubular dysfunction from
natriuretic peptides (15). This phenomenon may also be encoun-
tered in SIADH and cerebral salt wasting (16). After levothyroxine
was started, the hyponatremia, hypouricemia and elevated frac-
tional excretion of urate resolved. This suggests that the hypona-
tremia was related to hypothyroidism. Thyroid autoantibodies and genetic testing were not performed. His levothyroxine requirements decreased overtime and at 2-year
follow-up, he was on 25 mcg daily. His development at 2 years
of age is almost normal with the exception of an oral aversion
requiring gastrostomy tube feedings and mild speech delay. Thyroid autoantibodies and genetic testing were not performed. His levothyroxine requirements decreased overtime and at 2-year
follow-up, he was on 25 mcg daily. His development at 2 years
of age is almost normal with the exception of an oral aversion
requiring gastrostomy tube feedings and mild speech delay. DISCUSSION This case report supports the association between hypothy-
roidism and hyponatremia and supports the physiologic hypoth-
esis of extracellular volume depletion with proximal tubular dys-
function. While hyponatremia may be an uncommon conse-
quence of severe hypothyroidism in infants, it is nevertheless
an important cause. Hypothyroidism in infancy can lead to sig-
nificant neurological damage and delays in intellectual devel-
opment, which can be prevented with appropriate thyroxine
treatment (20, 21). Therefore, if not for the development of
hyponatremia in this patient, the diagnosis of hypothyroidism
would likely have been missed or delayed leading to possible
irreversible neurological damage and developmental delay in our
patient. We therefore recommend that thyroid function tests be
included in the evaluation of unexplained hyponatremia, partic-
ularly in the pediatric population. The test is of minimal cost
and could rule out an easily treatable condition with significant
morbidity. There are two possible mechanisms described in the literature
in which hypothyroidism could lead to hyponatremia; our patient
had features of both. The prevailing view is that of a prerenal
mechanism and compensatory dilutional hyponatremia (8). It
has been shown that hypothyroidism initially causes a significant
increase in peripheral vascular resistance, decrease in cardiac
output, and concomitant decrease in glomerular filtration rate
(8). There is also evidence to suggest impaired sodium reabsorp-
tion in the proximal and distal tubules as a result of decreased
Na–K ATPase activity; this also would lead to volume depletion
and aggravate the pre-renal mechanisms (9, 10). Supporting this
hypothesis are studies, which have demonstrated a relationship
between hypothyroidism and subclinical and clinical pre-renal
acute kidney injury (11). Effective circulating volume deple-
tion from hypothyroidism would lead to up-regulation of both
the rennin–angiotensin–aldosterone system and AVP, leading to
hyponatremia. ETHICS STATEMENT Another
possible
mechanism
of
hyponatremia
due
to
hypothyroidism is the SIADH. Some studies have demonstrated
inappropriately elevated AVP levels, possibly due to impaired
osmoregulation or decreased metabolic clearance of AVP in
hypothyroid patients (12). A formal consent is not required for case reports at our institution. Verbal consent was obtained prior to working on the case and was
again obtained after completion of the report. Frontiers in Pediatrics | www.frontiersin.org REFERENCES of antidiuretic hormone. Am J Med (1957) 23(4):529–42. doi:10.1016/0002-
9343(57)90224-3 of antidiuretic hormone. Am J Med (1957) 23(4):529–42. doi:10.1016/0002-
9343(57)90224-3 14. Passwell JH, Modan M, Brish M, Orda S, Boichis H. Fractional excretion of
uric acid in infancy and childhood. Index of tubular maturation. Arch Dis Child
(1974) 49(11):878–82. doi:10.1136/adc.49.11.878 1. Macaron C, Famuyiwa O. Hyponatremia of hypothyroidism. Appropriate sup-
pression of antidiuretic hormone levels. Arch Intern Med (1978) 138(5):820–2. doi:10.1001/archinte.138.5.820 1. Macaron C, Famuyiwa O. Hyponatremia of hypothyroidism. Appropriate sup-
pression of antidiuretic hormone levels. Arch Intern Med (1978) 138(5):820–2. doi:10.1001/archinte.138.5.820 15. Espiner EA. Physiology of natriuretic peptides. J Intern Med (1994)
235(6):527–41. doi:10.1111/j.1365-2796.1994.tb01261.x 2. Nakano M, Higa M, Ishikawa R, Yamazaki T, Yamamuro W. Hyponatremia with
increased plasma antidiuretic hormone in a case of hypothyroidism. Intern Med
(2000) 39(12):1075–8. doi:10.2169/internalmedicine.39.1075 16. Berendes E, Walter M, Cullen P, Prien T, Van Aken H, Horsthemke J, et al. Secretion of brain natriuretic peptide in patients with aneurysmal subarachnoid
haemorrhage. Lancet (1997) 349(9047):245–9. doi:10.1016/S0140-6736(96)
08093-2 3. Chelimsky G, Davis ID, Kliegman RM. Neonatal hyponatremia associated
with congenital hypothyroidism. Clin Pediatr (1997) 36(3):177–80. doi:10.1177/
000992289703600310 17. Croal BL, Blake AM, Johnston J, Glen AC, O’Reilly DS. Absence of relation
between hyponatraemia and hypothyroidism. Lancet (1997) 350(9088):1402. doi:10.1016/S0140-6736(05)65181-1 4. Schutt-Aine JC. Hypothyroid myxedema and hyponatremia in an eight-year-
old child: a case report. J Natl Med Assoc (1980) 72(7):705–8. 5. Buhler UK, Savary A, Krauer B, Stalder GR. Water intoxication in a cretinoid
infant. J Clin Endocrinol Metab (1966) 26(1):111–6. doi:10.1210/jcem-26-1-111 18. Asami T, Uchiyama M. Sodium handling in congenitally hypothyroid
neonates. Acta Paediatr (2004) 93(1):22–4. doi:10.1111/j.1651-2227.2004. tb00668.x 6. Robles-Valdes C, Ramirez Mayans JA, Alcantara Lomeli JI. Severe hypona-
tremia in congenital hypothyroidism. J Pediatr (1979) 94(4):631–2. doi:10.1016/
S0022-3476(79)80037-2 19. Warner MH, Holding S, Kilpatrick ES. The effect of newly diagnosed hypothy-
roidism on serum sodium concentrations: a retrospective study. Clin Endocrinol
(2006) 64(5):598–9. doi:10.1111/j.1365-2265.2006.02489.x 7. Léger J, Olivieri A, Donaldson M, Torresani T, Krude H, van Vliet G, et al. Euro-
pean Society for Paediatric Endocrinology consensus guidelines on screening,
diagnosis, and management of congenital hypothyroidism. J Clin Endocrinol
Metab (2014) 99(2):363–84. doi:10.1210/jc.2013-1891 20. Oerbeck B, Sundet K, Kase BF, Heyerdahl S. Congenital hypothyroidism: influ-
ence of disease severity and L-thyroxine treatment on intellectual, motor, and
school-associated outcomes in young adults. Pediatrics (2003) 112(4):923–30. doi:10.1542/peds.112.4.923 8. Ojamaa K, Balkman C, Klein IL. Acute effects of triiodothyronine on arterial
smooth muscle cells. ACKNOWLEDGMENTS The pathogenesis of hyponatremia in this patient appears to be
most consistent with the theory of a pre-renal state and concomi-
tant proximal tubular dysfunction. Our patient’s FENa was low NA performed this work as a medical student at New York
University. November 2015 | Volume 3 | Article 96 3 Hyponatremia due to Hypothyroidism Agathis et al. Frontiers in Pediatrics | www.frontiersin.org November 2015 | Volume 3 | Article 96 REFERENCES Ann Thorac Surg (1993) 56(1 Suppl):S61–6. doi:10.1016/
0003-4975(93)90556-W discussion S66-67, 21. Van Vliet G. Neonatal hypothyroidism: treatment and outcome. Thyroid (1999)
9(1):79–84. doi:10.1089/thy.1999.9.79 9. Bautista AA, Duya JE, Sandoval MA. Salt-losing nephropathy in hypothy-
roidism. BMJ Case Rep (2014) 2014:1–4. doi:10.1136/bcr-2014-203895 Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. 10. Holmes EW Jr, DiScala VA. Studies on the exaggerated natriuretic response
to a saline infusion in the hypothyroid rat. J Clin Invest (1970) 49(6):1224–36. doi:10.1172/JCI106336 11. Hanna FW, Scanlon MF. Hyponatraemia, hypothyroidism, and role of arginine-
vasopressin. Lancet (1997) 350(9080):755–6. doi:10.1016/S0140-6736(05)
62563-9 Copyright © 2015 Agathis, Libman and Moritz. This is an open-access article dis-
tributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms. 12. Kimura T. Potential mechanisms of hypothyroidism-induced hyponatremia. Intern Med (2000) 39(12):1002–3. doi:10.2169/internalmedicine.39.1002 13. Schwartz WB, Bennett W, Curelop S, Bartter FC. A syndrome of renal
sodium loss and hyponatremia probably resulting from inappropriate secretion November 2015 | Volume 3 | Article 96 Frontiers in Pediatrics | www.frontiersin.org
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A multimodal neuroimaging study of brain abnormalities and clinical correlates in post treatment Lyme disease
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PloS one
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cc-by
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A multimodal neuroimaging study of brain
abnormalities and clinical correlates in post
treatment Lyme disease Cherie L. MarvelID1,2*, Kylie H. Alm2, Deeya Bhattacharya1, Alison W. Rebman3,
Arnold Bakker2, Owen P. Morgan1¤, Jason A. Creighton1, Erica A. Kozero3,
Arun Venkatesan1, Prianca A. Nadkarni1, John N. Aucott3 Cherie L. MarvelID1,2*, Kylie H. Alm2, Deeya Bhattacharya1, Alison W. Rebman3,
Arnold Bakker2, Owen P. Morgan1¤, Jason A. Creighton1, Erica A. Kozero3,
Arun Venkatesan1, Prianca A. Nadkarni1, John N. Aucott3 1 Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, United States of
America, 2 Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of
Medicine, Baltimore, MD, United States of America, 3 Division of Rheumatology, Department of Medicine,
Lyme Disease Research Center, Johns Hopkins University School of Medicine, Baltimore, MD, United States
of America ¤ Current address: Department of Psychology, Cornell University, Martha Van Rensselaer Hall, Ithaca, NY,
United States of America
* cmarvel1@jhmi.edu Abstract Citation: Marvel CL, Alm KH, Bhattacharya D,
Rebman AW, Bakker A, Morgan OP, et al. (2022) A
multimodal neuroimaging study of brain
abnormalities and clinical correlates in post
treatment Lyme disease. PLoS ONE 17(10):
e0271425. https://doi.org/10.1371/journal. pone.0271425 Lyme disease is the most common vector-borne infectious disease in the United States. Post-treatment Lyme disease (PTLD) is a condition affecting 10–20% of patients in which
symptoms persist despite antibiotic treatment. Cognitive complaints are common among
those with PTLD, suggesting that brain changes are associated with the course of the ill-
ness. However, there has been a paucity of evidence to explain the cognitive difficulties
expressed by patients with PTLD. This study administered a working memory task to a care-
fully screened group of 12 patients with well-characterized PTLD and 18 healthy controls
while undergoing functional MRI (fMRI). A subset of 12 controls and all 12 PTLD participants
also received diffusion tensor imaging (DTI) to measure white matter integrity. Clinical vari-
ables were also assessed and correlated with these multimodal MRI findings. On the work-
ing memory task, the patients with PTLD responded more slowly, but no less accurately,
than did controls. FMRI activations were observed in expected regions by the controls, and
to a lesser extent, by the PTLD participants. The PTLD group also hypoactivated several
regions relevant to the task. Conversely, novel regions were activated by the PTLD group
that were not observed in controls, suggesting a compensatory mechanism. Notably, three
activations were located in white matter of the frontal lobe. DTI measures applied to these
three regions of interest revealed that higher axial diffusivity correlated with fewer cognitive
and neurological symptoms. Whole-brain DTI analyses revealed several frontal lobe regions
in which higher axial diffusivity in the patients with PTLD correlated with longer duration of ill-
ness. Together, these results show that the brain is altered by PTLD, involving changes to
white matter within the frontal lobe. Higher axial diffusivity may reflect white matter repair
and healing over time, rather than pathology, and cognition appears to be dynamically
affected throughout this repair process. PLOS ONE PLOS ONE Introduction Lyme disease is a vector-borne infectious disease initiated by the bite of a tick infected with
various genospecies of the bacteria Borrelia burgdorferi sensu lato [1]. In recent decades, both
the geographic range and the number of cases have increased significantly, and the Centers for
Disease Control and Prevention (CDC) recently estimated an incidence of 476,000 cases annu-
ally in the US [2]. Untreated Lyme disease can manifest clinically as the skin rash erythema
migrans, or cause cardiac, neurologic, or joint signs of infection resulting from the dissemina-
tion of the bacteria [1,3]. Lyme disease is treated with antibiotics, after which such symptoms
typically resolve. However, a subset of patients (10–20%) who are appropriately treated for Lyme disease
develop a chronic illness consisting of persistent or recurrent symptoms [4,5]. A specific,
research-based definition for post-treatment Lyme disease (PTLD) has been operationalized to
identify patients with symptoms linked temporally to strong evidence of prior exposure to B. burgdorferi [5–7]. Although fatigue, widespread musculoskeletal pain, and cognitive difficul-
ties are the most prominent symptoms of PTLD, patients also often report a constellation of
other neurologic, sleep, ocular, mood, and other symptoms [7–9]. Symptom severity and
course can be variable, yet PTLD often significantly impacts cognition and health-related qual-
ity of life [7,10–13]. There is currently no sensitive or specific test to aid diagnosis of PTLD,
nor are there FDA-approved treatment options for patients. Research in individuals suffering
from PTLD has been relatively sparse, in part, due to the complexity of the disease and the dif-
ficulty in confirming a PTLD diagnosis in the absence of additional underlying or co-morbid
diseases that would complicate the interpretation of research results. The underlying mechanisms of brain changes that may impact cognition in people with
PTLD are largely unknown. Few neuroimaging studies that have been reported in people with
PTLD, and there is a lack of consistent findings explaining neurological deficits [14–17]. Sev-
eral brain perfusion and metabolism studies have shown abnormal patterns in patients who
underwent antibiotic treatment [14–17]. It has been noted that brain changes associated with
Lyme disease may involve abnormal white matter perfusion that impacts cognition [18,19]. Moreover, microglial activation of patients with PTLD has been suggested as a contributing
factor of PTLD-related neurological deficits [20]. Introduction However, state-of-the-art magnetic reso-
nance imaging (MRI) methods that measure the structural and functional integrity of gray and
white matter in PTLD have not been reported to date, limiting our understanding of neuro-
logic deficits related to PTLD. The aim of the current study was to use functional magnetic resonance imaging (fMRI) and
diffusion tensor imaging (DTI) methods to examine brain function and structure in people
with PTLD. We sought to test the hypothesis that people with PTLD show altered task-related
activations as revealed by fMRI, and white matter abnormalities as revealed by DTI. Editor: Yangming Ou, Harvard Medical School,
UNITED STATES Editor: Yangming Ou, Harvard Medical School,
UNITED STATES
Received: June 29, 2022
Accepted: September 15, 2022
Published: October 26, 2022
Copyright: © 2022 Marvel 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. Received: June 29, 2022
Accepted: September 15, 2022
Published: October 26, 2022 Copyright: © 2022 Marvel 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. Data Availability Statement: All data and analysis
code are available on the Open Science Framework
(https://osf.io/kshq7). Funding: Funding for this project was generously
provided by an anonymous donor to JNA. The
funders 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. 1 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Participants Twelve adult participants with PTLD ( 18 years) were originally recruited from a referral-
based clinic population. Those providing permission to contact for future studies as part of the
consent process were approached for the current MRI study. The median time between partic-
ipation in both studies was approximately 2.5 weeks (range <1 week– 9.4 weeks). Study participant selection methods replicated many of the criteria set forth in the Infec-
tious Diseases Society of America’s (IDSA) proposed case definition for PTLD [6]. A rigorous
chart review process confirmed study eligibility to validate PTLD in the absence of PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 2 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease confounding factors. Confirmation of PTLD was determined by: 1) physician-documented
erythema migrans rash, or 2) evidence of new-onset objective signs (e.g., joint swelling, facial
palsy) and laboratory evidence of infection following CDC recommendations for test interpre-
tation [21], or 3) evidence of new-onset symptoms not attributable to another cause and labo-
ratory evidence of infection following CDC recommendation for test interpretation. Along
with PTLD confirmation, eligibility required a history of appropriate antibiotic treatment and
post treatment symptoms specified in the IDSA case definition; fatigue, musculoskeletal pain,
and/or cognitive difficulty. Additionally, at least one symptom had been experienced in the
past two weeks that limited daily functioning at least half the time when present. Further eligi-
bility inclusion and exclusion criteria for this study have been published previously [7]. Participants were excluded for a range of specific, co-morbid conditions with significant
symptom overlap with PTLD such as fibromyalgia, chronic fatigue syndrome, major psychiat-
ric disease conditions (except non-suicidal depression that manifested after Lyme infection),
malignancy, and autoimmune disease. Exclusion criteria also consisted of: history of Lyme
vaccine, sleep apnea, cirrhosis, hepatitis B/C, HIV, dementia, cancer (past 2 years), illicit sub-
stance abuse, prescription drug abuse, and alcoholism. A total of 18 adult control participants were recruited through community flyers and
included in the fMRI analysis. Control participants were additionally screened for any co-mor-
bid conditions with significant symptom overlap with PTLD, exclusion criteria as described
above, or a past diagnosis of Lyme disease. Participants In a final screening stage, both participants with PTLD and controls were excluded from
study participation if they endorsed the following characteristics that might confound data
interpretation: major neurologic disorders (including stroke and seizures); head injury result-
ing in loss of consciousness of > 5 minutes, significant learning disability, left-handedness, or
being a non-native English speaker (i.e., acquired English post puberty). Participants were also
excluded for reasons of safety concerns within the MRI environment, such as: current or possi-
ble pregnancy, metal inside or attached to the body, and claustrophobia. Demographic and clinical characteristics are summarized in Table 1. A subset of 12 controls
who were matched demographically to the PTLD group also had DTI data available and were
included in the DTI analysis. The Institutional Review Board of the Johns Hopkins University School of Medicine
approved this study, and written informed consent was obtained from all study participants
prior to initiation of study activities. The planning and conduct of this research were in accor-
dance with the Helsinki Declaration as revised in 2013. PLOS ONE f symptoms reported at the moderate or severe level over the past two weeks on the Post-Lyme Questionnaire of Symptoms
interpreted using CDC criteria for positivity, which incorporates duration of illness at the time of the test. bThe total number of symptoms reported at the moderate or severe level over the past two weeks on the Post-Lyme Questionnaire of Symptoms (range of 0 to 36). cTwo-tier tests were interpreted using CDC criteria for positivity, which incorporates duration of illness at the time of the test. dConfirmed through medical record review. Participants presenting with erythema migrans rash were not required to have a concurrent positive two-tier serology. Those with neurologic disease (n = 2 with Bell’s Palsy, n = 1 with meningitis/encephalitis), late Lyme arthritis, or an initial flulike illness were required to have a
concurrent positive two-tier test. Two tier tests were interpreted using CDC criteria for positivity, which incorporates duration of illness at the time of the test. dConfirmed through medical record review. Participants presenting with erythema migrans rash were not required to have a concurrent positive two-tier serology. Those with neurologic disease (n = 2 with Bell’s Palsy, n = 1 with meningitis/encephalitis), late Lyme arthritis, or an initial flulike illness were required to have a
concurrent positive two-tier test. eBeck Depression Inventory: Cognitive/Affective Subscale score. https://doi.org/10.1371/journal.pone.0271425.t001 binary responses for the following 3 items only; memory impairment, difficulty finding words,
and difficulty focusing or concentrating (range 0–3). binary responses for the following 3 items only; memory impairment, difficulty finding words,
and difficulty focusing or concentrating (range 0–3). Clinical data collection Participants were asked to complete a self-administered 36-item post-Lyme questionnaire of
symptoms (PLQS) which was developed based on prior clinical and research experience
among patients with PTLD. The list of individual symptom items has been previously pub-
lished [22]. For each item, participants indicated severity over the past 2 weeks (0 = absent,
1 = mild, 2 = moderate, 3 = severe), and a binary response was created (absent/mild = 0 vs. moderate/severe = 1). A total symptom score was generated by summing each binary item
(range 0–36). Additionally, in order to focus on specific symptoms of interest to the current
study, a ‘neurologic’ symptom score was generated a priori by summing the binary responses
for the following 12 symptoms; fatigue, numbness in hands/feet, numbness in face/scalp, head-
ache, photophobia, drooping facial muscle, drooping eyelid, neck pain, poor coordination,
memory impairment, difficulty finding words, and difficulty focusing or concentrating (range
0–12). Finally, a more narrow ‘cognitive’ symptom score was generated by summing the PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 3 / 22 Neuroimaging of brain abnormalities in post treatment Lyme disease PLOS ONE PLOS ONE Table 1. Baseline demographic and clinical characteristics. N (%) are presented for categorical variable; mean (standard deviation), 95% confidence interval [lower
limit, upper limit] are presented for continuous variables; Shapiro-Wilk tests were used for tests of normality. Significant values of the Shapiro-Wilk tests are denoted in
bold, p .05, two-tailed. Control groups did not differ from PTLD for age, education, or gender. Group
PTLD
n = 12
Controls
(for fMRI)
n = 18
Matched Controls
(for DTI)
n = 12
Age (years)
45.16 (13.62)
[36.51, 53.82], W = .96, p = .72
47.01 (13.10)
[40.49, 53.52], W = .86, p = .01
45.33 (13.76)
[36.60, 54.08], W = .91, p = .20
Male Gender
7 (58.33%)
6 (33.33%)
4 (33.33%)
Education (years)
16.17 (2.21)
[14.76, 17.57], W = .97, p = .92
16.44 (2.01)
[15.45, 17.44], W = .90, p = .06
16.00 (1.91)
[14.79, 17.21], W = .86, p = .05
Duration of illnessa (days)
944.83 (1043.13)
[282.06, 1607.61], W = .75, p = .003
Antibiotic exposurea (days)
190.17 (309.37)
[-6.40, 386.73], W = .55, p < .001
Number of symptoms at MRI scanb
8.17 (4.39)
[5.38, 10.95], W = .94, p = .48
Neurologic symptoms
4.50 (2.02
[3.21, 5.79], W = .90, p = .16
Cognitive symptoms
2.00 (1.35)
[1.14, 2.86], W = .71, p = .001
Two-tier antibody positive at MRI scanc
4 (33.33%)
Initial Lyme disease clinical presentationd
Erythema migrans rash
4 (33.33%)
Neurologic Lyme disease
3 (25.00%)
Late Lyme arthritis
1 (8.33%)
Flulike Illness
4 (33.33%)
Beck Depression Inventorye
18.42 (12.05)
[10.76, 26.07], W = .79, p = .008 Table 1. Baseline demographic and clinical characteristics. N (%) are presented for categorical variable; mean (standard deviation), 95% confidence interval [lower
limit, upper limit] are presented for continuous variables; Shapiro-Wilk tests were used for tests of normality. Significant values of the Shapiro-Wilk tests are denoted in
bold, p .05, two-tailed. Control groups did not differ from PTLD for age, education, or gender. aTotal days from Lyme disease onset (or start of antibiotics) until MRI scan. aTotal days from Lyme disease onset (or start of antibiotics) until MRI scan. aTotal days from Lyme disease onset (or start of antibiotics) until MRI scan. MRI procedures The num-
ber of target letters (1 or 2), rehearsal duration (4 or 6 seconds), expected response (yes or no),
and duration of ITI (6–9 seconds) were pseudorandomized so that presentation of identical
parameters was limited to three consecutive trials. Participants were instructed to respond as quickly and accurately as possible while complet-
ing all operations silently ‘‘in your head.” Button press responses were recorded if conducted
within six seconds following probe onset (yes = right index finger; no = right middle finger). Trials were jittered with an inter-trial interval (ITI) of six to nine seconds. Response time (RT)
and accuracy were recorded for each trial. In order to familiarize subjects with the rules of the
task, subjects practiced 10 trials of each condition prior to entering the MRI environment. The control and forward conditions were completed during two separate blocks, with the
order counter-balanced across participants. Each block contained 64 trials (~ 16 minutes). Each trial consisted of pseudorandom presentations of targets such that letters were unique
within a trial. Probes matched a target (or newly derived target) on 50% of the trials. The num-
ber of target letters (1 or 2), rehearsal duration (4 or 6 seconds), expected response (yes or no),
and duration of ITI (6–9 seconds) were pseudorandomized so that presentation of identical
parameters was limited to three consecutive trials. An additional event-related finger tapping task was administered in order to compute indi-
vidualized hemodynamic response functions (HRFs) [23,25]. This task consisted of a button
press with the right index finger every 29–31 seconds upon presentation of a 1-second cue to
“tap” followed by “rest”, which lasted for 10 minutes total. The individualized HRFs were used
in the convolution step of MRI processing, rather than a canonical HRF, in case the HRFs in
the PTLD group differed from that of control participants. Stimuli were delivered using E-Prime 2.0 software (Psychology Software Tools, Pittsburgh,
PA) on a Dell Optiplex SX9202 workstation running Windows 7. The stimuli were rear-pro-
jected onto a screen in the MRI scanner, which was then reflected into a head coil-mounted
mirror within the participant’s line of sight. Responses were collected using two fiber optic
button boxes (MRA, Inc., Washington, PA) that were held in the participant’s right hand. MRI data acquisition. MRI data were acquired on a Philips 3 Tesla scanner using a
32-channel head coil. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 MRI procedures Behavioral task. Participants were asked to perform a working memory task in the MRI
scanner consisting of two conditions, previously described in detail [23,24]. Briefly, in the con-
trol condition, participants viewed one or two uppercase consonants (one second), followed
by a blank screen (four or six seconds). Participants held these letters in mind through silent
rehearsal. Finally, a single lowercase letter was presented (one second). Participants indicated
via button press whether the single probe item matched either of the targets presented at the
start of the trial. In the “forward” condition, rather than rehearse the original target letters pre-
sented at the start of the trial, participants were required to count two alphabetical letters PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 4 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease forward of each target letter(s) and hold the new letters in mind. For example, if the target let-
ters were ‘‘f” and ‘‘q”, participants would count forward to the letters ‘‘h” and ‘‘s”. When the
probe letter appeared, participants indicated whether the probe matched the newly derived let-
ters instead of the original target letters. Therefore, the two conditions differed specifically dur-
ing the rehearsal phase of the trial, in which target letters were simply rehearsed as presented
(control condition) or rehearsed by counting two alphabetical letters forward (forward
condition). Participants were instructed to respond as quickly and accurately as possible while complet-
ing all operations silently ‘‘in your head.” Button press responses were recorded if conducted
within six seconds following probe onset (yes = right index finger; no = right middle finger). Trials were jittered with an inter-trial interval (ITI) of six to nine seconds. Response time (RT)
and accuracy were recorded for each trial. In order to familiarize subjects with the rules of the
task, subjects practiced 10 trials of each condition prior to entering the MRI environment. The control and forward conditions were completed during two separate blocks, with the
order counter-balanced across participants. Each block contained 64 trials (~ 16 minutes). Each trial consisted of pseudorandom presentations of targets such that letters were unique
within a trial. Probes matched a target (or newly derived target) on 50% of the trials. MRI procedures Structural images were collected using a sagittal magnetization prepared
gradient-echo (MPRAGE) sequence aligned to the anterior-posterior commissure (AC-PC)
axis: repetition time (TR)/echo time (TE) = 6.9/3.3 ms; field of view = 240 x 240; 170 slices;
slice thickness 1.0 mm; 0 mm gap; flip angle = 8 degrees; voxel size = 0.75 x 0.75 x 1.0 mm. The
total scan duration was 6 minutes. FMRI data were collected using a T2-weighted gradient
echo EPI pulse sequence (TR = 1000 msec; TE = 30 ms; flip = 61; in-plane resolution = 3.75
mm; slice thickness = 6 mm with a 1 mm gap; 20 oblique-axial slices; FOV = 240 mm). T2-weighted images were acquired in the oblique-axial plane rotated 25 degrees clockwise
with respect to the AC-PC line in order to optimize imaging of the cerebellum and neocortex. The number of acquired volumes within each block ranged from 917 to 922 for the working
memory tasks and 600 for the tapping task. The start of the fMRI scan was triggered by E-
prime software at the beginning of each block. Functional MRI data analysis. The SPM12 software package (Wellcome Department of
Cognitive Neurology) was used for preprocessing and statistical computations. High temporal
resolution fMRI in conjunction with neocortical-specific HRFs were used to ensure maximum
accuracy in characterizing phase-specific blood oxygen level dependent (BOLD) responses
[23,25]. Individual HRF regressors were convolved with reference waveforms for the target 5 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease encoding (1 sec), rehearsal (4 or 6 sec) and probe retrieval (6–9 sec) phases of the task for each
subject within the first-level event-related analysis. In this report, we only focus on the
rehearsal phase of analyses. Standard image preprocessing steps were performed, including
slice timing correction (reference = middle slice), motion correction, anatomical co-registra-
tion, normalization to the Montreal Neurological Institute (MNI) stereotaxic space, and spatial
smoothing (FWHM = 8 mm). Due to a technical error, one PTLD participant’s functional
MRI data was corrupted and could not be processed. It was excluded from the functional
imaging stages of analysis. Individual statistical maps were computed for each subject using
the general linear model approach as implemented in SPM12, with high pass filtering of 128 s. MRI procedures A random effects analysis was then performed to map the average responses to the rehearsal
phase of the task on correct trials only. Incorrect trials were not given a regressor and were
considered as residual variance. This analysis was performed by computing a contrast volume
per subject and using these volumes to calculate one-sample t-test values at every voxel. Of par-
ticular interest were within-group contrasts comparing the BOLD signal difference between
the 2-target forward minus 2-target control working memory conditions that were then com-
pared between groups. MNI coordinates were transformed into the coordinate system of the
Talairach and Tourneaux stereotaxic atlas [26] using the MNI to Talairach transformation
described by Lancaster et al. [27] in order to make anatomical determinations of the activa-
tions. However, MNI coordinates are reported in the tables and figures. Significance levels
were set to p < .005, uncorrected, with a minimum cluster size of 10 voxels. Functionally defined regions of interest (ROIs) were circumscribed on each participant’s
scan based on the activation clusters observed in the between-groups contrast using the Mars-
BaR toolbox for SPM [28]. The resultant contrast values per participant were then entered into
subsequent analyses to test for correlations with behavioral task performance and diffusion
weighted imaging (DTI) measures. DWI data acquisition and preprocessing. Abnormal white matter fMRI findings led us
to investigate the relationship to white matter structural integrity by adding diffusion tensor
imaging (DTI) methods to the protocol already in progress, which was administered to a sub-
set of participants (n = 12 per group). Diffusion-weighted images (DWI) were acquired using a spin echo sequence with
TR = 7012 ms, TE = 75 ms, FOV = 212 x 212 mm2, 0.83 x 0.83 x 2.2 mm voxels, flip
angle = 90˚, b-value = 700, number of gradients = 33, and 70 axial slices. Two sequences were
acquired. The DWI data were preprocessed using FSL [29] to correct for eddy current-induced
distortions and subject motion using affine registration. The b-vector matrix was adjusted
based on rigid body registration and skull stripping was performed using FSL’s automated
brain extraction tool (BET) to remove non-brain tissue. A standard least squares diffusion ten-
sor fitting model was applied to the data to derive whole brain maps for the following diffusion
tensor imaging (DTI) metrics: fractional anisotropy (FA), mean diffusivity (MD), radial diffu-
sivity (RD), and axial diffusivity (AD). MRI procedures These estimates were computed on a voxel-by-voxel
basis using a three-dimensional Gaussian distribution model that yielded a single mean ellip-
soid for each voxel. For each participant, the two runs of DWI data were preprocessed sepa-
rately, and the scalar maps resulting from each run were averaged to improve signal-to-noise
ratio. Tissue class segmentation analysis. To determine the proportion of white matter within
each significant cluster of activation derived from the fMRI analysis, a tissue class segmenta-
tion analysis was used. First, a study-specific T1 modal model template was created from all
participants in the study using Advanced Normalization Tools (ANTs), and a 3D vector field
transformation for each subject was calculated to align the individual’s structural scan to the
template modal model based on the entire sample [30,31]. Tissue class segmentation analysis PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 6 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease was then completed on the template modal model using FSL’s FAST automated segmentation
tool [32], a well-validated automated tissue segmentation tool [33]. FAST utilizes a hidden
Markov random field model and an associated expectation-maximization algorithm to seg-
ment brain images into three tissue classes: gray matter, white matter, and cerebrospinal fluid. To compute the overlap between the resulting binary white matter segmentation mask and
each significant functional activation cluster, each cluster of activation was first transformed
into a binary ROI. The binary ROIs were subsequently resampled into a 1 x 1 x 1 mm space
using nearest neighbor interpolation, given that both the tissue class segmentation analysis
and the DTI analyses were completed in this space. Following registration, each functional
ROI mask was multiplied by the binary white matter segmentation mask to identify voxels in
each ROI that fell within the white matter segmentation mask. The number of voxels in the
subsequent overlap image was then calculated as a percentage relative to the ROI size, yielding
the percentage of voxels within a particular ROI that were classified as white matter. This
method was repeated for each of the significant activation clusters from the fMRI analysis. ROIs classified as greater than 50% white matter were selected for further analysis. g
y
DTI data analysis. FSL’s Tract-based Spatial Statistics (TBSS) pipeline [34] was used to
derive a mean white matter skeleton from participants’ DTI data that represents the center of
all white matter tracts common to the sample. MRI procedures First, each participant’s FA map was registered
to every other participant’s FA map using the nonlinear registration tool, FNIRT [35,36]. The
“most representative” image, i.e. the image that required the least warping to align every other
image to it, was chosen as the target for registration and affine-transformed into MNI152 stan-
dard 1 x 1 x 1 mm space. Then, all other FA maps were transformed into this standard space
by combining the individual nonlinear transforms to the target FA map with the affine trans-
form from the target to MNI space. Next, the co-registered FA maps were merged into one 4D
image, where each volume is a specific participant’s standard space FA map. From this 4D
image, the mean FA image was computed and thinned using an FA threshold of 0.2, which
retained only the center of all fiber pathways common to the group, generating a mean white
matter skeleton. This skeletonization process ensures that subsequent analyses are restricted to
tracts that are well-aligned across participants, thereby reducing potential misregistrations as a
source of false positives. It also ensures subsequent analyses are less susceptible to partial vol-
ume effects. Finally, each participant’s aligned FA map was projected onto the mean skeleton
to generate skeletonized FA data for each participant. The previously computed warps and
skeleton projections were also applied to MD, RD, and AD maps in order to align them into
MNI152 1 x 1 x 1 mm standard space and create participant level skeletonized MD, RD, and
AD data. The entire TBSS process was repeated for a patient-only whole brain analysis to examine
whether voxel-wise DTI metrics correlated with duration of illness (DOI) in the patients with
PTLD. We held an agnostic interest in DOI due to the practical question of whether longer
exposure to PTLD led to relevant brain changes. The TBSS processing steps were the same as
detailed above, with only the PTLD patient group included in this iteration. The resulting skel-
etonized DTI data were used to generate voxel-wise cross-subject statistics using “randomise”
[37], FSL’s tool for nonparametric permutation inference testing (using 2000 permutations). GLM contrasts were constructed to test for both positive and negative correlations between
the voxel-wise skeletonized DTI data and the DOI variable. Family-wise error (FWE) correc-
tion was performed using threshold-free cluster enhancement [38], which avoids the use of an
arbitrary threshold for the initial cluster formation. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 MRI procedures A p-value < .05, FWE-corrected for multi-
ple comparisons was considered statistically significant. For visualization of results, the
“tbss_fill” script was used to enhance ease of viewing. 7 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Mean white matter microstructure was computed for the functional activation ROIs that
consisted of greater than 50% white matter, as determined by the tissue class segmentation
analysis. Using each binarized functional activation ROI as a mask, mean FA, MD, RD, and
AD values were extracted from each individual participant’s skeletonized DTI data. To compute the percentage of overlap between the functional activation ROIs and the
white matter skeleton, each binarized functional activation ROI mask was multiplied by the
mean white matter skeleton generated from the TBSS pipeline to isolate the voxels from the
functional ROIs that fell within the white matter skeleton. The number of overlapping voxels
for each ROI was then computed, and results were expressed as a percentage relative to the
ROI size. This analysis was only computed for ROIs consisting of greater than 50% white mat-
ter, as determined by the tissue class segmentation analysis. To investigate the white matter surrounding the frontal lobe activation ROIs, we sought to
quantify overlap between the observed functional ROIs and long-range white matter pathways. For this analysis, we examined the 42 standard white matter tracts generated from the
XTRACT atlas [39]. Each tract mask was binarized and then multiplied by each binarized
functional activation ROI to isolate ROI voxels that fell within the associated long-range white
matter tract. The percent overlap for each ROI with each long-range tract was then computed
and expressed as a percentage relative to ROI size. Statistical analyses of clinical and behavioral variables. The clinical, behavioral, MRI,
and DTI data collected in this study contained continuous variables, with the exception of gen-
der (categorical data). T-tests were used to compare continuous variables (e.g., age and educa-
tion), and Pearson chi-square tests were used to compare categorical data (e.g., gender). Shapiro-Wilk tests were used to determine if continuous variables followed a normal distribu-
tion. If a variable was not normally distributed, Mann-Whitney U tests were conducted to
compare groups. Mixed-design ANOVAs were used to compare repeated measures between
the two groups (e.g., fMRI task performance). Results The total healthy control group did not differ from the PTLD group in terms of age,
(Mdn = 51.1), U = 101, p = .76, r = 18.4, or education level, t(22) = .35, p = .730, d = .133. The
matched control group also did not differ from the PTLD group in terms of age, t(22) = .03, p
= .975, d = .013, or education level, (Mdn = 16.0), U = 74.0, p = .91., r = 15.1. Pearson’s Chi
Square tests were used to determine that gender counts also did not differ between the healthy
control and PTLD groups, χ2(1, 30) = 1.83, p = .176, or between the matched control and
PTLD groups, χ2(1, 24) = 1.51, p = .219. MRI procedures [No ANOVAs contained a within-subjects fac-
tor with more than two levels; sphericity corrections were, therefore, not needed.] Pearson cor-
relations were used when the Shapiro-Wilk’s normality tests indicated a normal data
distribution (e.g., correlating fMRI data with the PLQS). Otherwise, Spearman’s rho non-
parametric correlations were used. All tests were two-tailed, with an alpha level .05 to define
statistical significance. Statistics were performed using IBM SPSS Statistics, Macintosh, version
27.0 (IBM Corp., Armonk, NY, USA). Behavioral results Mean accuracy and RT (for accurate trials only) were computed for the following trial types:
control condition, 1 stimulus; control condition, 2 stimuli; forward condition, 1 stimulus; and
forward condition, 2 stimuli for each group. Due to a technical error, we were unable to collect
behavioral data from one PTLD participant, which also removed them from the fMRI analyses,
but their data were included in the DTI analyses. (S3 File, Table 1) A 2(condition: control vs. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 8 / 22 Neuroimaging of brain abnormalities in post treatment Lyme disease PLOS ONE Fig 1. Behavioral performance on the fMRI working memory task. (A) Accuracy performance is presented, broken down by condition (control vs. forward),
stimulus number (1 vs. 2 letters), and study group. Accuracy was particularly low for participants in the forward condition with two letters. However, accuracy
performance did not differ overall between groups. (B) Response times for accurate trials are presented, as in (A). Response times were slowest in the forward
condition with two letters. Overall, the PTLD group responded more slowly than did controls, as indicated by the overhead bracket. indicates condition x
stimulus interaction, p < .001; bracket indicates overall group difference, p = .037. Error bars denote one standard error. https://doi org/10 1371/journal pone 0271425 g001 Fig 1. Behavioral performance on the fMRI working memory task. (A) Accuracy performance is presented, broken down by condition (control vs. forward),
stimulus number (1 vs. 2 letters), and study group. Accuracy was particularly low for participants in the forward condition with two letters. However, accuracy
performance did not differ overall between groups. (B) Response times for accurate trials are presented, as in (A). Response times were slowest in the forward
condition with two letters. Overall, the PTLD group responded more slowly than did controls, as indicated by the overhead bracket. indicates condition x
stimulus interaction, p < .001; bracket indicates overall group difference, p = .037. Error bars denote one standard error. https://doi.org/10.1371/journal.pone.0271425.g001 https://doi.org/10.1371/journal.pone.0271425.g001 forward) x 2(stimulus number: 1 vs. 2) x 2(group: controls vs. PTLD) mixed-design ANOVA
yielded a main effect of condition F(1, 27) = 6.51, p = .017, ηp
2 = .019, and stimulus number F
(1, 27) = 20.6, p < .001, ηp
2 = .43, indicating that participants’ accuracy decreased as a function
of higher working memory load requirements. Behavioral results This was confirmed by an interaction of stimu-
lus number x condition, F(1, 27) = 13.9, p = .001, ηp
2 = .34, indicating that trials were least
accurate in the forward, 2 stimuli trial type. There were no main effect or interactions involv-
ing group, all p-values > .416 (Fig 1A). A 2(condition) x 2(stimulus number) x 2(group)
mixed-design ANOVA was also conducted for the RT measure. As with the accuracy measure,
there were main effects of condition, F(1, 27) = 51.3, p < .001, ηp
2 = .66, and stimulus number,
F(1, 27) = 60.9, p < .001, ηp
2 = .69. There was also an interaction of condition x stimulus num-
ber, F(1, 27) = 35.3, p < .001, ηp
2 = .57. There were no interactions involving group. However,
there was a main effect of group, showing that the PTLD group responded more slowly overall
than did controls, F(1, 27) = 4.80, p = .037, ηp
2 = .15 (Fig 1B). Thus, participants found the for-
ward, 2 stimuli trial type to be disproportionately more difficult than other trial types, as evi-
denced by decreased accuracy and slowed RTs. Moreover, the PTLD group showed general
motor slowing. Functional MRI results Functional imaging analyses focused on the rehearsal phase of each trial. Within this phase, we
focused on activations during the most difficult condition (2 stimuli, forward condition). To
do so, we computed the contrast values of the “2-target forward” minus “2-target control” con-
ditions for each participant. To validate the results of our task, we first examined the healthy
control fMRI data and compared it to a prior study that characterized this task in young,
healthy adults [23] (S1 Table). Results generally overlapped with those original findings (the
current study included healthy participants who were about 20 years older), with increased
BOLD signal in association with verbal working memory rehearsal in the frontal lobe (BA 9
and 32), premotor cortex, caudate, thalamus, inferior parietal lobe, and superior cerebellum. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 9 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease The large degree of overlap with the original study supported the fMRI task’s validity. The
PTLD group also revealed regions of overlap with those original findings (S2 Table), with
increased BOLD signal in the frontal lobe (BA 9, supplementary motor area, and left inferior
frontal gyrus BA 45), premotor cortex, caudate, and precuneus. A number of activated regions,
however, were observed in the PTLD group but not observed in the controls’ data or in the
original study. This suggested that the PTLD group was unable to fully utilize a typical verbal
working memory circuit and compensated to maintain high accuracy. Notably, some frontal
lobe activations appeared to be primarily in white matter, surpassing a threshold of p < .001
uncorrected, which warranted further examination when it appeared again in the between-
groups comparison, as described below. We applied a double subtraction approach to compare BOLD signal activations between
the groups. We used the contrast values obtained from the within-groups comparison (first
subtraction) to compare differences between groups (second subtraction). Fig 2 shows positive
BOLD signals that represented greater “forward minus control” activation differentials in the
PTLD group than in the control group (exceptions are noted in Table 2 where activations were
higher in the control condition for the control group, yielding a “false” hyperactivation in the
PTLD group). PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease PLOS ONE Table 2. Task-related BOLD activations between PTLD and control participants. Regions are based on Talairach coordinates27 and listed anterior-posterior (y-plane). Cluster size
T-value
x, y, z (MNI)
Brain region
GM/WM
p-value
Controls > PTLD
17
3.14
58, 14, 26
Right inferior frontal gyrus (BA 9)
GM
< .005
10
3.01
40, 12, 30
Right inferior frontal gyrus (BA 9)
GM
< .005
124
3.69
48, 0, 44
Right precentral gyrus (BA 6)
WM (64%)
< .001
95
3.68
8, -24, 8
Right thalamus
GM
< .001
21
3.08
44, -36, 44
Right inferior parietal lobule (BA 40)
GM
< .005
PTLD > Controls
120
3.37
0, 50, 24
Left medial frontal gyrus (BA 9)
GM
< .005
33
3.22
-18, 48, 26
Left superior frontal gyrus (BA 9)
WM (89%)
< .005
40
3.82
-22, 38, 36
Left middle frontal gyrus (BA 8)
WM (41%)
< .001
56
3.97
-18, 24, 40
Left middle frontal gyrus (BA 8)
WM (80%)
< .001
46
3.72
-34, 18, 38
Left precentral gyrus (BA 9)
WM (99%)
< .001
26
3.31
-56, -4, 6
Left precentral gyrus (BA 6)
GM
< .005
45
3.61
62, -10, 8
Right superior temporal gyrus (BA 42)
GM
< .001
18
3.15
2, -14, -4
Right thalamus
GM
< .005
23
3.33
-62, -16, 6
Left superior temporal gyrus (BA 41)
GM
< .005 A
Brodmann Area; GM
gray matter; WM
white matter. = positive BOLD signal indicates greatest activity in the control participants during the control condition, which reversed the BOLD signal interpretation in this
double subtraction method. The percentage of WM computed within a cluster is noted in parentheses in the GM/WM column (reporting those with WM > 50%). Cluster information is reported at p < .005, uncorrected. that group differences were due to the PTLD group showing hypo-activation (or not activating
at all) in brain regions normally associated with the task, even though their accuracy was
normal. Increased task-related activity in PTLD participants versus controls was observed in the
frontal lobe (BA 8 and 9). While frontal lobe involvement would be expected in this working
memory task, the clusters of task-related activation were located primarily within white matter,
as opposed to the regions of relative hypoactivation noted above which were predominantly in
gray matter. PLOS ONE One white matter region was activated more robustly in controls than in the
PTLD group (in BA 6). Closer inspection of this region at the more conservative p < .001
threshold, however, indicated that it was actually comprised of two smaller gray matter activa-
tions within close proximity that, when smoothed, bridged white matter. Additional analyses
were conducted to compute the percentage of white matter included in each ROI for these
four frontal lobe activations, as described below. Based on these findings, masks were created
for each ROI that allowed us to compute MRI signal contrast values within these circum-
scribed regions for correlations with DTI and clinical variables (described below). A = Brodmann Area; GM = gray matter; WM = white matter. https://doi.org/10.1371/journal.pone.0271425.t002 g
y
= positive BOLD signal indicates greatest activity in the control participants during the control condition, which reversed the BOLD signal interpretation in this
double subtraction method. The percentage of WM computed within a cluster is noted in parentheses in the GM/WM column (reporting those with WM > 50%).
Cluster information is reported at p < .005, uncorrected. Functional MRI results Increased task-related activity in the controls versus PTLD participants was observed in
regions that were consistent with the original study and other similar paradigms, such as the
premotor cortex, thalamus, and inferior parietal lobe [23–25,40]. These results also indicated Fig 2. FMRI activation differences between study groups during the working memory task. Activations represent a double
subtraction between groups (i.e., the difference between-groups of the difference within-groups [(forward, 2 stimulus) minus (control, 2
stimulus)]). Red indicates activity in PTLD > controls, except for where indicated in Table 2. Blue indicates activity in controls > PTLD. Areas of greater activation in control participants (blue) compared to PTLD participants were consistent with localized activity
previously documented as relevant to task performance and reflected hypoactivity in these regions by the PTLD group [23]. Unexpectedly, three frontal lobe activations demonstrated by the PTLD group (red) were located primarily within white matter. Numbers denote y-axis on the MNI template. Color scale represents .005 < p < .0005. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g002 Fig 2. FMRI activation differences between study groups during the working memory task. Activations represent a double
subtraction between groups (i.e., the difference between-groups of the difference within-groups [(forward, 2 stimulus) minus (control, 2
stimulus)]). Red indicates activity in PTLD > controls, except for where indicated in Table 2. Blue indicates activity in controls > PTLD. Areas of greater activation in control participants (blue) compared to PTLD participants were consistent with localized activity
previously documented as relevant to task performance and reflected hypoactivity in these regions by the PTLD group [23]. Unexpectedly, three frontal lobe activations demonstrated by the PTLD group (red) were located primarily within white matter. Numbers denote y-axis on the MNI template. Color scale represents .005 < p < .0005. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g002 10 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 Tissue class segmentation results Neuroimaging of brain abnormalities in post treatment Lyme diseas PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Fig 3. Localization of frontal task-related fMRI activations in white matter. Significant frontal clusters of elevated
activation in PTLD participants compared to controls from the contrast values of the forward minus control
conditions were transformed into binary regions of interest (ROIs) labeled by their Brodmann area location. Blue:
Brodmann area 8 (BA 8) ROI, red: Anterior Brodmann area 9 (BA 9 anterior) ROI, pink: Posterior Brodmann area 9
(BA 9 posterior) ROI. Labeled ROIs were used to compute the percent overlap with each of the following measures. (A) White matter mask (white) derived from a study-specific T1 template using tissue class segmentation analysis. The
BA 8 ROI showed 80.06% overlap, the BA 9 anterior ROI showed 89.39% overlap, and the BA 9 posterior ROI showed
99.73% overlap with white matter. (B) Mean fractional anisotropy (FA) and white matter skeleton (green) maps
derived using diffusion tensor imaging (DTI) analysis showing overlap with fMRI ROIs. The BA 8 ROI showed 25.00% Fig 3. Localization of frontal task-related fMRI activations in white matter. Significant frontal clusters of elevated
activation in PTLD participants compared to controls from the contrast values of the forward minus control
conditions were transformed into binary regions of interest (ROIs) labeled by their Brodmann area location. Blue:
Brodmann area 8 (BA 8) ROI, red: Anterior Brodmann area 9 (BA 9 anterior) ROI, pink: Posterior Brodmann area 9
(BA 9 posterior) ROI. Labeled ROIs were used to compute the percent overlap with each of the following measures. (A) White matter mask (white) derived from a study-specific T1 template using tissue class segmentation analysis. The
BA 8 ROI showed 80.06% overlap, the BA 9 anterior ROI showed 89.39% overlap, and the BA 9 posterior ROI showed
99.73% overlap with white matter. (B) Mean fractional anisotropy (FA) and white matter skeleton (green) maps
derived using diffusion tensor imaging (DTI) analysis showing overlap with fMRI ROIs. The BA 8 ROI showed 25.00%
overlap, the BA 9 anterior ROI showed 22.35% overlap, and the BA 9 posterior ROI showed 24.46% overlap with
skeletonized white matter. For reference, the white matter skeleton accounts for 34.47% of the overall white matter
mask from the tissue class segmentation. (C) Mean FA map overlaid with three long range white matter pathways
obtained from DTI. Tissue class segmentation results Calculation of the overlap between the white matter mask derived from the tissue segmenta-
tion analysis and the ROIs derived from the fMRI working memory contrast (forward minus
control conditions) resulted in the identification of four significant functional activation clus-
ters that were primarily localized to white matter (i.e. over 50% of the voxels within the func-
tional ROI mask overlapped with the white matter tissue segmentation mask). In three frontal
activation areas, the patients with PTLD showed elevated activation compared to controls (Fig
3A) localized to the left BA 8 ROI (80.06% white matter), the left BA 9 anterior ROI (89.39% PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 11 / 22 Fig 3. Localization of frontal task-related fMRI activations in white matter. Significant frontal clusters of elevated
activation in PTLD participants compared to controls from the contrast values of the forward minus control
conditions were transformed into binary regions of interest (ROIs) labeled by their Brodmann area location. Blue:
Brodmann area 8 (BA 8) ROI, red: Anterior Brodmann area 9 (BA 9 anterior) ROI, pink: Posterior Brodmann area 9
(BA 9 posterior) ROI. Labeled ROIs were used to compute the percent overlap with each of the following measures. (A) White matter mask (white) derived from a study-specific T1 template using tissue class segmentation analysis. Th
BA 8 ROI showed 80.06% overlap, the BA 9 anterior ROI showed 89.39% overlap, and the BA 9 posterior ROI showed
99.73% overlap with white matter. (B) Mean fractional anisotropy (FA) and white matter skeleton (green) maps
derived using diffusion tensor imaging (DTI) analysis showing overlap with fMRI ROIs. The BA 8 ROI showed 25.00%
overlap, the BA 9 anterior ROI showed 22.35% overlap, and the BA 9 posterior ROI showed 24.46% overlap with
skeletonized white matter. For reference, the white matter skeleton accounts for 34.47% of the overall white matter
mask from the tissue class segmentation. (C) Mean FA map overlaid with three long range white matter pathways
obtained from DTI. The BA 8 ROI showed 41.57% overlap with the frontal aslant tract (FAT; orange), the BA 9
anterior ROI showed 49.62% overlap with anterior thalamic radiation (ATR; yellow), and the BA 9 posterior ROI
showed 57.61% overlap with the superior longitudinal fasciculus 2 (SLF 2; cyan). L = left, R = right hemispheres. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 Tissue class segmentation results The BA 8 ROI showed 41.57% overlap with the frontal aslant tract (FAT; orange), the BA 9
anterior ROI showed 49.62% overlap with anterior thalamic radiation (ATR; yellow), and the BA 9 posterior ROI
showed 57.61% overlap with the superior longitudinal fasciculus 2 (SLF 2; cyan). L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g003 https://doi.org/10.1371/journal.pone.0271425.g003 12 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease white matter), and the left BA 9 posterior ROI (99.73% white matter). By contrast, in one sig-
nificant cluster, controls showed increased activation relative to patients localized to the right
BA 6 ROI (63.56% white matter). The remaining 10 activation clusters obtained from func-
tional imaging analysis were categorized as non-majority white matter, with eight of the ROIs
ranging from 0% - 11.81% white matter and two with 40.63% and 41.30% white matter
respectively. DTI results A whole-brain between-groups analysis did not show significant group differences (FWE-cor-
rected p > .05). However, we were specifically interested in examining the integrity of white
matter within the regions identified in the fMRI analysis. Overlap with white matter skeleton. Overlap with the white matter skeleton derived
from the TBSS analysis was computed for ROIs obtained from the fMRI analysis that exhibited
greater than 50% white matter as determined by the tissue segmentation analysis (see above). The area of task-related activation in the left BA 8 showed 25.00% overlap with the white mat-
ter skeleton, while left BA 9 anterior ROI showed 22.35% overlap, and the left BA 9 posterior
ROI showed 24.46% overlap (Fig 3B). Overlap with the white matter skeleton was substantially
lower for the right BA 6 ROI (13.46%). Overlap with long range white matter tracts. For the three white matter frontal activa-
tion ROIs that exhibited elevated task activation in patients, a follow-up analysis was com-
pleted to identify long-range fiber pathways that showed overlap with the task-related
activation ROIs. The left BA 8 ROI exhibited 41.57% overlap with the frontal aslant tract
(FAT, Fig 3C) and 17.70% overlap with the first branch of the superior longitudinal fasciculus
(SLF 1). The left BA 9 anterior ROI exhibited 49.62% overlap with the anterior thalamic radia-
tion (ATR, Fig 3C), 12.12% overlap with the dorsal cingulum, and 13.26% overlap with the for-
ceps minor. Finally, the left BA 9 posterior ROI showed 57.61% overlap with the second
branch of the superior longitudinal fasciculus (SLF 2, Fig 3C). Relationship between white matter microstructure and duration of illness. Within the
PTLD group, an exploratory whole brain analysis was conducted to identify areas that demon-
strated a significant relationship between DTI microstructural measures (FA, MD, AD, and
RD) and DOI. Significant positive correlations with DOI were observed in right frontal regions
(FWE-corrected p < .05) for both MD and AD (Fig 4). Across both DTI measures, the correla-
tions were found in regions consistent with the right ATR and SLF 3. In MD and AD, voxels
with a significant positive correlation with DOI primarily overlapped with the right ATR
(60.55% and 17.73%, respectively) and the right SLF 3 (23.39% and 54.29%, respectively). No
significant correlations emerged for FA or RD, and no significant negative correlations were
found. Relationship among fMRI, DTI, and clinical variables In the PTLD group, we explored the relation among the three clusters of activation localized to
the white matter and their respective DTI axial diffusivity measures, using fMRI-derived ROI
masks applied to the fMRI and DTI skeletonized maps (Fig 5). The fMRI and DTI BA 9 ROIs
marginally and negatively correlated, r(11) = -.55, p = .077 (Table 3 in S1 File). Thus, greater
axial diffusivity was tentatively associated with less white matter activation in this region. DTI skeletonized axial diffusivity within the three ROIs were correlated with the sum of
symptoms on the PLQS (total, cognitive, and neurologic) to assess the clinical relevance of
these regions. Axial diffusivity in the BA 9 anterior ROI negatively correlated with all three
clinical measures (total: r(12) = -.76, p = .004; neurological: r(12) = -.78, p = .003; cognitive: r In the PTLD group, we explored the relation among the three clusters of activation localized to
the white matter and their respective DTI axial diffusivity measures, using fMRI-derived ROI
masks applied to the fMRI and DTI skeletonized maps (Fig 5). The fMRI and DTI BA 9 ROIs
marginally and negatively correlated, r(11) = -.55, p = .077 (Table 3 in S1 File). Thus, greater
axial diffusivity was tentatively associated with less white matter activation in this region. DTI skeletonized axial diffusivity within the three ROIs were correlated with the sum of
symptoms on the PLQS (total, cognitive, and neurologic) to assess the clinical relevance of
these regions. Axial diffusivity in the BA 9 anterior ROI negatively correlated with all three
clinical measures (total: r(12) = -.76, p = .004; neurological: r(12) = -.78, p = .003; cognitive: r PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 13 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Fig 4. Relationship between diffusion tensor imaging (DTI) metrics and duration of illness (DOI) in PTLD. (A)
Mean diffusivity (MD) results. Top: Area of significant correlations between MD and DOI overlaid on the mean
fractional anisotropy (FA) map and white matter skeleton (green). Positive correlations are displayed in red (FWE-
corrected p < .05). There were no significant negative correlations. Bottom: MD results overlaid with two long range
white matter pathways for visualization showing overlap with anterior thalamic radiation (ATR; yellow) and superior
longitudinal fasciculus 3 (SLF 3; blue). (B) Axial diffusivity (AD) results. Relationship among fMRI, DTI, and clinical variables Top: Area of significant correlations between
AD and DOI overlaid on the mean FA map and white matter skeleton (green). Positive correlations are displayed in
red (FWE-corrected p < .05). There were no significant negative correlations. Bottom: AD results overlaid with ATR
(yellow) and SLF 3 (blue) showing overlap for localization. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g004 Fig 4. Relationship between diffusion tensor imaging (DTI) metrics and duration of illness (DOI) in PTLD. (A)
Mean diffusivity (MD) results. Top: Area of significant correlations between MD and DOI overlaid on the mean
fractional anisotropy (FA) map and white matter skeleton (green). Positive correlations are displayed in red (FWE-
corrected p < .05). There were no significant negative correlations. Bottom: MD results overlaid with two long range
white matter pathways for visualization showing overlap with anterior thalamic radiation (ATR; yellow) and superior
longitudinal fasciculus 3 (SLF 3; blue). (B) Axial diffusivity (AD) results. Top: Area of significant correlations between
AD and DOI overlaid on the mean FA map and white matter skeleton (green). Positive correlations are displayed in
red (FWE-corrected p < .05). There were no significant negative correlations. Bottom: AD results overlaid with ATR
(yellow) and SLF 3 (blue) showing overlap for localization. L = left, R = right hemispheres. https://doi.org/10.1371/journal.pone.0271425.g004 (12) = -.81, p = .001), showing that greater diffusivity was associated with fewer symptoms (Fig
5). The two other ROIs did not correlate with sum of symptoms [all p-values > .29] (Table 4 in
S1 File). A similar comparison between the fMRI beta weight contrast values within these PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 14 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Fig 5. Correlation matrix of the relationship among fMRI, DTI, and clinical variables. Brain regions correspond to the regions of interest revealed by fMRI
between-groups contrasts that were located in white matter. FMRI beta values and DTI axial diffusivity measures were correlated with clinical variables. Notably, DTI measures of the BA 9 anterior region negatively correlated with PLQS scores (i.e., higher axial diffusivity was associated with fewer symptoms). PLQS = post-Lyme questionnaire of symptoms, fMRI ACC = accuracy on the fMRI task, fMRI RT = response times on the fMRI task, BDI = Beck Depression
Inventory; = p .05; = p .01 (two-tailed). Fig 5. Relationship among fMRI, DTI, and clinical variables Correlation matrix of the relationship among fMRI, DTI, and clinical variables. Brain regions correspond to the regions of interest revealed by fMRI
between-groups contrasts that were located in white matter. FMRI beta values and DTI axial diffusivity measures were correlated with clinical variables. Notably, DTI measures of the BA 9 anterior region negatively correlated with PLQS scores (i.e., higher axial diffusivity was associated with fewer symptoms). PLQS = post-Lyme questionnaire of symptoms, fMRI ACC = accuracy on the fMRI task, fMRI RT = response times on the fMRI task, BDI = Beck Depression
Inventory; = p .05; = p .01 (two-tailed). https://doi.org/10.1371/journal.pone.0271425.g005 ROIs and the sum of PLQS measures also did not correlate [all p-values > .45] (Table 4 in S1
File). Thus, these associations point to frontal lobe axial diffusivity, specifically, as a potential
indicator of healthy outcomes in PTLD (i.e., higher axial diffusivity with fewer symptoms). We also compared the three ROIs from fMRI beta contrast and DTI axial diffusivity values
to accuracy and RT performance on the working memory task. FMRI values and DTI values
did not correlate with accuracy or response times [all p-values > .10] (Table 4 in S1 File). Axial
diffusivity in these three regions also did not correlate with accuracy [all p-values > .22]
(Table 4 in S1 File). Higher axial diffusivity in the left BA 9 posterior ROI correlated with
lower (faster) response times, Spearman’s r(11) = -.63, p = .039 [the two other p-values > .85]. Given that depressive symptoms can accompany PTLD [7,41], we probed for the influence
of depressive symptoms on fMRI, DTI, and clinical variables using the Beck Depression Inven-
tory (BDI) total score [42]. The BDI total score correlated only with fMRI BA 6 beta values,
Spearman’s r(11) = -.767, p = .006, and did not correlate with any other fMRI ROI values (gray
or white matter) or axial diffusivity measures [all p-values > .05] (Table 4 in S1 File). However,
a higher BDI total score positively correlated with the total sum of symptoms, Spearman’s r
(12) = .59, p = .044 and sum of neurological symptoms, Spearman’s r(12), p = .024 [but not
with cognitive symptoms, p = .24]. The BDI total score did not correlate with accuracy or
response times on the working memory task [both p-values > .66] (Table 4 in S1 File). Relationship among fMRI, DTI, and clinical variables Taken together, these associations suggest that increased axial diffusivity impacts clinical
variables in a positive way. Notably, white matter axial diffusivity may be a marker of healing
during PTLD and represent a healthier outcome. Discussion This study applied multimodal neuroimaging methods to examine brain structure and func-
tion in a carefully selected sample of people with well-characterized PTLD in the absence of
co-morbid diseases and other factors that could otherwise explain the results. The original
hypothesis that the PTLD group would show altered task-related activations, as revealed by
fMRI, was supported. The PTLD group activated different gray brain regions relative to con-
trols, some of which were not previously associated with this task in a prior study of healthy
adults [23]. Moreover, the PTLD group hypoactivated other areas that were relevant to the PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 15 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease task, relative to controls, suggesting that the PTLD group relied on compensatory mechanisms
to complete the task, given that they performed as well as controls did. Unexpectedly, three of the PTLD group’s activated regions found in the frontal lobe were
located in white matter. Event-related white matter findings are distinctly unusual in fMRI
studies because of the relatively low energy demands and blood volume in white matter [43–
46]. These factors render the white matter BOLD signal comparably much less detectable than
in gray matter. However, evidence has accumulated in recent years suggesting that hemody-
namic changes can be detected in white matter using high magnetic field strength fMRI meth-
ods [45,47]. Explanations for the BOLD signal detection in white matter, especially in an
event-related manner, range from reduced physiological noise relative to signal, to visualiza-
tion of action potentials, to the energy associated with neurovascular coupling of astrocytes
[48]. It is notable that the oxygen extraction fraction in white matter has been reported to be
comparable to that of gray matter, a finding that may be explained by the need to maintain
resting membrane potentials in white matter oligodendrocytes [46,49]. Thus, white matter
changes may signify dysfunction- or excessive function- of glial cells [2]. Because this study
was not originally designed to examine white matter function, additional studies are needed to
further examine the white matter activations observed here. We scrutinized further the locations of three white matter frontal lobe fMRI-guided ROIs
using tissue segmentation analyses. Results showed that the left BA 9 anterior, BA 8, and BA 9
posterior ROIs contained 80% or more overlap with white matter tissue. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 Discussion 16 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease The relationship of these unexpected white matter findings to the clinical features of PTLD
suggest that white matter abnormalities may have an important role in the symptomatology of
PTLD. Prior studies have reported white matter abnormalities in PTLD. Notably, Fallon et al. (2003) reported brain perfusion abnormalities in white matter regions, including increased
blood flow to the frontal lobe [18]. The current findings supported those of Fallon et al. using
DTI methods that measured white matter integrity, guided by fMRI activations. The conver-
gence of data across imaging modalities underscore the role and vulnerability of the frontal
lobe in PTLD symptoms. This study was limited by the small sample size. First, this sample size potentially intro-
duced a risk of false positive findings by limited statistical power. However, several measures
were employed to mitigate this risk by independently confirming our results in multiple ways. We identified white matter activations that passed a threshold of p < .001, uncorrected
(Table 2 and S2 Table), a commonly used threshold for fMRI data reporting. We confirmed
that the white matter activations from the fMRI analyses were, indeed, located in white matter
using a follow-up segmentation analysis. We then created localized regions of interest specifi-
cally from these fMRI-guided activations and used these regions exclusively in our correlations
with clinical variables. Given that these regions passed several thresholds, using independent
multimodal imaging approaches, the converging findings suggest plausible results in this rela-
tively small sample size. Second, generalization of these findings to the larger PTLD community is also mitigated by
the relatively small sample size and stringent inclusion criteria (e.g., exclusion of atypical early
presentation of Lyme disease that did not meet CDC criteria). The homogeneity of the demo-
graphics within our sample further limited generalizability of the findings, given that the study
included people with a relatively advanced education level, high socioeconomic status, residing
within the mid-Atlantic region of the United States. Third, due to the small sample size, we
could not statistically control for the time between infection and antibiotic treatment, antibi-
otic dose, and duration of treatment across participants. Such factors may have influenced
healing and recovery processes that could not be fully accounted for in this study. Discussion Overlaying these
three ROIs onto a skeletonized DTI map, which is a highly conservative mapping that accounts
for 34% of the overall white matter mask, indicated 22% or more intersection. These results
confirmed that task-related activity can be localized to the white matter of the frontal lobe in
people with PTLD. These three ROIs were subsequently examined in more detail exploring their white matter
structural integrity using DTI methods (i.e., diffusivity measures). We found that, as a group,
the PTLD participants’ DTI measures did not differ from that of controls. When we compared
clinical and neurologic symptoms measured outside of the scanner environment to DTI mea-
sures in the PTLD group, we found that greater axial diffusivity was associated with fewer
symptoms. Thus, the white matter DTI changes observed in this study may represent a healthy
marker of the neurological repair process. We found that axial and mean diffusivity increased with DOI in regions within the right
frontal lobe. It should be noted that DOI per se did not correlate with clinical measures from
the PLQS, behavioral measures from the fMRI task, or BDI scores (all p-values > .42), indicat-
ing that diffusivity measures were the driving factor behind the correlations. The regions iden-
tified by the diffusivity/DOI correlation were discrete from the white matter ROIs derived
from the fMRI task, which were instead located in the left frontal lobe. Importantly, both mea-
sures implicated white matter changes within the frontal lobe. Cognitive difficulties referable
to the frontal lobe are commonly reported in patients with PTLD [7,13,50]. The measure of axial diffusivity is thought to be related to axonal properties, such as diame-
ter, count, and density [51–55]. However, it is important to note that the literature reflects
ambiguity with respect to whether increased or decreased axial diffusivity is related to axonal
injury. Some studies have reported axonal damage associated with axial diffusivity increases
[56–59], while others have reported axonal damage associated with axial diffusivity decreases
[60]. Moreover, studies have shown that axial diffusivity patterns can differ by region of inter-
est [61–64]. While it is not possible to fully elucidate the biological basis of the altered diffusion
signal in the data reported here, our findings demonstrate an important link between axial dif-
fusivity and clinical outcomes, as well as DOI. Acknowledgments We thank Bronte Wen for assistance with fMRI data processing and Cheryl Novak and Susan
Joseph for the recruitment of study participants. Discussion Fourth, this
was a cross-sectional study with a wide ranging DOI across participants. Ideally, participants
would be followed longitudinally from disease onset in order to better monitor the changes
associated with PTLD directly over time. Nonetheless, given the careful selection of partici-
pants with PTLD and little else to medically explain these results, these data provide an impor-
tant preliminary look at structural and functional brain changes associated with PTLD and
guide future neurologically-based research in the field. This study represents an in-depth
examination of the integrity of brain structure and function in people with PTLD using more
sophisticated neuroimaging measures than has been reported to date. The findings provide
quantitative, objective measures of brain changes that can be associated with clinical and cog-
nitive measures. Importantly, these findings support and validate PTLD patient reports of cog-
nitive difficulties [7]. Future studies will need to be conducted to replicate these results, given
the small sample size. Additionally, longitudinal tracking of brain changes from initial infec-
tion through development of PTLD will be needed to characterize changes in gray and white
matter over time. Results reported here may have implications for other diseases in which white matter
pathology has been demonstrated (e.g., multiple sclerosis) or in illnesses in which cognitive
complaints follow disease onset in the absence of objective methods to confirm neuropathol-
ogy (e.g., chronic fatigue syndrome, fibromyalgia, post acute COVID) [65–68]. The use of
multimodal neuroimaging methods, like the ones used in the current study, may be a viable
approach for obtaining information on brain function and structure to identify biomarkers of
disease burden. 17 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Supporting information S1 Table. FMRI task BOLD activations within healthy controls. Brain regions are based on
Talairach coordinates. Bold indicates activations that overlapped with a prior study in young,
healthy adults using the same fMRI task by Marvel & Desmond, 2012. All regions met thresh-
old criteria for p < .001, uncorrected. (DOCX) S2 Table. FMRI task BOLD activations within PTLD. Brain regions are based on Talairach
coordinates. Bold indicates activations that overlapped with a prior study in young, healthy
adults using the same fMRI task by Marvel & Desmond, 2012. ^ = regions that were primarily
located in white matter. All regions met threshold criteria for p < .001, uncorrected. (DOCX) S2 Table. FMRI task BOLD activations within PTLD. Brain regions are based on Talairach
coordinates. Bold indicates activations that overlapped with a prior study in young, healthy
adults using the same fMRI task by Marvel & Desmond, 2012. ^ = regions that were primarily
located in white matter. All regions met threshold criteria for p < .001, uncorrected. (DOCX) S1 File. FMRI, DTI, and clinical variables. Table 1 in S1 File shows descriptive statistics for
fMRI, DTI, and clinical variables. Table 2 in S1 File shows descriptive statistics for gray matter
fMRI data and scores on the Beck Depression Inventory (BDI). In both tables, data are shown
as: mean (standard deviation), 95% confidence interval [lower limit, upper limit]. Shapiro-
Wilk tests were used for tests of normality. Significant values of the Shapiro-Wilk tests are
denoted in bold, p .05, two-tailed. Table 3 in S1 File reports correlations between the fMRI
ROIs and their respective DTI ROIs. Pearson’s tests were used for bivariate correlations
involving BA 9 values because they have a normal distribution. A Spearman’s test was used for
bivariate correlations involving BA 8 values because they had a non-normal distribution. Table 4 in S1 File reports correlations between fMRI and DTI ROIs and fMRI Task Accuracy,
RT, Symptoms, and BDI. Spearman’s tests were used for bivariate correlations involving Task
Accuracy, RT, Cognitive Symptoms, and BDI because they had a non-normal distribution. Pearson’s tests were used for bivariate correlations involving Total Clinical Symptoms and
Neurological Symptoms because they had a normal distribution. (DOCX) Author Contributions Conceptualization: Cherie L. Marvel, Alison W. Rebman, Arun Venkatesan, John N. Aucott. Conceptualization: Cherie L. Marvel, Alison W. Rebman, Arun Venkatesan, John N. Aucott. Data curation: Kylie H. Alm, Deeya Bhattacharya, Alison W. Rebman, Erica A. Kozero. Formal analysis: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Owen P. Morgan,
Prianca A. Nadkarni. Formal analysis: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Owen P. Morgan,
Prianca A. Nadkarni. Formal analysis: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Owen P. Morgan,
Prianca A. Nadkarni. References 1. Steere AC, Strle F, Wormser GP, Hu LT, Branda JA, Hovius JWR, et al. Lyme borreliosis. Nat Rev Dis
Primers. 2016; 2:16090. https://doi.org/10.1038/nrdp.2016.90 PMID: 27976670 2. Kugeler K, Schwartz A, Delorey M, Mead P, Hinckley A. Estimating the Frequency of Lyme Disease
Diagnoses, United States, 2010–2018. Emerging Infectious Disease journal. 2021; 27(2):616. https://
doi.org/10.3201/eid2702.202731 PMID: 33496229 3. Steere AC. Lyme Disease. New England Journal of Medicine. 2001 2001/07/12; 345(2):115–25. https://
doi.org/10.1056/NEJM200107123450207 PMID: 11450660 4. Mac S, Bahia S, Simbulan F, Pullenayegum EM, Evans GA, Patel SN, et al. Long-Term Sequelae and
Health-Related Quality of Life Associated With Lyme Disease: A Systematic Review. Clinical infectious
diseases: an official publication of the Infectious Diseases Society of America. 2020; 71(2):440–52. https://doi.org/10.1093/cid/ciz1158 PMID: 31773171 5. Rebman AW, Aucott JN. Post-treatment Lyme Disease as a Model for Persistent Symptoms in Lyme
Disease. Frontiers in medicine. 2020; 7:57. https://doi.org/10.3389/fmed.2020.00057 PMID: 32161761 6. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, et al. The Clinical
Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and
Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America. Clinical Infec-
tious Diseases. 2006; 43(9):1089–134. https://doi.org/10.1086/508667 PMID: 17029130 7. Rebman AW, Bechtold KT, Yang T, Mihm EA, Soloski MJ, Novak CB, et al. The Clinical, Symptom, and
Quality-of-Life Characterization of a Well-Defined Group of Patients with Post treatment Lyme Disease
Syndrome. Front Med (Lausanne). 2017; 4:224. https://doi.org/10.3389/fmed.2017.00224 PMID:
29312942 8. Lobraico J, Butler A, Petrini J, Ahmadi R. New Insights Into Stages of Lyme Disease Symptoms From a
Novel Hospital-Based Registry. Journal of Primary Care & Community Health. 2014 2014/10/01; 5
(4):284–7. https://doi.org/10.1177/2150131914540693 PMID: 24970880 9. Zomer TP, Barendregt JNM, van Kooten B, van Bemmel T, Landman GW, van Hees BC, et al. Non-
specific symptoms in adult patients referred to a Lyme centre. Clinical Microbiology and Infection. 2019
2019/01/01/; 25(1):67–70. https://doi.org/10.1016/j.cmi.2018.09.016 PMID: 30287411 10. Klempner MS, Hu LT, Evans J, Schmid CH, Johnson GM, Trevino RP, et al. Two Controlled Trials of
Antibiotic Treatment in Patients with Persistent Symptoms and a History of Lyme Disease. New
England Journal of Medicine. 2001 2001/07/12; 345(2):85–92. https://doi.org/10.1056/
NEJM200107123450202 PMID: 11450676 11. Chandra AM, Keilp JG, Fallon BA. Correlates of perceived health-related quality of life in post-treatment
Lyme encephalopathy. Psychosomatics. 2013 Nov-Dec; 54(6):552–9. https://doi.org/10.1016/j.psym. 2013.04.003 PMID: 23845316 12. Rebman AW, Aucott JN, Weinstein ER, Bechtold KT, Smith KC, Leonard L. Funding acquisition: John N. Aucott. Funding acquisition: John N. Aucott. Investigation: Cherie L. Marvel, Deeya Bhattacharya, Owen P. Morgan, Jason A. Creighton,
Erica A. Kozero, John N. Aucott. Methodology: Cherie L. Marvel, Kylie H. Alm, Alison W. Rebman, Arnold Bakker, John N. Aucott. Project administration: Cherie L. Marvel, Deeya Bhattacharya, Jason A. Creighton. Resources: Cherie L. Marvel, Arnold Bakker, John N. Aucott. 18 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease Software: Kylie H. Alm, Owen P. Morgan. Software: Kylie H. Alm, Owen P. Morgan. Supervision: Cherie L. Marvel, Arnold Bakker, John N. Aucott. Software: Kylie H. Alm, Owen P. Morgan. Supervision: Cherie L. Marvel, Arnold Bakker, John N. Aucott. Writing – original draft: Cherie L. Marvel, Kylie H. Alm, Deeya Bhattacharya, Alison W. Reb-
man, Arnold Bakker, John N. Aucott. Writing – review & editing: Cherie L. Marvel, Kylie H. Alm, Deeya Bhattacharya, Alison W. Rebman, Arnold Bakker, Owen P. Morgan, Jason A. Creighton, Erica A. Kozero, Arun
Venkatesan, Prianca A. Nadkarni, John N. Aucott. References Living in Limbo: Contested
Narratives of Patients With Chronic Symptoms Following Lyme Disease. Qualitative Health Research. 2015 2017/03/01; 27(4):534–46. 13. Touradji P, Aucott JN, Yang T, Rebman AW, Bechtold KT. Cognitive Decline in Post-treatment Lyme
Disease Syndrome. Archives of Clinical Neuropsychology. 2019; 34(4):455–65. https://doi.org/10.1093/
arclin/acy051 PMID: 29945190 14. Newberg A, Hassan A, Alavi A. Cerebral metabolic changes associated with Lyme disease. Nucl Med
Commun. 2002 Aug; 23(8):773–7. https://doi.org/10.1097/00006231-200208000-00011 PMID:
12124483 15. Fallon BA, Das S, Plutchok JJ, Tager F, Liegner K, Van Heertum R. Functional brain imaging and
neuropsychological testing in Lyme disease. Clin Infect Dis. 1997 Jul; 25 Suppl 1:S57–63. https://doi. org/10.1086/516175 PMID: 9233666 19 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease 16. Sumiya H, Kobayashi K, Mizukoshi C, Aoki T, Koshino Y, Taki J, et al. Brain perfusion SPECT in Lyme
neuroborreliosis. J Nucl Med. 1997 Jul; 38(7):1120–2. PMID: 9225802 17. Westervelt HJ, McCaffrey RJ. Neuropsychological functioning in chronic Lyme disease. Neuropsychol
Rev. 2002 Sep; 12(3):153–77. https://doi.org/10.1023/a:1020381913563 PMID: 12428915 18. Fallon BA, Keilp J, Prohovnik I, Heertum RV, Mann JJ. Regional cerebral blood flow and cognitive defi-
cits in chronic lyme disease. J Neuropsychiatry Clin Neurosci. 2003 Summer; 15(3):326–32. https://doi. org/10.1176/jnp.15.3.326 PMID: 12928508 19. Fernandez RE, Rothberg M, Ferencz G, Wujack D. Lyme disease of the CNS: MR imaging findings in
14 cases. AJNR Am J Neuroradiol. 1990 May; 11(3):479–81. PMID: 2112310 20. Coughlin JM, Yang T, Rebman AW, Bechtold KT, Du Y, Mathews WB, et al. Imaging glial activation in
patients with post-treatment Lyme disease symptoms: a pilot study using [(11)C]DPA-713 PET. J Neu-
roinflammation. 2018; 15(1):346. https://doi.org/10.1186/s12974-018-1381-4 PMID: 30567544 21. Centers for Disease Control and Prevention [Internet]. Lyme Disease (Borrelia burgdorferi) 2022 Case
Definition. [Accessed August 23, 2022]. Available from: https://ndc.services.cdc.gov/case-definitions/
lyme-disease-2022. 22. Rebman AW, Yang T, Aucott JN. Symptom heterogeneity and patient subgroup classification among
US patients with post-treatment Lyme disease: an observational study. BMJ Open. 2021; 11(1):
e040399. https://doi.org/10.1136/bmjopen-2020-040399 PMID: 33441353 23. Marvel CL, Desmond JE. From storage to manipulation: How the neural correlates of verbal working
memory reflect varying demands on inner speech. Brain Lang. 2012 Jan; 120(1):42–51. https://doi.org/
10.1016/j.bandl.2011.08.005 PMID: 21889195 24. Marvel CL, Faulkner ML, Strain EC, Mintzer MZ, Desmond JE. An fMRI investigation of cerebellar func-
tion during verbal working memory in methadone maintenance patients. Cerebellum. 2012 Mar; 11
(1):300–10. https://doi.org/10.1007/s12311-011-0311-0 PMID: 21892700 25. References Chen SH, Desmond JE. Temporal dynamics of cerebro-cerebellar network recruitment during a cogni-
tive task. Neuropsychologia. 2005; 43(9):1227–37. https://doi.org/10.1016/j.neuropsychologia.2004.12. 015 PMID: 15949507 26. Talairach J, & Tournoux P. Co-planar Stereotaxic atlas of the human brain 3-D proportional system, An
approach to cerebral imaging (M. Rayport, Trans.): New York: Thieme Medical Publishers, Inc.; 1988. 27. Lancaster JL, Rainey LH, Summerlin JL, Freitas CS, Fox PT, Evans AC, et al. Automated labeling of
the human brain: a preliminary report on the development and evaluation of a forward-transform
method. Hum Brain Mapp. 1997 1997; 5(4):238–42. https://doi.org/10.1002/(SICI)1097-0193(1997)
5:4<238::AID-HBM6>3.0.CO;2-4 PMID: 20408222 28. Brett M, Anton J-L, Valabregue R, Poline J-B. Region of interest analysis using an SPM
toolbox [abstract]. Presented at the 8th International Conference on Functional Mapping of the Human
Brain. NeuroImage. 2002;16. 29. Smith SM, Jenkinson M, Woolrich MW, Beckmann CF, Behrens TEJ, Johansen-Berg H, et al. Advances in functional and structural MR image analysis and implementation as FSL. NeuroImage. 2004 2004/01/01/; 23:S208–S19. https://doi.org/10.1016/j.neuroimage.2004.07.051 PMID: 15501092 30. Klein A, Andersson J, Ardekani BA, Ashburner J, Avants B, Chiang M-C, et al. Evaluation of 14 nonlin-
ear deformation algorithms applied to human brain MRI registration. NeuroImage. 2009; 46(3):786–
802. https://doi.org/10.1016/j.neuroimage.2008.12.037 PMID: 19195496 31. Yushkevich PA, Avants BB, Pluta J, Das S, Minkoff D, Mechanic-Hamilton D, et al. A high-resolution
computational atlas of the human hippocampus from postmortem magnetic resonance imaging at 9.4
T. NeuroImage. 2009; 44(2):385–98. https://doi.org/10.1016/j.neuroimage.2008.08.042 PMID:
18840532 32. Zhang Y, Brady M, Smith S. Segmentation of brain MR images through a hidden Markov random field
model and the expectation-maximization algorithm. IEEE Transactions on Medical Imaging. 2001; 20
(1):45–57. https://doi.org/10.1109/42.906424 PMID: 11293691 33. Eggert LD, Sommer J, Jansen A, Kircher T, Konrad C. Accuracy and Reliability of Automated Gray Mat-
ter Segmentation Pathways on Real and Simulated Structural Magnetic Resonance Images of the
Human Brain. PLoS One. 2012; 7(9):e45081. https://doi.org/10.1371/journal.pone.0045081 PMID:
23028771 34. Smith SM, Jenkinson M, Johansen-Berg H, Rueckert D, Nichols TE, Mackay CE, et al. Tract-based
spatial statistics: Voxelwise analysis of multi-subject diffusion data. NeuroImage. 2006 2006/07/15/; 31
(4):1487–505. https://doi.org/10.1016/j.neuroimage.2006.02.024 PMID: 16624579 35. Andersson J, Jenkinson M, editors. Non-linear optimisation FMRIB Technial Report TR 07 JA 1. 2007. 36. Jenkinson M, editor. Non-linear registration aka Spatial normalisation. 2007. 35. Andersson J, Jenkinson M, editors. Non-linear optimisation FMRIB Technial Report TR 07 JA 1. 2007. 36. Jenkinson M, editor. Non-linear registration aka Spatial normalisation. 2007. References 20 / 22 PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease 37. Winkler AM, Ridgway GR, Webster MA, Smith SM, Nichols TE. Permutation inference for the general
linear model. NeuroImage. 2014; 92(100):381–97. https://doi.org/10.1016/j.neuroimage.2014.01.060
PMID: 24530839 38. Smith SM, Nichols TE. Threshold-free cluster enhancement: Addressing problems of smoothing,
threshold dependence and localisation in cluster inference. NeuroImage. 2009 2009/01/01/; 44(1):83–
98. https://doi.org/10.1016/j.neuroimage.2008.03.061 PMID: 18501637 39. Warrington S, Bryant KL, Khrapitchev AA, Sallet J, Charquero-Ballester M, Douaud G, et al. XTRACT
—Standardised protocols for automated tractography in the human and macaque brain. NeuroImage. 2020 2020/08/15/; 217:116923. https://doi.org/10.1016/j.neuroimage.2020.116923 PMID: 32407993 40. Marvel CL, Desmond JE. The contributions of cerebro-cerebellar circuitry to executive verbal working
memory. Cortex. 2010 Jul-Aug; 46(7):880–95. https://doi.org/10.1016/j.cortex.2009.08.017 PMID:
19811779 41. Bransfield RC. Neuropsychiatric Lyme Borreliosis: An Overview with a Focus on a Specialty Psychia-
trist’s Clinical Practice. Healthcare (Basel). 2018; 6(3):104. https://doi.org/10.3390/healthcare6030104
PMID: 30149626 42. Beck A, Steer R, Brown G. Beck Depression Inventory—Second Edition Manual. San Antonio: The
Psychological Corporation; 1996. 43. Logothetis NK, Wandell BA. Interpreting the BOLD signal. Annu Rev Physiol. 2004; 66:735–69. https://
doi.org/10.1146/annurev.physiol.66.082602.092845 PMID: 14977420 44. Rostrup E, Law I, Blinkenberg M, Larsson HBW, Born AP, Holm S, et al. Regional Differences in the
CBF and BOLD Responses to Hypercapnia: A Combined PET and fMRI Study. NeuroImage. 2000
2000/02/01/; 11(2):87–97. https://doi.org/10.1006/nimg.1999.0526 PMID: 10679182 45. Gore JC, Li M, Gao Y, Wu T-L, Schilling KG, Huang Y, et al. Functional MRI and resting state connectiv-
ity in white matter—a mini-review. Magnetic resonance imaging. 2019; 63:1–11. https://doi.org/10. 1016/j.mri.2019.07.017 PMID: 31376477 46. Harris JJ, Attwell D. The Energetics of CNS White Matter. The Journal of Neuroscience. 2012; 32
(1):356. https://doi.org/10.1523/JNEUROSCI.3430-11.2012 PMID: 22219296 47. Wu X, Yang Z, Bailey SK, Zhou J, Cutting LE, Gore JC, et al. Functional connectivity and activity of
white matter in somatosensory pathways under tactile stimulations. NeuroImage. 2017 2017/05/15/;
152:371–80. https://doi.org/10.1016/j.neuroimage.2017.02.074 PMID: 28284801 48. Gawryluk JR, Mazerolle EL, D’Arcy RCN. Does functional MRI detect activation in white matter? A
review of emerging evidence, issues, and future directions. Front Neurosci. 2014; 8:239. https://doi.org/
10.3389/fnins.2014.00239 PMID: 25152709 49. Raichle ME, MacLeod AM, Snyder AZ, Powers WJ, Gusnard DA, Shulman GL. A default mode of brain
function. Proceedings of the National Academy of Sciences. 2001; 98(2):676. 50. Keilp JG, Corbera K, Slavov I, Taylor MJ, Sackeim HA, Fallon BA. WAIS-III and WMS-III performance
in chronic Lyme disease. References J Int Neuropsychol Soc. 2006 Jan; 12(1):119–29. https://doi.org/10.1017/
S1355617706060231 PMID: 16433951 51. Beaulieu C. What Makes Diffusion Anisotropic in the Nervous System? Diffusion MRITheory, Methods,
and Applications. Oxford, UK: Oxford University Press; 2012. 52. Song SK, Sun SW, Ju WK, Lin SJ, Cross AH, Neufeld AH. Diffusion tensor imaging detects and differ-
entiates axon and myelin degeneration in mouse optic nerve after retinal ischemia. Neuroimage. 2003
Nov; 20(3):1714–22. https://doi.org/10.1016/j.neuroimage.2003.07.005 PMID: 14642481 53. Budde MD, Kim JH, Liang HF, Schmidt RE, Russell JH, Cross AH, et al. Toward accurate diagnosis of
white matter pathology using diffusion tensor imaging. Magn Reson Med. 2007 Apr; 57(4):688–95. https://doi.org/10.1002/mrm.21200 PMID: 17390365 54. Budde MD, Xie M, Cross AH, Song S-K. Axial Diffusivity Is the Primary Correlate of Axonal Injury in the
Experimental Autoimmune Encephalomyelitis Spinal Cord: A Quantitative Pixelwise Analysis. The Jour-
nal of Neuroscience. 2009; 29(9):2805. https://doi.org/10.1523/JNEUROSCI.4605-08.2009 PMID:
19261876 55. DeBoy CA, Zhang J, Dike S, Shats I, Jones M, Reich DS, et al. High resolution diffusion tensor imaging
of axonal damage in focal inflammatory and demyelinating lesions in rat spinal cord. Brain. 2007 Aug;
130(Pt 8):2199–210. https://doi.org/10.1093/brain/awm122 PMID: 17557778 56. Bosch B, Arenaza-Urquijo EM, Rami L, Sala-Llonch R, Junque´ C, Sole´-Padulle´s C, et al. Multiple DTI
index analysis in normal aging, amnestic MCI and AD. Relationship with neuropsychological perfor-
mance. Neurobiol Aging. 2012 Jan; 33(1):61–74. https://doi.org/10.1016/j.neurobiolaging.2010.02.004
PMID: 20371138 57. Alves GS, Oertel Kno¨chel V, Kno¨chel C, Carvalho AF, Pantel J, Engelhardt E, et al. Integrating retro-
genesis theory to Alzheimer’s disease pathology: insight from DTI-TBSS investigation of the white PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 21 / 22 PLOS ONE Neuroimaging of brain abnormalities in post treatment Lyme disease matter microstructural integrity. Biomed Res Int. 2015; 2015:291658. https://doi.org/10.1155/2015/
291658 PMID: 25685779 matter microstructural integrity. Biomed Res Int. 2015; 2015:291658. h
291658 PMID: 25685779 matter microstructural integrity. Biomed Res Int. 2015; 2015:291658. https://doi.org/10.1155/2015/
291658 PMID: 25685779 58. 58. Schmierer K, Wheeler-Kingshott CA, Tozer DJ, Boulby PA, Parkes HG, Yousry TA, et al. Quantitative
magnetic resonance of postmortem multiple sclerosis brain before and after fixation. Magn Reson Med. 2008 Feb; 59(2):268–77. https://doi.org/10.1002/mrm.21487 PMID: 18228601 59. Newcombe V, Chatfield D, Outtrim J, Vowler S, Manktelow A, Cross J, et al. Mapping traumatic axonal
injury using diffusion tensor imaging: correlations with functional outcome. PLoS One. 2011; 6(5):
e19214–e. https://doi.org/10.1371/journal.pone.0019214 PMID: 21573228 60. Wang X, Cusick MF, Wang Y, Sun P, Libbey JE, Trinkaus K, et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0271425
October 26, 2022 68.
Glass JM. Fibromyalgia and cognition. J Clin Psychiatry. 2008; 69 Suppl 2:20–4. PMID: 18537459 References Diffusion basis spectrum imaging
detects and distinguishes coexisting subclinical inflammation, demyelination and axonal injury in experi-
mental autoimmune encephalomyelitis mice. NMR Biomed. 2014 Jul; 27(7):843–52. https://doi.org/10. 1002/nbm.3129 PMID: 24816651 61. Bennett IJ, Madden DJ, Vaidya CJ, Howard DV, Howard JH Jr., Age-related differences in multiple
measures of white matter integrity: A diffusion tensor imaging study of healthy aging. Hum Brain Mapp. 2010; 31(3):378–90. 62. Burzynska AZ, Preuschhof C, Ba¨ckman L, Nyberg L, Li SC, Lindenberger U, et al. Age-related differ-
ences in white matter microstructure: region-specific patterns of diffusivity. Neuroimage. 2010 Feb 1; 49
(3):2104–12. https://doi.org/10.1016/j.neuroimage.2009.09.041 PMID: 19782758 63. Isaac Tseng WY, Hsu YC, Chen CL, Kang YJ, Kao TW, Chen PY, et al. Microstructural differences in
white matter tracts across middle to late adulthood: a diffusion MRI study on 7167 UK Biobank partici-
pants. Neurobiol Aging. 2021 Feb; 98:160–72. https://doi.org/10.1016/j.neurobiolaging.2020.10.006
PMID: 33290993 64. Madden DJ, Bennett IJ, Burzynska A, Potter GG, Chen NK, Song AW. Diffusion tensor imaging of cere-
bral white matter integrity in cognitive aging. Biochim Biophys Acta. 2012 Mar; 1822(3):386–400. https://doi.org/10.1016/j.bbadis.2011.08.003 PMID: 21871957 65. Freeman CW, Masur J, Hassankhani A, Wolf RL, Levine JM, Mohan S. Coronavirus Disease (COVID-
19)-Related Disseminated Leukoencephalopathy: A Retrospective Study of Findings on Brain MRI. American Journal of Roentgenology. 2021 2021/04/01; 216(4):1046–7. https://doi.org/10.2214/AJR.20. 24364 PMID: 32903058 66. Hampshire A, Trender W, Chamberlain SR, Jolly AE, Grant JE, Patrick F, et al. Cognitive deficits in peo-
ple who have recovered from COVID-19. EClinicalMedicine. 2021 Sep; 39:101044. https://doi.org/10. 1016/j.eclinm.2021.101044 PMID: 34316551 67. Morris G, Berk M, Puri BK. A Comparison of Neuroimaging Abnormalities in Multiple Sclerosis, Major
Depression and Chronic Fatigue Syndrome (Myalgic Encephalomyelitis): is There a Common Cause? Mol Neurobiol. 2018 Apr; 55(4):3592–609. https://doi.org/10.1007/s12035-017-0598-z PMID:
28516431 68. Glass JM. Fibromyalgia and cognition. J Clin Psychiatry. 2008; 69 Suppl 2:20–4. PMID: 18537459 22 / 22
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Produtos de Grafos Z_m-bem-cobertos
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Trends in Computational and Applied Mathematics
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Produtos de Grafos Zm-bem-cobertos R.M. BARBOSA1, M.R.C. SANTANA2, Instituto de Informática, INF, UFG - Uni-
versidade Federal de Goiás, 74001-970 Goiânia, GO, Brasil. R.M. BARBOSA1, M.R.C. SANTANA2, Instituto de Informática, INF, UFG - Uni-
versidade Federal de Goiás, 74001-970 Goiânia, GO, Brasil. Resumo. Um grafo é Zm-bem-coberto se |I| ≡|J| (mod m), m ≥2, para todo I,
J conjuntos independentes maximais em V (G). Um grafo G é fortemente Zm-bem-
coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm-bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto e G\{v} é Zm-bem-
coberto, ∀v ∈V (G). Mostramos que os grafos 1-Zm-bem-cobertos, bem como os
fortemente Zm-bem-cobertos, com exceção de K1 e K2, têm cintura ≤5. Mos-
tramos uma condição necessária e suficiente para que produtos lexicográficos de
grafos sejam Zm-bem-cobertos e algumas propriedades para o produto cartesiano
de ciclos. Palavras-chave. Teoria dos Grafos, Conjuntos Independentes em Grafos, Produ-
tos de Grafos. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83.
doi: 10.5540/tema.2012.013.01.0075
c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83. doi: 10.5540/tema.2012.013.01.0075
c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83. doi: 10.5540/tema.2012.013.01.0075
c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. 1rommel@inf.ufg.br
2marcia@inf.ufg.br Recebido em 02 Maio 2011; Aceito em 24 Fevereiro 2012. TEMA Tend. Mat. Apl. Comput., 13, No. 1 (2012), 75-83.
doi: 10.5540/tema.2012.013.01.0075
c⃝Uma Publicação da Sociedade Brasileira de Matemática Aplicada e Computacional. 1.
Introdução De forma similar, foi definido em [2, 3] que um grafo
G é fortemente Zm-bem-coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm-
bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto
e G\{v} é Zm-bem-coberto, ∀v ∈V (G). Uma folha é um vértice de grau 1, um
talo é um vértice adjacente a uma folha e um arbusto é um subgrafo induzido por
um talo e suas folhas. Vértices x e y de um grafo G são ditos ser conectados por
uma 2-ponte se existem vértices u e v ∈V (G) com grau de u e v igual a 2 e com
N(u) = {x, v} e N(v) = {u, y}. para qualquer e ∈E(G). De forma similar, foi definido em [2, 3] que um grafo
G é fortemente Zm-bem-coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm-
bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto
e G\{v} é Zm-bem-coberto, ∀v ∈V (G). Uma folha é um vértice de grau 1, um
talo é um vértice adjacente a uma folha e um arbusto é um subgrafo induzido por
um talo e suas folhas. Vértices x e y de um grafo G são ditos ser conectados por
uma 2-ponte se existem vértices u e v ∈V (G) com grau de u e v igual a 2 e com
N(u) = {x, v} e N(v) = {u, y}. O Teorema 1.1, provado em [10], fornece uma caracterização de grafos Zm-bem-
cobertos de cintura > 5. O Teorema 1.1, provado em [10], fornece uma caracterização de grafos Zm-bem-
cobertos de cintura > 5. Teorema 1.1. [10] Um grafo é Zm-bem-coberto conexo de cintura > 5 se e somente
se G é K1, C7 ou um grafo conexo de cintura pelo menos seis, que consiste de uma
união finita de arbustos Bi, cada um com talo xi, onde cada talo tem ri congruente
a 1 (mod m) folhas e onde, para cada i e j, uma e somente uma das seguintes
condições é satisfeita: 1. xi e xj são unidos por uma aresta e qualquer outro caminho, caso exista,
unindo xi e xj deve incluir pelo menos um talo diferente de xi e xj; 2. 1.
Introdução Os grafos aqui considerados são grafos simples. As definições e notações utilizadas
seguem [7]. Denotamos o conjunto de vértices de um grafo G por V (G) e o conjunto de
arestas por E(G). N(v) é o conjunto de vértices adjacentes a v em G. Um con-
junto I ⊆V (G) é independente se quaisquer dois vértices de I não são adjacentes. Denotamos por α(G) a cardinalidade do maior conjunto independente de vértices
de G. A cintura de um grafo com um ciclo é o tamanho de seu menor ciclo. Um
grafo sem ciclo tem cintura infinita. Um grafo é bem-coberto se todo conjunto in-
dependente maximal de vértices em G tiver mesma cardinalidade. Um grafo G é
um grafo Zm-bem-coberto, m ≥2, se |I| ≡|J| (mod m), para todos I, J conjuntos
independentes maximais em V (G). Estes grafos foram introduzidos em [9]. Ca-
racterizações de grafos Zm-bem-cobertos cúbicos foram dadas em [4], com cintura
≥6 em [10], cordais, simpliciais e arco-circulares em [6] e livres de K1,3 em [5]. O
problema de determinação do número de independência de um grafo é um problema
NP-Completo [14] para grafos em geral. Para grafos bem-cobertos este problema
torna-se mais simples, pois é suficiente encontrar qualquer conjunto independente
maximal, visto que todos tem a mesma cardinalidade. Caro [8] provou que o pro-
blema de reconhecimento de grafos bem-cobertos é Co-NP-completo mesmo para
grafos Zm-bem-cobertos que são livres de K1,3m+1. Pinter [16] definiu um grafo for-
temente bem-coberto G como um grafo que é bem-coberto e G\{e} é bem-coberto Recebido em 02 Maio 2011; Aceito em 24 Fevereiro 2012. 76 Barbosa e Santana Barbosa e Santana Barbosa e Santana para qualquer e ∈E(G). De forma similar, foi definido em [2, 3] que um grafo
G é fortemente Zm-bem-coberto se G é um grafo Zm-bem-coberto e G\{e} é Zm-
bem-coberto, ∀e ∈E(G). Um grafo G é 1-Zm-bem-coberto se G é Zm-bem-coberto
e G\{v} é Zm-bem-coberto, ∀v ∈V (G). Uma folha é um vértice de grau 1, um
talo é um vértice adjacente a uma folha e um arbusto é um subgrafo induzido por
um talo e suas folhas. Vértices x e y de um grafo G são ditos ser conectados por
uma 2-ponte se existem vértices u e v ∈V (G) com grau de u e v igual a 2 e com
N(u) = {x, v} e N(v) = {u, y}. para qualquer e ∈E(G). 1.
Introdução xi e xj são conectados por km 2-pontes, k ∈N, e qualquer outro caminho
unindo xi e xj deve incluir outro talo além de xi e xj; 3. Todo caminho unindo xi e xj contém pelo menos um talo diferente de xi e
xj. 3. Todo caminho unindo xi e xj contém pelo menos um talo diferente de xi e
xj. Na figura 1 temos um exemplo de um grafo Z3-bem-coberto conexo de cintura
6, com conjuntos independentes maximais de cardinalidades 4, 7 e 10. 3 2-pontes
Figura 1: Grafo Z3-bem-coberto de cintura 6 Figura 1: Grafo Z3-bem-coberto de cintura 6 A classe de grafos Zm-bem-cobertos inclui todos os grafos bem-cobertos, já que
em todo grafo bem-coberto os conjuntos independentes maximais têm a mesma
cardinalidade e, portanto, são congruentes módulo qualquer natural m. Alguns
resultados sobre grafos bem-cobertos podem ser estendidos para os grafos Zm-bem-
cobertos. Como principais contribuições, mostramos que se G é 1-Zm-bem-coberto e
não é K1 nem K2, então G tem cintura ≤5 e apresentamos também uma condição
necessária e suficiente para que produtos lexicográficos de grafos sejam Zm-bem-
cobertos. Ainda, mostramos que o produto cartesiano de dois ciclos Cn e Cm é
Zm-bem-coberto se e somente se ele é bem-coberto. 77 Produtos de Grafos Zm-bem-cobertos Outros resultados válidos para grafos bem-cobertos não podem ser estendidos
aos grafos Zm-bem-cobertos. Na Proposição 3.2, mostramos que podemos construir
infinitos grafos fortemente Zm-bem-cobertos que sejam planares. Há apenas 4 grafos
planares fortemente bem-cobertos, o que foi provado por Pinter [16]. Para um subconjunto S de V (H ◦{G1, G2, . . . , G|V (H)|}), denotamos XH(S) =
{x ∈V (H) : ∃y ∈V (Gx)(x, y) ∈S} e XGx(S) = {y ∈V (Gx) : (x, y) ∈S} para
todo x ∈XH(S). No grafo da figura 2 (b), se S = {(1, a), (3, c)}, XH(S) = {1, 3},
XG1(S) = {a} e XG3(S) = {c}. 2.
Produto Lexicográfico , G|V (H)|)), S é um conjunto
independente maximal em H ◦(G1, . . . , G|V (H)|), se e somente se, XH(S) é um
conjunto independente maximal em H, e para todo v ∈XH(S), o conjunto XGv(S)
é um conjunto independente maximal em Gv. Proposição 2.1. [17] Dados S ⊂V (H ◦(G1, . . . , G|V (H)|)), S é um conjunto
independente maximal em H ◦(G1, . . . , G|V (H)|), se e somente se, XH(S) é um
conjunto independente maximal em H, e para todo v ∈XH(S), o conjunto XGv(S)
é um conjunto independente maximal em Gv. De forma similar a apresentada por Topp e Volkmann [17], o Teorema 2.2 nos
fornece uma condição necessária e suficiente para o produto de grafos ser Zm-bem-
coberto. Teorema 2.2. Seja H um grafo e {G1, G2, . . . , G|V (H)|} uma família de grafos
não vazios. O produto lexicográfico H ◦{G1, G2, . . . , G|V (H)|} é um grafo Zm-bem-
coberto, para um dado m ≥2, se e somente se H e {G1, G2, ..., G|V (H)|} satisfazem
as seguintes condições: 1. Gi é Zm-bem-coberto para i = 1, . . . , |V (H)|, para um m qualquer ≥2. 2. P
v∈I α(Gv) ≡P
u∈J α(Gu) (mod m), ∀I, J, conjuntos independentes maxi-
mais de H. Demonstração. (=⇒)Suponha que H ◦{G1, G2, . . . , G|V (H)|} é Zm-bem-coberto e
que existe Gv0 que não é Zm-bem-coberto. Então Gv0 tem dois conjuntos inde-
pendentes maximais de vértices Iv0 e Jv0 tais que |Iv0| ̸≡|Jv0| (mod m). Es-
tenda {v0} a um conjunto independente maximal L em V (H). Para qualquer
v ∈L\{v0}, seja Iv um conjunto independente maximal em Gv. Então, pela
Proposição 2.1, A = ∪v∈L\{v0}{(v, x) : x ∈Iv} ∪{(v0, y) : y ∈Iv0} e B =
∪v∈L\{v0}{(v, x) : x ∈Iv}∪{(v0, t) : t ∈Jv0} são conjuntos independentes maximais
em H ◦{G1, G2, . . . , G|V (H)|} tal que |A| ̸≡|B| (mod m). Isto é uma contradição. Logo, Gi deve ser Zm-bem-coberto para todo i = 1, 2, . . . , |V (H)|. Demonstração. (=⇒)Suponha que H ◦{G1, G2, . . . , G|V (H)|} é Zm-bem-coberto e
que existe Gv0 que não é Zm-bem-coberto. Então Gv0 tem dois conjuntos inde-
pendentes maximais de vértices Iv0 e Jv0 tais que |Iv0| ̸≡|Jv0| (mod m). 2.
Produto Lexicográfico , G|V (H)|} são congruentes (mod m) e portanto H ◦{G1, G2, . . . , G|V (H)|} é um
grafo Zm-bem-coberto. 2.
Produto Lexicográfico Es-
tenda {v0} a um conjunto independente maximal L em V (H). Para qualquer
v ∈L\{v0}, seja Iv um conjunto independente maximal em Gv. Então, pela
Proposição 2.1, A = ∪v∈L\{v0}{(v, x) : x ∈Iv} ∪{(v0, y) : y ∈Iv0} e B =
∪v∈L\{v0}{(v, x) : x ∈Iv}∪{(v0, t) : t ∈Jv0} são conjuntos independentes maximais
em H ◦{G1, G2, . . . , G|V (H)|} tal que |A| ̸≡|B| (mod m). Isto é uma contradição. Logo, Gi deve ser Zm-bem-coberto para todo i = 1, 2, . . . , |V (H)|. Sejam I e J dois conjuntos independentes maximais de vértices em H. Provare-
mos que P
v∈I α(Gv) ≡P
v∈J α(Gv) (mod m). Seja Rv um conjunto independente
maximal em Gv, ∀v ∈I∪J. Então, S1 = S
v∈I{(v, x) : x ∈Rv} e S2 = S
v∈J{(v, x) :
x ∈Rv} são conjuntos independentes maximais em H ◦{G1, G2, . . . , G|V (H)|}. En-
tão |S1| ≡|S2| (mod m). Mas, {|(v, x) : x ∈Rv}| ≡α(Gv)(mod m), ∀u, v ∈I ∪J,
segue que P
v∈I α(Gv) ≡|S1| ≡|S2| ≡P
v∈J α(Gv) (mod m). v∈I
v∈J
(⇐=)Seja I um conjunto independente maximal em H ◦{G1, G2, . . . , G|V (H)|}. Pela Proposição 2.1, XH(I) é um conjunto independente maximal em H e XGv(I)
é um conjunto independente maximal em Gv para todo v ∈XH(I). Já que
I = S
v∈XH(I){(v, x) : x ∈XGv(I)} e |XGv(I)| ≡α(Gv) (mod m), temos |I| =
P
v∈XH(I) |{[v, x] : x ∈XGv(I)}| = P
v∈XH(I) |XGv(I)| = P
v∈XH(I) α(Gv). Então,
pela condição (2), quaisquer dois conjuntos independentes maximais em H◦{G1, G2,
. . . , G|V (H)|} são congruentes (mod m) e portanto H ◦{G1, G2, . . . , G|V (H)|} é um
grafo Zm-bem-coberto. ∈
∈J
(⇐=)Seja I um conjunto independente maximal em H ◦{G1, G2, . . . , G|V (H)|}. Pela Proposição 2.1, XH(I) é um conjunto independente maximal em H e XGv(I)
é um conjunto independente maximal em Gv para todo v ∈XH(I). Já que
I = S
v∈XH(I){(v, x) : x ∈XGv(I)} e |XGv(I)| ≡α(Gv) (mod m), temos |I| =
P
v∈XH(I) |{[v, x] : x ∈XGv(I)}| = P
v∈XH(I) |XGv(I)| = P
v∈XH(I) α(Gv). Então,
pela condição (2), quaisquer dois conjuntos independentes maximais em H◦{G1, G2,
. . . 2.
Produto Lexicográfico Propriedades relacionadas a produtos lexicográficos de grafos foram provadas para
problemas de coloração [1] e cobertura por ciclos [15]. Alguns resultados sobre
produtos de grafos e algumas aplicações podem ser encontradas em [12]. Topp e Volkmann [17] mostram quando o produto lexicográfico de grafos é bem-
coberto. Como uma generalização deste resultado, apresentamos uma condição ne-
cessária e suficiente para construção de grafos Zm-bem-cobertos a partir do produto
lexicográfico de grafos. Dados um grafo H e uma família de grafos não vazios {G1, G2, . . . , G|V (H)|}
indexados pelos vértices de H, o produto lexicográfico H◦{G1, G2, . . . , G|V (H)|} de H
e {G1, G2, . . . , G|V (H)|} é o grafo tendo o conjunto de vértices S
v∈V (H){v}×V (Gv),
sendo que dois vértices (v1, v2) e (u1, u2) são adjacentes se {v1, u1} ∈E(H) ou
(v1 = u1 e {v2, u2} ∈E(Gvi)). Se todos os grafos da família G são isomorfos entre
si, escrevemos o produto lexicográfico entre H e a família G como H ◦G. Um exemplo de produto lexicográfico H ◦{G1, G2, G3} pode ser visto na figura
2, onde H = P3, G1 = K2, G2 = C4 e G3 = C3. 1
2
3
G1
G2
G3
(1, a) (1, b)
(3, a) (3, b) (3, c)
(2, a) (2, b) (2, c) (2, d)
K2
C3
C4
(b)
(a)
Figura 2: (a) Grafo P3 e (b) Produto lexicográfico P3 ◦{K2, C4, C3} 1
2
3
G1
G2
G3
(1, a) (1, b)
(3, a) (3, b) (3, c)
(2, a) (2, b) (2, c) (2, d)
K2
C3
C4
(b)
(a) (a) (b) Figura 2: (a) Grafo P3 e (b) Produto lexicográfico P3 ◦{K2, C4, C3} Para um subconjunto S de V (H ◦{G1, G2, . . . , G|V (H)|}), denotamos XH(S) =
{x ∈V (H) : ∃y ∈V (Gx)(x, y) ∈S} e XGx(S) = {y ∈V (Gx) : (x, y) ∈S} para
todo x ∈XH(S). No grafo da figura 2 (b), se S = {(1, a), (3, c)}, XH(S) = {1, 3},
XG1(S) = {a} e XG3(S) = {c}. Os conjuntos independentes maximais nos produtos lexicográficos de grafos po-
dem ser descritos como na Proposição 2.1. 78 Barbosa e Santana Barbosa e Santana Proposição 2.1. [17] Dados S ⊂V (H ◦(G1, . . . 3.
Grafos Zm-bem-cobertos Conforme a Cintura Pinter [16] provou que há somente 4 grafos fortemente bem-cobertos que são pla-
nares. Diferentemente, podemos construir infinitos grafos fortemente Zm-bem-
cobertos que sejam planares e de cintura 4, para l e m inteiros naturais, m ≥2, 79 Produtos de Grafos Zm-bem-cobertos através do produto (K1,ml+1) ◦2K1. Na figura 3. podemos observar a forma geral
destes grafos (a) e em (b) temos um grafo K1,3 ◦2K1 que é fortemente Z2-bem-
coberto planar. (b) K1 K1
2ml + 2
. . . (a)
(b)
Figura 3: (a) Grafo K1,ml+1 ◦2K1
(b) Grafo K1,3 ◦2K1 K1 K1
2ml + 2
. . . (a) (b) (a) Figura 3: (a) Grafo K1,ml+1 ◦2K1
(b) Grafo K1, (b) Grafo K1,3 ◦2K1 Proposição 3.2. Há um número infinito de grafos planares fortemente Zm-bem-
cobertos com cintura 4. Proposição 3.2. Há um número infinito de grafos planares fortemente Zm-bem-
cobertos com cintura 4. Demonstração. Sejam l e m inteiros naturais, m ≥2. Pela forma de construção,
os grafos (K1,ml+1) ◦2K1 são fortemente Zm-bem-cobertos, planares e com cintura
4. Os grafos Zm-bem-cobertos de cintura ≥6 caracterizados no Teorema 1.1,
com exceção de K1 e K2, não são 1-Zm-bem-cobertos e nem fortemente Zm-bem-
cobertos, e, portanto, os outros grafos com estas propriedades devem ter cintura ≤
5. Teorema 3.3. K1 e K2 são os únicos grafos conexos fortemente Zm-bem-cobertos
com cintura ≥6. Demonstração. Se G é um grafo Zm-bem-coberto com cintura ≥6, G atende ao
Teorema 1.1. C7 não é um grafo fortemente Zm-bem-coberto e K1 é. Suponha
que G seja diferente de C7 e K1. Logo, G é uma união finita de arbustos. Se G
consiste de apenas um talo com uma folha, G ∼= K2 que é um grafo fortemente
Zm-bem-coberto. Considere que G é diferente de C7, K1 e K2. Ao removermos
uma aresta que une uma folha x a um talo vi, teremos um componente H em G,
H = G −{x} com o talo vi e ml folhas, l ≥0, e então H não é Zm-bem-coberto e
portanto G também não é. o e Teorema 3.4. K1 e K2 são os únicos grafos conexos 1-Zm-bem-cobertos com cin-
tura ≥6. Demonstração. Seja G um grafo Zm-bem-coberto com cintura ≥6. Pelo Teorema
1.1, G é K1, C7 ou uma união finita de arbustos sendo que cada talo tem 1 (mod
m) folhas. Por inspeção, C7 não é 1-Zm-bem-coberto e K1 é 1-Zm-bem-coberto. Se
G consiste de apenas um talo com uma folha, G ∼= K2 que é um grafo 1-Zm-bem-
coberto. Suponha, então, que G é uma união finita de arbustos. Ao removermos 80 Barbosa e Santana uma folha x de um arbusto B qualquer com talo vi, este terá apenas ml folhas l ≥0
e não mais satisfaz ao Teorema 1.1. Portanto, G não é 1-Zm-bem-coberto. 4.
Produto Cartesiano O produto cartesiano G1 ×G2 de dois grafos G1 e G2 é o grafo contendo conjunto de
vértices V (G1 × G2) = V (G1)× V (G2), e dois vértices (v1, v2) e (u1, u2) de G1 × G2
são adjacentes se ou [(v1, u1) ∈E(G1) e v2 = u2] ou [(v2, u2) ∈E(G2) e v1 = u1]. Na figura 4. temos um grafo C3 × C4. Figura 4: Grafo C3 × C4 Figura 4: Grafo C3 × C4 Figura 4: Grafo C3 × C4 Uma questão sobre o produto cartesiano de dois grafos G e H é saber se é possí-
vel que G × H seja bem-coberto quando G e H não são bem-cobertos. Fradkin [11]
respondeu parcialmente esta questão para uma grande classe de grafos que inclui
todos os grafos não-bem-cobertos livres de triângulo. Topp e Volkman [17] apre-
sentaram alguns resultados sobre o produto cartesiano de alguns grafos, incluindo
os grafos bipartidos e ciclos. Teorema 4.5. [17] O produto cartesiano G1 × G2 de grafos G1 e G2 bipartidos
diferentes de K1 é bem-coberto se e somente se G1 = G2 = K2. Proposição 4.3. [17] O produto cartesiano Cn×Ck de ciclos Cn e Ck é bem-coberto
se e somente se n = 3 ou k = 3. O produto cartesiano de um ciclo Cn × K2 é um grafo cúbico que é Z2-bem-
coberto e todos os seus conjuntos independentes maximais têm cardinalidade par. Os grafos cúbicos Zm-bem-cobertos foram caracterizados por Barbosa e Ellingham
[6]. [ ]
Na Observação 1, temos uma forma de obter um conjunto independente maximal
em um grafo Cn × Ck. Observação 1. [17] Sejam Cn e Ck dois ciclos com vértices x1, x2, . . . , xn e y1,
y2, . . . , yk, respectivamente, e arestas x1x2, x2x3 . . . , xnx1 e y1y2, y2y3 . . . , yky1. Seja
In,k o conjunto dos vértices (xi, yj) de Cn × Ck tal que i = 1, . . . , 2 ⌊n/2⌋, j =
1, . . . , 2 ⌊k/2⌋, e i + j é um inteiro par. Se n e k são ambos ímpares, acrescente a
In,k o vértice (xn, yk). O conjunto In,k é independente maximal em Cn × Ck. Observação 1. [17] Sejam Cn e Ck dois ciclos com vértices x1, x2, . . . , xn e y1,
y2, . 4.
Produto Cartesiano Novamente, vamos considerar dois casos. Primeiro, considere que
k é ímpar. Podemos verificar que C3 × C3 não é 1-Zm-bem-coberto. Para k ≥5,
seja J1 = I3,k\{(x1, y1)} que é maximal em G\{(x1, y1)}. Agora, seja o conjunto
J2 = I3,k\{(x1, y1), (x2, y2), (x1, y3)} ∪{(x1, y2), (x2, y1), (x3, y3)}. J2 também é
maximal em G\{(x1, y1)}. J1 e J2 tem cardinalidades consecutivas. Considere, agora, que k é par. Sejam J1 = I3,k\{(x1, y1), } ∪{(x3, y1)} e
J2 = J1\{(x2, y4), (x3, y1)} ∪{(x3, y4)}. Logo, J2 e J1 são independentes maximais
em G\{(x1, y1)}, com cardinalidades consecutivas e, portanto, G não é Zm-bem-
coberto. 4.
Produto Cartesiano . . , yk, respectivamente, e arestas x1x2, x2x3 . . . , xnx1 e y1y2, y2y3 . . . , yky1. Seja
In,k o conjunto dos vértices (xi, yj) de Cn × Ck tal que i = 1, . . . , 2 ⌊n/2⌋, j =
1, . . . , 2 ⌊k/2⌋, e i + j é um inteiro par. Se n e k são ambos ímpares, acrescente a
In,k o vértice (xn, yk). O conjunto In,k é independente maximal em Cn × Ck. O produto cartesiano de dois ciclos Cn e Ck é bem-coberto se e somente se
n = 3 ou k = 3. Estes grafos possuem tanto C3 quanto C4 induzidos e estão numa 81 Produtos de Grafos Zm-bem-cobertos classe de grafos que ainda não foi caracterizada para grafos bem-cobertos e Zm-
bem-cobertos. Este resultado pode ser estendido para os grafos Zm-bem-cobertos,
como mostramos na Proposição 4.4. classe de grafos que ainda não foi caracterizada para grafos bem-cobertos e Zm-
bem-cobertos. Este resultado pode ser estendido para os grafos Zm-bem-cobertos,
como mostramos na Proposição 4.4. Proposição 4.4. O produto cartesiano Cn×Ck de ciclos Cn e Ck é Zm-bem-coberto
se e somente se ele é bem-coberto. Demonstração. O produto Cn × Ck, quando n = 3 ou k = 3 é bem-coberto pela
Proposição 4.3 e portanto, Zm-bem-coberto. Demonstração. O produto Cn × Ck, quando n = 3 ou k = 3 é bem-coberto pela
Proposição 4.3 e portanto, Zm-bem-coberto. É necessário considerar apenas n ou k ímpares, já que, pelo Teorema 4.5, quando
os grafos são ambos bipartidos, seu produto cartesiano não é bem-coberto e por-
tanto, não é Zm-bem-coberto. Restam, então, dois casos a considerar: se n e k são
ímpares e se apenas um deles é ímpar. É necessário considerar apenas n ou k ímpares, já que, pelo Teorema 4.5, quando
os grafos são ambos bipartidos, seu produto cartesiano não é bem-coberto e por-
tanto, não é Zm-bem-coberto. Restam, então, dois casos a considerar: se n e k são
ímpares e se apenas um deles é ímpar. Caso n e k sejam ímpares, podemos encontrar um conjunto independente maxi-
mal J1 = In,k\{(x1, y1), (x1, y3), (x2, y2)}∪{(x1, y2), (xn, y3)} em que |J1|=|In,k|−1
e portanto, |J1| e |In,k| não são congruentes módulo m, pois são consecutivos. 4.
Produto Cartesiano Caso n e k sejam ímpares, podemos encontrar um conjunto independente maxi-
mal J1 = In,k\{(x1, y1), (x1, y3), (x2, y2)}∪{(x1, y2), (xn, y3)} em que |J1|=|In,k|−1
e portanto, |J1| e |In,k| não são congruentes módulo m, pois são consecutivos. ,
Para o segundo caso, exatamente um de n e k é ímpar. Neste caso, como
Cn × Ck é isomorfo a Ck × Cn, vamos assumir que k é ímpar. Sejam J2 =
In,k\{(x1, y1), (x1, y3), (x2, y2), (xn, y2)} ∪{(x1, y2), (x1, yk)} e J3 = J2\{(xn−1, y3),
(xn, y4)} ∪{(xn, y3)}. Logo, J3 e J2 são independentes maximais em Cn × Ck com
cardinalidades consecutivas e, portanto, G não é Zm-bem-coberto. Para o segundo caso, exatamente um de n e k é ímpar. Neste caso, como
Cn × Ck é isomorfo a Ck × Cn, vamos assumir que k é ímpar. Sejam J2 =
In,k\{(x1, y1), (x1, y3), (x2, y2), (xn, y2)} ∪{(x1, y2), (x1, yk)} e J3 = J2\{(xn−1, y3),
(xn, y4)} ∪{(xn, y3)}. Logo, J3 e J2 são independentes maximais em Cn × Ck com
cardinalidades consecutivas e, portanto, G não é Zm-bem-coberto. Embora os grafos Cn × Ck Zm-bem-cobertos sejam também bem-cobertos, mos-
tramos na Proposição 4.5 que eles não são 1-Zm-bem-cobertos. Embora os grafos Cn × Ck Zm-bem-cobertos sejam também bem-cobertos, mos-
tramos na Proposição 4.5 que eles não são 1-Zm-bem-cobertos. Proposição 4.5. Se G é o produto cartesiano Cn × Ck de ciclos Cn e Ck, então
G não é 1-Zm-bem-coberto. Demonstração. Pela Proposição 4.4, Cn × Ck é Zm-bem-coberto se e somente se
n = 3 ou k = 3. Vamos mostrar que ao removermos um vértice deste grafo, o grafo
resultante não é Zm-bem-coberto. Considere o grafo G = C3 × Ck, já que ele é isomorfo a Ck × C3. Remova de G o
vértice (x1, y1). Novamente, vamos considerar dois casos. Primeiro, considere que
k é ímpar. Podemos verificar que C3 × C3 não é 1-Zm-bem-coberto. Para k ≥5,
seja J1 = I3,k\{(x1, y1)} que é maximal em G\{(x1, y1)}. Agora, seja o conjunto
J2 = I3,k\{(x1, y1), (x2, y2), (x1, y3)} ∪{(x1, y2), (x2, y1), (x3, y3)}. J2 também é
maximal em G\{(x1, y1)}. J1 e J2 tem cardinalidades consecutivas. Considere o grafo G = C3 × Ck, já que ele é isomorfo a Ck × C3. Remova de G o
vértice (x1, y1). 5.
Considerações Finais They are also the only ones with girth ≥6 and strongly Zm-well-covered. We show
a necessary and sufficient condition for the lexicographic product of graphs to be a
Zm-well-covered one and some properties for the cartesian product of cycles. Keywords. Graph theory, independent sets in graphs, graph products. Keywords. Graph theory, independent sets in graphs, graph products. 5.
Considerações Finais Apresentamos uma forma de construção de grafos Zm-bem-cobertos a partir do
produto lexicográfico de um grafo H e uma família de grafos Zm-bem-cobertos. Para os grafos bem-cobertos, o problema foi resolvido por Topp e Volkmann [17]. 82 Barbosa e Santana Há um número finito de grafos planares fortemente bem-cobertos, como provado
por Pinter [16]. Porém, mostramos que existe um número infinito de grafos planares
que são fortemente Zm-bem-cobertos que tem, ainda, cintura 4 (Proposição 3.2). (
)
Mostramos que um grafo 1-Zm-bem-coberto, diferente de K1 e K2 tem cintura ≤
5, porém ainda não é conhecida uma caracterização destes grafos. Especificamente
para os grafos com cintura 5, o problema é saber se existe um grafo 1-Zm-bem-
coberto que não seja 1-bem-coberto. Caso este grafo não exista, vale a conjectura
5.1. Outro problema é saber se há um grafo fortemente Zm-bem-coberto com cintura
5 que não seja fortemente bem-coberto (conjectura 5.1). Conjectura 5.1. Se G é um grafo 1-Zm-bem-coberto com cintura 5, então G é
1-bem-coberto. Conjectura 5.1. Se G é um grafo fortemente Zm-bem-coberto com cintura 5, então
G é fortemente bem-coberto. Se G é o produto cartesiano de dois ciclos Cn e Ck, mostramos que G é Zm-bem-
coberto se e somente se G é bem-coberto. Estes grafos não são 1-Zm-bem-cobertos. Abstract. A graph is Zm-well-covered if |I| ≡|J| (mod m), for all I, J maximal
independent sets in V (G). A graph G is strongly Zm-well-covered if G is a Zm-
well-covered graph and G\{e} is Zm-well-covered, ∀e ∈E(G). A graph G is 1-Zm-
well-covered if G is Zm-well-covered and G\{v} is Zm-well-covered, ∀v ∈V (G). We prove that K1 and K2 are the only 1-Zm-well-covered graphs with girth ≥6. They are also the only ones with girth ≥6 and strongly Zm-well-covered. We show
a necessary and sufficient condition for the lexicographic product of graphs to be a
Zm-well-covered one and some properties for the cartesian product of cycles. Abstract. A graph is Zm-well-covered if |I| ≡|J| (mod m), for all I, J maximal
independent sets in V (G). A graph G is strongly Zm-well-covered if G is a Zm-
well-covered graph and G\{e} is Zm-well-covered, ∀e ∈E(G). A graph G is 1-Zm-
well-covered if G is Zm-well-covered and G\{v} is Zm-well-covered, ∀v ∈V (G). We prove that K1 and K2 are the only 1-Zm-well-covered graphs with girth ≥6. Referências [1] M. Asté, F. Havet, C.L. Sales, Grundy number and products of graphs, Discrete
Mathematics, 310 (2010), 1482–1490. [2] R.M. Barbosa, On 1-Zm-well-covered graphs and strongly Zm-well-covered
graphs, Ars Combinatoria, 57 (2000), 225–232. [3] R.M. Barbosa, “Sobre Conjuntos Independentes Maximais em Grafos”, Tese de
Doutorado, COPPE-UFRJ, 1999. [4] R.M. Barbosa, M.N. Ellingham, A characterisation of cubic parity graphs,
Australasian Journal of Combinatorics, 28 (2003), 273–293. [5] R.M. Barbosa, B. Hartnell, Almost parity graphs and claw-free parity graphs,
J. Combin. Math. Combin. Comput., 27 (1998), 117–122. [6] R.M. Barbosa, B. Hartnell, Characterization of Zm-well-covered graphs for
some classes of graphs, Discrete Mathematics, 233 (2001), 293–297. Produtos de Grafos Zm-bem-cobertos 83 [7] J.A. Bondy, U.S.R. Murty, “Graph Theory”, Graduate Texts in Mathematics,
Springer, 2008. [8] Y. Caro, Subdivisions, parity and well-covered graphs, J. Graph Theory, 25
(1997), 85–94. [9] Y. Caro, M. Ellingham, J. Ramey, Local structure when all maximal inde-
pendent sets have equal weight, SIAM J. Discrete Mathematics, 11 (1998),
644–654. [10] Y. Caro, B. Hartnell, A Characterization of Zm-well-covered graphs of girth 6
or more, J. Graph Theory, 33 (2000), 246–255. [11] A.O. Fradkin, On the well-coveredness of Cartesian products of graphs, Dis-
crete Mathematics, 309 (2009), 238–246. [12] R. Hammack, W. Imrich, S. Klavzar “Handbook of Product Graphs”, Second
Edition, CRC Press, 2011. [13] B. Hartnell, Well-covered graphs, J. Combin. Math. Combin. Comput., 29
(1999), 107–115. [14] R.M. Karp, Reducibility among combinatorial problems, em “Complexity of
Computer Computations” (Yorktown Heights), pp. 85-104, Nova York, 1972. [15] R.J. Nowakowski, K. Seyffarth, Small cycle double covers of products I: Lexi-
cographic product with paths and cycles, J. Graph Theory, 57 (2008), 99–123. [16] M.R. Pinter, Strongly well-covered graphs, Discrete Mathematics, 132 (1994),
231–246. [17] J. Topp, L. Volkmann, On the well coveredness of Products of Graphs, Ars
Combinatoria, 33 (1992), 199–215. 84
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English
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The use of endoluminal techniques in the revision of primary bariatric surgery procedures: a systematic review
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Surgical endoscopy/Surgical endoscopy and other interventional techniques
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cc-by
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The use of endoluminal techniques in the revision of primary bariatric
surgery procedures: a systematic review Yan Mei Goh1,2 · Nicole Ellen James1 · En Lin Goh1,3 · Achal Khanna2 Received: 16 November 2019 / Accepted: 19 February 2020 / Published online: 28 February 2020
© The Author(s) 2020, corrected publication 2020 * Yan Mei Goh
yanmei.goh@doctors.org.uk and Other Intervent and Other Intervent Surgical Endoscopy (2020) 34:2410–2428
https://doi.org/10.1007/s00464-020-07468-w REVIEW ARTICLE Abstract Background Weight regain following primary bariatric surgery is attributed to anatomical, behavioural and hormonal fac-
tors. Dilation of the gastrojejunal anastomosis is a possible cause of weight regain after roux-en-Y gastric bypass (RYGB). However, surgical revision has significant risks with limited benefits. Endoluminal procedures have been suggested to manage
weight regain post-surgery. This systematic review aims to assess efficacy of endoluminal procedures. Methods Studies where endoluminal procedures were performed following primary bariatric surgery were identified Main Background Weight regain following primary bariatric surgery is attributed to anatomical, behavioural and hormonal fac-
tors. Dilation of the gastrojejunal anastomosis is a possible cause of weight regain after roux-en-Y gastric bypass (RYGB). However, surgical revision has significant risks with limited benefits. Endoluminal procedures have been suggested to manage
weight regain post-surgery. This systematic review aims to assess efficacy of endoluminal procedures. Methods Studies where endoluminal procedures were performed following primary bariatric surgery were identified. Main
outcome measures were mean weight loss pre- and post-procedure, excess weight loss, recurrence rates, success rates and
post procedure complications fi
Methods Studies where endoluminal procedures were performed following primary bariatric surgery were identified. Main
outcome measures were mean weight loss pre- and post-procedure, excess weight loss, recurrence rates, success rates and
post-procedure complications.i Results Twenty-six studies were included in this review. Procedures identified were (i) endoluminal plication devices (ii)
other techniques e.g. sclerotherapy, mucosal ablation, and Argon Plasma Coagulation (APC) and (iii) combination therapy
involving sclerotherapy/mucosal ablation/APC and endoscopic OverStitch device. Endoluminal plication devices show great-
est initial weight loss within 12 months post-procedure, but not sustained at 18 months. Only one study utilising sclerotherapy
showed greater sustained weight loss with peak EWL (19.9%) at 18 months follow-up. Combination therapy showed the
greatest sustained EWL (36.4%) at 18 months. Endoluminal plication devices were more successfully performed in 91.8%
of patients and had lower recurrence rates (5.02%) compared to sclerotherapy and APC, with 46.8% success and 21.5%
recurrence rates. Both procedures demonstrate no major complications and low rates of moderate complications. Only mild
complications were noted for combination therapy.fi Conclusions The paucity of good quality data limits our ability to demonstrate and support the long-term efficacy of endo-
luminal techniques in the management of weight regain following primary bariatric surgery. Future work is necessary to
not only clarify the role of endoluminal plication devices, but also combination therapy in the management of weight regain
following primary bariatric surgery. 2
Department of General Surgery, Milton Keynes University
Hospital, Milton Keynes, UK 1
Imperial College London, London, UK 3
Oxford University Hospitals NHS Foundation Trust, Oxford,
UK Abstract Non-surgical management of
weight regain following bariatric surgery requires the input
of the multidisciplinary team. Despite this, a proportion of
patients still experience weight regain following bariatric
surgery [9]. Dilatation of the gastrojejunal anastomosis or
the gastric pouch is a well-recognised post-operative occur-
rence. On the basis that gastric pouch size, distension and
transit time following RYGB is a surgical mechanism for
early satiety and weight loss, this post-operative event may
reduce the restrictive and malabsorptive effects of RYGB. Surgical revision of the gastrojejunal anastomosis is contro-
versial as most patients are exposed to major post-operative
complications, higher readmission rates and morbidity [9]
but do not achieve significant weight reduction [10, 11]. Data extraction and outcome measures Two independent reviewers (YMG, NEJ) screened all titles
and abstract manually for inclusion. A third reviewer (ELG)
was consulted in the case of a disagreement. Relevant data
were entered into Review manager 5.4 (Cochrane Collabo-
ration, Oxford, United Kingdom). The following data items
were extracted: year of publication, country of origin, study
design, number of participants, type of primary procedure,
type of endoluminal procedure performed, patient demo-
graphics, mean time since initial procedure, selection crite-
ria in each study, mean pre-revision weight and BMI, mean
weight loss post-procedure, complications post-procedure,
average length of procedure, average stoma diameter at the
end of the procedure, excess weight loss, length of follow-up
and number of successful endotherapy. Literature search The following databases were searched: (a) Medline
(1946—present) via OvidSP, (b) MEDLINE Epub ahead of
print, in process and other non-indexed citations (latest issue
via Ovid SP, last search 19th July 2019); (c) Ovid Embase
(1947—19th July 2019). Additionally, all references of
included articles were manually reviewed to identify addi-
tional studies. Three strings were utilised; these terms were
“bariatric surg*.mp. OR metabolic surg*.mp. OR weight
loss surg*.mp.”, “revision*”.mp., “endoscopic procedure. mp. OR endosco*.mp.” and truncated search terms using
wild card character and “related articles” function were used
to broaden search. The references of included articles were
also hand-searched to identify any additional studies. i
Hence, endoluminal revision procedures have been devel-
oped to address this gap. These techniques come in various
forms: endoluminal plication devices and other techniques
like sclerotherapy, mucosal ablation and argon plasma
coagulation. Endoluminal plication devices work by taking
superficial or full-thickness bites of the intraluminal pouch
mucosa or at the gastrojejunal anastomosis. Sutures or clips
are then deployed via endoscope. Meanwhile, other tech-
niques like sclerotherapy, mucosal ablation and APC induce
scarring at the gastrojejunal anastomosis thus reducing its
size. As the number of patients undergoing bariatric surgery
continues to grow, the need to consider endoluminal revision
procedures becomes increasingly important. Thus, this sys-
tematic review aims to assess efficacy of endoluminal tech-
niques that attempt to revise primary bariatric procedures. Criteria for considering studies for this review Studies were appraised for rigorousness in methodology
using the Newcastle–Ottawa Quality Assessment Scale
[12] and risk of bias assessed using the National Institute
of Health (NIH) Quality Assessment Tool for Case Series
Studies [13]. All published studies that utilised endoluminal or endo-
scopic techniques following primary bariatric surgery were
evaluated. Inclusion criteria are as follows: (a) studies inves-
tigating patients who had undergone endoscopic procedures
following a primary bariatric surgery procedure (b) weight
regain after surgery (c) presence or recurrence of comor-
bidities (d) post-operative complications (e) presence of
anatomical cause for weight regain. Exclusion criteria are
as follows: studies that did not include revision surgery,
endoluminal procedures used in the management of com-
plications following primary surgery, articles that assess
primary bariatric surgery, non-endoluminal interventions,
review articles, studies not written in the English language,
animal studies, comment, opinions or letters, case reports Abstract Keywords Endoluminal techniques · Revision surgery · Bariatric surgery The role of bariatric surgery has grown significantly over the
past decade, with an additional 10,000 procedures performed per year from 2011 to 2015, and an increase of 20,000 proce-
dures from 2015 to 2016 in the USA [1]. In particular, gastric
bypass, sleeve gastrectomy, adjustable gastric banding and
biliopancreatic diversion with duodenal switch are frequently
performed. These procedures are associated with significant
long-term weight loss as well as alterations in gut hormone
production and metabolism that suppress appetite and pro-
mote satiety. However, weight regain following primary bari-
atric surgery remains an ongoing problem. It is estimated that
clinically significant weight regain occurs in up to one-third
of patients [2–6] who have undergone a Roux-en-Y gastric
bypass (RYGB) or vertical banded gastroplasty (VBG). Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00464-020-07468-w) contains
supplementary material, which is available to authorized users. There are several factors that weight regain post-RYGB
can be attributed to; notably a combination of lifestyle, 3
Oxford University Hospitals NHS Foundation Trust, Oxford,
UK :.(123456789
3 Surgical Endoscopy (2020) 34:2410–2428 2411 and technical articles with no evidence of patient follow-up
post-procedure, and conference abstracts. mental health, hormonal/metabolic and surgical factors. Thus, the need to understand and address these issues with
patients in the pre- and post-operative stage is crucial in pre-
venting the reemergence of obesity related comorbidities and
impaired quality of life [7, 8]. Non-surgical management of
weight regain following bariatric surgery requires the input
of the multidisciplinary team. Despite this, a proportion of
patients still experience weight regain following bariatric
surgery [9]. Dilatation of the gastrojejunal anastomosis or
the gastric pouch is a well-recognised post-operative occur-
rence. On the basis that gastric pouch size, distension and
transit time following RYGB is a surgical mechanism for
early satiety and weight loss, this post-operative event may
reduce the restrictive and malabsorptive effects of RYGB. Surgical revision of the gastrojejunal anastomosis is contro-
versial as most patients are exposed to major post-operative
complications, higher readmission rates and morbidity [9]
but do not achieve significant weight reduction [10, 11]. mental health, hormonal/metabolic and surgical factors. Thus, the need to understand and address these issues with
patients in the pre- and post-operative stage is crucial in pre-
venting the reemergence of obesity related comorbidities and
impaired quality of life [7, 8]. Results Twenty-six studies comprising a total of 1835 patients who
had undergone endoluminal procedure following initial pri-
mary bariatric procedure were included in this study (Fig. 1). Endoluminal plication devices were used in 1087 patients,
other techniques in 721 patients, and a combination of the
two types of procedures in 27 patients. All studies were 1 3 1 3 3 2412 Surgical Endoscopy (2020) 34:2410–2428 published over a period of twelve years from 2007 to 2019. There were eight prospective case series and one prospec-
tive multicentre randomised control trial. Of the 26 studies,
19 were performed in USA, one in Brazil, two in centres
located in USA and Brazil, one in Belgium, one in France
and one in Canada. Mean age of patients included in the
review was 51.5 years old (range 22.0–71.4 years). The
mean time since initial bariatric procedure was 86.7 months
(range 12.0–222 months) (Table 1).i • BMI 30–60 kg/m2 greater than six months after RYGB
[21] published over a period of twelve years from 2007 to 2019. There were eight prospective case series and one prospec-
tive multicentre randomised control trial. Of the 26 studies,
19 were performed in USA, one in Brazil, two in centres
located in USA and Brazil, one in Belgium, one in France
and one in Canada. Mean age of patients included in the
review was 51.5 years old (range 22.0–71.4 years). The
mean time since initial bariatric procedure was 86.7 months
(range 12.0–222 months) (Table 1). • BMI 30–60 kg/m2 greater than six months after RYGB
[21] • BMI 30–60 kg/m2 greater than six months after RYGB
[21] One study [38] did not detail the inclusion nor exclusion
criteria in patient selection. Endoluminal bariatric procedures The endoluminal procedures identified were (i) endolumi-
nal plication devices e.g. StomaphyX™, Restorative Obesity
Surgery Endoluminal (ROSE) procedure, Incisionless Oper-
ating Platform (IOP), Over-The-Scope Clip (OTSC-Clip),
e.g. sutured Transoral Outlet Reduction (TORe), Endoscopic
Overstitch device and Endoscopic Gastrojejunal Revision
(EJGR) and (ii) other techniques e.g. sclerotherapy, mucosal
ablation, and Argon Plasma Coagulation (APC). Initial
bariatric procedures performed were the roux-en-Y gastric
bypass (RYGB), transected vertical gastric bypass (TVGB),
vertical banded gastroplasty (VBG) and laparoscopic sleeve
gastrectomy (LSG) (Tables 1,2). Twenty-five of the 26 included studies had clear selection
criteria for all patients included in their study. These are as
follows: • Greater than 18 months following initial bariatric proce-
dure [14–20]fi • Weight regain or failure to lose sufficient weight [17,
19–35] • Aged between 18 to 65 years old [16, 18, 32] • Decreased satiety [19, 29, 30] • Dilated gastrojejunal anastomosis and gastric pouch
[19–21, 25, 30, 32, 33, 36, 37] • Reappearing comorbidities [27] Fig. 1 PRISMA chart of the
study selection process
Records idenfied through
EMBASE (n = 486)
Screening
Included
Eligibility
Idenficaon
Records aer duplicates removed
(n = 511)
Records screened
(n = 511)
Records excluded:
Non-English (n = 7)
Conference abstracts (n = 156)
Reviews (n = 51)
Arcles assessing primary
bariatric surgery (n = 64)
Non-endoluminal revision
surgery
(n = 103)
Management of complicaons
Full-text arcles
assessed for eligibility
(n = 34)
Full-text arcles excluded due
to duplicate paent cohort or
on further assessment did not
meet inclusion criteria (n = 8)
Studies included in
qualitave synthesis
(n = 26)
Records idenfied through
Ovid Medline
(n = 128) Fig. 1 PRISMA chart of the
study selection process Idenficaon Records idenfied through
Ovid Medline
(n = 128) Records idenfied through
EMBASE (n = 486) Records aer duplicates removed
(n = 511) Records excluded:
Non-English (n = 7)
Conference abstracts (n = 156)
Reviews (n = 51)
Arcles assessing primary
bariatric surgery (n = 64)
Non-endoluminal revision
surgery
(n = 103)
Management of complicaons Full-text arcles
assessed for eligibility
(n = 34) Studies included in
qualitave synthesis
(n = 26) 1 3 Surgical Endoscopy (2020) 34:2410–2428 2413 1
Table 1 Patient and study demographics
Study
Country
Study type
No
Primary
Operation
Procedure
M:F
Mean age
(years)
Mean time
since initial
procedure
(months)
Selection criteria
Mean pre-
revision weight
(kg)
Mean pre-
revision
BMI (kg/m2)
Mikami et al. Endoluminal bariatric procedures [14]
USA
Retro case
series
39
RYGB
StomaphyX™3:36
47.8 (29–64)
> 2 years post-
op > 10% of
nadir weight
108 (65.9–
172.2)
39.8 (22.7–
63.2)
Manouchehri
et al. [26]
Canada
Pros case
series
14
Vertical
Banded
Gastro-
plasty
StomaphyX™1:13
47.3 ± 7.9
116.4 ± 73.2
Persistent WG
119.5 ± 25.9
43.4 ± 9.7
Ong’Uti
et al. [15]
USA
Retro case
series
27
RYGB
StomaphyX™2:25
49 (44–54)
72 (60–96)
> 2 years post-op
103 (88.5–
115)**
37 (32–40)
Goyal et al. (39)
USA
Retro case
series
55
RYGB
StomaphyX™1:53
49.6 (30–68)
68.4 (12–156)
96.6
36.1
Mullady
et al. [29]
USA
Pros case
series
20
RYGB
ROSE
1:19
48 (36–62)
63 (24–117)
WR/ no WL,
satiety
36.7 (28,4–
48.8)
Horgan et al. [18]
USA
Pros case
series
116
RYGB
ROSE via
Incisionless
Operating
Platform
(IOP)
15:101
45.6 ± 8.7
> 2yrs post-
op, > 50% EWL
after RYGB
110.8 ± 20.5
39.9 ± 6.7
Ryou et al. [30]
USA
Pros case
series
5
RYGB
ROSE
0:5
48 (41–55)
56 (24–76)
WR, satiety,
dilated pouch/
GJA
100.4
36.3
Gallo et al. [17]
USA
Retro case
series
27
RYGB
ROSE
2:25
49.2 ± 9.6
(26–68)
142.8 ± 51.6
> 50% EWL, sig
WG 2 years
post-op
106.2 ± 21.2**
40.6 (30–67)
Buttelmann
et al. [31]
USA
Retro case
series
8
RYGB
ROSE
48
Inadequate/
failure to lose
weight
43.7
Thompson
et al. [18]
USA
Retro case
series
116
RYGB
IOP
15:101
46 ± 9
> 2yrs after
RYGB
110.5 ± 20.5
39.9 ± 6.7
Heylen et al. [27]
Belgium
Pros case
series
46
TVGB
OTSC-clip
19:75
> 10% WG
2yrs post-op,
reappearing
comorbidi-
ties, volume/
frequency of
meals
32.8 1 Surgical Endoscopy (2020) 34:2410–2428 2414 414
Surgical Endoscopy (2020) 34:2410 2
1 3
Table 1 (continued)
Study
Country
Study type
No
Primary
Operation
Procedure
M:F
Mean age
(years)
Mean time
since initial
procedure
(months)
Selection criteria
Mean pre-
revision weight
(kg)
Mean pre-
revision
BMI (kg/m2)
Patel et al. [32]
USA
Retro case
series
50
RYGB
EGJR
IST- 2:32
IST
48.6 ± 10.3
IST
115.2 ± 39.6
WR > 2yrs,
stoma dila-
tion > 15 mm
IST
114.5 ± 20.5
IST 41/7 ± 6/4
PST- 2:14
PST
55.8 ± 10.8
PST 114 ± 42
PST
110.2 ± 22.6
PST 40.7 ± 8.7
Tsai et al. Endoluminal bariatric procedures [22]
Switzerland
Retro case
series
81
RYGB
EGJR
(OverStitch
device)
22:59
48.0 (26.8–
71.4)
84 (12–222)
> 15 kg increase
from nadir
weight, 10 kg
increase within
6–12 months
post-op
127.1 (96–225)
44.7 (35.3–67)
Catalano
et al. [33]
USA
Retro case
series
28
RYGB
Sclerotherapy
(sodium
morrhuate)
10:18
41.1 (27–58)
Stoma
size > 1.2 cm,
WR after
RYGB
112
Loewen and
Barba [34]
USA
Retro case
series
71
RYGB
Sclerotherapy
(sodium
morrhuate)
4:67
45 (30–64)
34.8 (34.8–66
> 5% WG,
inadequate
WL < 50%
EWL
98.1 ± 21.6**
35.5
Jirapinyo
et al. [24]
USA
Pros case
series
43
RYGB
Sclerotherapy
(sodium
morrhuate)
(34)
3:31
47 ± 9
72 ± 60
> 1 yr post-
op > 20% of
nadir weight
TORe (9)
1:8
47 ± 13
84 ± 48
Thompson
et al. [21]
USA
Pros, multi-
centre RCT
50
RYGB
TORe
3:47
47.6 ± 9.46
58
BMI 30–60
at > 6 months
post-op,
Inadequate
WL > 50%
EWL/
WR > 5% EWL,
GJA > 2 cm
101.5 ± 16.4
37.6 ± 4.9
de Moura
et al. [39]
USA
Retro case
report
1
RYGB
TORe
0:1
55
144
73.35**
27.9
Kumar and
Thompson
[37]
USA
Retro case
series
59
RYGB
ST TORe
3:56
48.8 ± 1.1
Stoma diam-
eter > 20 mm
40.4 ± 1.0
59
FT TORe
15:44
49.9 ± 1.3
41.1 ± 1.3
Kumar and
Thompson
[36]
USA
Pros case
series
150
RYGB
TORe
(OverStitch
device)
27:123
51.2 ± 0.8
103.2 ± 3.6
GJA > 15 mm
110.7 ± 2.2
40.1 ± 0.7 Surgical Endoscopy (2020) 34:2410–2428 2415 1 3
Table 1 (continued)
Study
Country
Study type
No
Primary
Operation
Procedure
M:F
Mean age
(years)
Mean time
since initial
procedure
(months)
Selection criteria
Mean pre-
revision weight
(kg)
Mean pre-
revision
BMI (kg/m2)
Jirapinyo
et al. [25]
USA
Retro case
series
25
RYGB
TORe
(OverStitch
device)
7:18
48 (34–69)
72 (24–120)
WR,
GJA > 15 mm
43
Vargas et al. [23]
USA, Brazil
Retro case
series
130
RYGB
TORe
(OverStitch
device)
16:114
47.12 ± 8.55
100.8 ± 57.4
WR
36.8 ± 6.84
Baretta et al. [20]
Brazil
Pros case
series
30
RYGB
APC
4:26
42.83
(22–59)
> 18 months
post-op, regain
of > 10% of
nadir weight,
stoma diam-
eter > 15 mm
121.77 ± 22.50
45.63 ± 7.63
Moon et al. Endoluminal bariatric procedures [19]
USA, Brazil
Retro case
series
558
RYGB
APC
103:455
40.9 ± 9.5
90 (60, 120)*
> 18 months
post-op, regain
of > 10% of
nadir weight,
satiety,
size of GJ
stoma > 15 mm
94.5 ± 18.6
34.0 ± 5.7
Riva et al. [35]
France
Retro case
series
22
RYGB
Mucosal abla-
tion + endo-
scopic
suturing
(OverStitch
device) (11)
5:17
52.2 ± 11.7
106.8 ± 99.6
Sig WG > 50%
104.3 ± 27.4
42.4 ± 9.05
Mucosal abla-
tion + endo-
scopic
sutur-
ing + scle-
rotherapy
(OverStitch
device) (11)
100.3 ± 27.0
42.4 ± 10.4
Eid [28]
USA
Retro case
series
5
LSG
APC + endo-
scopic
suturing
(OverStitch
device)
4:1
59.2 (48–63)
37.4 (32.2–
48.2)
WR
110.25 (85.05–
130.50)**
37 1 3 Surgical Endoscopy (2020) 34:2410–2428 2416 416
Surgical Endoscopy (2020) 34:2410–2428
ab e
(co t ued)
Study
Mean post-surgical weight (kg)
Mean post-revision BMI (kg/m2)
Complication
post-procedure
3 months
6 months
1 year
2 years
3 years
3 months
6 months
1 year
2 years
3 years
Mikami et al. [14]
101.3
99.3
98
Minor: sore
throat (87.1),
epigastric pain
(76.9)
Manouchehri
et al. [26]
109.6 ± 24.4
(4 months)
39.8 ± 9.1
(4 months)
Minor headache,
back pain
Ong’Uti et al. [15]
101.3
94.5
93.9
(81.6 ± 102)**
33 (29–36)
(0 months)
Goyal et al. (39)
92.9
(1 month)
92.8
94.9
Nil
Mullady et al. [29]
Minor: abdomi-
nal bloating,
mild sore
throats
Horgan et al. [18]
103
Mild: pharyh-
gitis 48 (41),
Nausea/vomit-
ing 14 (12),
Abdo pain 13
(11) Moderate:
superficial dis-
tal oesophagus
tear 3(2.9)
Ryou et al. [30]
92.6
33.4
Nil
Gallo et al. [17]
39.2 ± 7
39.9 ± 10.1
37.7 ± 6.3
Nil
Buttelmann
et al. [31]
40.6
40.7
39
38.9
Nil
Thompson
et al. [18]
104.6
Heylen et al. [27]
29.7
27.4
Mild: sore
throat Moder-
ate: 5(10.9)
dysphagia
(repeat OGD),
2 persistent
dysphagia had
endoscopic
dilatation) 416
Surgical Endoscopy (2020) 3
(
)
Study
Mean post-surgical weight (kg)
Mean post-revision BMI (kg/m2)
Complication
post-procedure
3 months
6 months
1 year
2 years
3 years
3 months
6 months
1 year
2 years
3 years
Mikami et al. [14]
101.3
99.3
98
Minor: sore
throat (87.1),
epigastric pain
(76.9)
Manouchehri
et al. [26]
109.6 ± 24.4
(4 months)
39.8 ± 9.1
(4 months)
Minor headache,
back pain
Ong’Uti et al. Endoluminal bariatric procedures [15]
101.3
94.5
93.9
(81.6 ± 102)**
33 (29–36)
(0 months)
Goyal et al. (39)
92.9
(1 month)
92.8
94.9
Nil
Mullady et al. [29]
Minor: abdomi-
nal bloating,
mild sore
throats
Horgan et al. [18]
103
Mild: pharyh-
gitis 48 (41),
Nausea/vomit-
ing 14 (12),
Abdo pain 13
(11) Moderate:
superficial dis-
tal oesophagus
tear 3(2.9)
Ryou et al. [30]
92.6
33.4
Nil
Gallo et al. [17]
39.2 ± 7
39.9 ± 10.1
37.7 ± 6.3
Nil
Buttelmann
et al. [31]
40.6
40.7
39
38.9
Nil
Thompson
et al. [18]
104.6
Heylen et al. [27]
29.7
27.4
Mild: sore
throat Moder-
ate: 5(10.9)
dysphagia
(repeat OGD),
2 persistent
d
h
i h d 104.6 Ryou et al. [30]
92.6
Gallo et al. [17]
Buttelmann
et al. [31]
Thompson
et al. [18]
Heylen et al. [27] Ryou et al. [30]
92.6
Gallo et al. [17]
Buttelmann
et al. [31]
Thompson
et al. [18]
Heylen et al. [27] 3 2417 Surgical Endoscopy (2020) 34:2410–2428 urgical Endoscopy (2020) 34:2410–2428
ued)
Mean post-surgical weight (kg)
Mean post-revision BMI (kg/m2)
Complication
post-procedure
3 months
6 months
1 year
2 years
3 years
3 months
6 months
1 year
2 years
3 years
122.6
121.1
119.1
Nil
92.1 (18 month)
Mild: 21 (75)
post-injection
pain. Complications Six studies reported no complications following proce-
dures involving endoluminal plication devices [17, 22, 30,
31, 38, 39]. Minor complications reported were abdominal
pain (22.5%), sore throat (49.4%), device failure (3.1%),
nausea and vomiting (11.0%). A greater range of moderate
complications was reported. Specifically, 9.02% of patients
reported moderate complications of mucosal tear or dam-
age, 4% reported haematemesis, 2.7% reported bleeding and
10.9% had dysphagia following endoluminal plication. No
major complications were reported by any study utilising
endoluminal plication devices. There were two studies in which endoluminal procedures
were performed in patients following VBG and TVGB,
respectively [26, 27]. One study utilised the StomaphyX™
for revision of VBG [26]. The authors demonstrated a weight
reduction of 9.9 kg at four months post-revisional procedure,
with a decrease in BMI of 3.6 kg/m2 (8.28% weight loss)
over the same time period [26]. The other study reported a
mean decrease in BMI of 3.1 kg/m2 (9.45% weight loss) fol-
lowing the use of the OTSC-clip at 3 months post-revisional
procedure in a group of TVGB patients [27]. This was sus-
tained at 7.01% at 12 months post-revisional procedure. On
review of both papers, neither study had reported the EWL
following endoluminal revision surgery. Studies utilising other techniques of sclerotherapy and
APC reported minor complication of post-injection pain,
abdominal pain and nausea (60%) and moderate complica-
tion of mucosal ulceration (35.7%). No major complications
were reported. 0
4
8
12
16
20
24
28
32
36
40
0
5
10
15
20
25
30
35
40
Months
% Excess
Weight
Loss
Endoluminal Plication
Device
Sclerotherapy and Argon
Plasma Coagulation
Fig. 2 Graph demonstrating percentage EWL over time in endolumi-
nal plication devices and others (sclerotherapy and APC) All endoluminal plication devices post-RYGB showed a
mean overall decrease in EWL over the first three months
of 13.9% [14–18, 21–25, 29–32, 36–39]. This EWL was
sustained at 13.7% at the 12-month follow-up (Fig. 2). Fol-
lowing this, the percentage EWL after 12 months post-pro-
cedure is demonstrated to show a steady decline to 8.5%
36 months post-procedure. Endoluminal plication devices
were shown to be successful in 91.8% of patients in studies
which provided data. Definitions of success in the various
procedures are outlined where data are available (Table 2). Weight loss Meanwhile, the other techniques used post-RYGB i.e. sclerotherapy and APC showed a much lower weight loss
compared to endoscopic plication devices with a 3.87%
EWL three months post-procedure [19, 20, 24, 33, 34]. A 19.9% EWL at 18 months post-procedure is reported in
Catalano et al.’s study utilising sclerotherapy [33], which
is greater than the EWL (13.0%) in endoluminal plication
devices. Sclerotherapy and APC were shown to be less suc-
cessful in 46.8% of patients when compared to utilisation
of endoluminal plication devices (91.8%), and had higher
recurrence rates (21.5%). Excess weight is defined as the difference between the
patient’s actual weight and ideal weight. The percentage
excess weight loss (EWL) is defined as the proportion of
weight loss after endoluminal procedures divided by the dif-
ference of regained weight from nadir weight. Revision surgery using endoluminal plication devices
were performed in 18 studies post-RYGB [14–18, 21–25,
29–32, 36–39]. Results of these studies were analysed
together. Of these, Stomaphyx™ was performed in three
studies [14, 15, 38], ROSE in five studies [16, 17, 29–31],
IOP in one study [18], TORe in seven studies [21, 23–25,
36, 39] and EJGR in two studies [22, 32]. These procedures
were performed a mean of 91.2 months (12.0–222 months)
after RYGB. Mean pre-revision weight was 105.6 kg
(65.9–225 kg). Mean weight loss (6.27 kg) was greatest
within the first 3 months post-procedure. This weight loss
was sustained for up to two years after the revision endolu-
minal procedure. Post-procedure BMI within the first three
months after the revision procedure had decreased by a mean
of 7.61%, but there are insufficient data to comment on mean
post-procedure BMI after two years. Mean EWL was sus-
tained at 19.3% six months following the initial procedure. However, this was not maintained in patients two years post-
procedure (EWL 10.3%). Endoluminal bariatric procedures Moderate:
10 (35.7) shal-
low circumfer-
ential ulcers at
stoma
Pain 1, heartburn
1, hypertensive
urgency 1,
bleeding 1
Pain 2
95.1 ± 15.22
Mod: gastric
mucosal tear,
pulmonary
oedema
55.8**
49.5**
21.2**
18.8**
Nil
Bleeding 1
Bleeding 1
101.1
100.1
100.2
90.7
91.5
36.6
36.3
36.3
36.8
36.7
Pain 6 (4.0),
bleeding 5
(3.3), nausea 3
(2.0)
Hematemesis
1, delayed GI
bleeding 1,
nausea 4,
severe emesis
with torn
stitches 2, ste-
nosis of GJA 1 36.7 1 Surgical Endoscopy (2020) 34:2410–2428 2418 g
)
Mean post-surgical weight (kg)
Mean post-revision BMI (kg/m2)
Complication
post-procedure
3 months
6 months
1 year
2 years
3 years
3 months
6 months
1 year
2 years
3 years
Nausea 18 (14),
Pain 23 (18),
Oesophageal
tear requiring
endoscopic
clipping 1
(< 1), balloon
dilation of
narrowed GJA
after TORe
5 (4)
83.29
(4 months)
78.87
31.14 ± 5.81
Severe stenosis
(stoma diam-
eter < 3 mm) 2,
ulcers at stoma
10
Stenosis 9,
GJ ulcer 3,
vomiting 3,
GJ leakage 2,
melena 1
36
34
Minor: nausea
and mild
abdominal pain
(44)
100.08
(75.15–
121.5)**
98.1 (72.9–
119.25)**
99.09 (74.25–
119.7)**
33.64
(26.7–44.9)
value, interquartile range, ** conversion from lb to kg (1 lb = 0.45)
ight loss, ROSE restorative obesity surgery, endoluminal (ROSE) procedure, EGJR endoscopic gastrojejunal revision, RYGB Roux-en-Y gastric bypass, LSG laparoscopic sleeve
TSC-clip over-the-scope clip, IOP incisionless operating platform, TORe sutured transoral outlet reduction, ST superficial-thickness, FT full-thickness, APC argon plasma coagu-
spective, retro: retrospective, GJA gastrojejunal anastomosis, WR weight regain, WL weight loss, WG weight gain, EWL excess weight loss Study
Mean post-surgical weight (kg)
Mean post-revision BMI (kg/m2)
Complication
post-procedure
3 months
6 months
1 year
2 years
3 years
3 months
6 months
1 year
2 years
3 years 31.14 ± 5.81 1 3 Surgical Endoscopy (2020) 34:2410–2428 2419 30, 38, 39]. Recurrence rates and need for further procedure
following endoluminal plication devices were 5.02%. Complications These include the ability to reduce the diameter of the gas-
trojejunal stoma and pouch length [16, 18, 21, 23, 25, 29,
30, 38], as well as weight loss post-procedure [16, 18, 29, Endoluminal Plication
Device Fig. 2 Graph demonstrating percentage EWL over time in endolumi-
nal plication devices and others (sclerotherapy and APC) 1 3 Surgical Endoscopy (2020) 34:2410–2428 2420 1 3
yp
(
)
Study
Procedure
Combination
therapy (Y/N)
Av. procedure
length (mins)
Av. stoma
diameter at end
of procedure
(mm)
% Excess weight loss
1 week
1 month
2 months
3 months
6 months
12 months
Mikami [14]
StomaphyX™
N
35 (16–62)
(2 weeks)
7.4
10.6
13.1
13.1
17.0
19.5
Ong-Uti [15]
StomaphyX™
N
20 (20–30)
(2 weeks)
24
33
47
20
Goyal [38]
StomaphyX™
N
24.1 (10–55)
12.8
7.3 ± 7.1
11.6 ± 12.1
11.5 (17.9)
Mullady et al. [29]
ROSE
N
103 (50–154)
5.8 kg*
8.8 kg**
Horgan et al. [16]
IOP
N
87
11.5
21.5 ± 15.3
Ryou et al. [30]
ROSE
N
80 (60–100)
4.2 kg**
7.8 kg**
Gallo et al. [17] ROSE
N
77 ± 30
8 ± 4
8.9
9.3
8
Buttelmann
[31]
ROSE
N
3.9***
4.1***
5.4***
Thompson [18]
IOP
N
87
11.5
14.5 ± 3.1
Patel 2017 [32]
EGJR
N
IST 50.4 ± 25.3
IST 6.6 ± 2.2
6 weeks*
15 (9–22)
19 (9–27)*
13 (5–32)*
10 (− 3.2 to
23.1)
PST
42.9 ± 18.1
PST 4.8 ± 1.8
Tsai [22]
EGJR
N
17.2 (12–33)
6 (4–14)
4.1**
5.8**
8.0**
Catalano [33]
Sclerotherapy
N
10 (8–15)
10.4
Loewen and
Barba [34]
Sclerotherapy
N
Jirapinyo [24]
Sclerotherapy
(sodium mor-
rhuate)
N
21 ± 6
2.7 ± 5.5****
6.1 ± 6.8 (9
months)****
TORe
23 ± 6
10.4 ± 2.2****
12.3 ± 12.6 (9
months)****
Thompson [21]
TORe
N
107 ± 182.9
15.9
de Moura [40]
TORe
N
12
20**
14**
Kumar and
Thompson
[37]
ST TORe
N
6.9 ± 0.2
8.1 ± 2.5
9.1 ± 2.3
FT TORe
7.1 ± 0.3
20.4 ± 3.3
18.9 ± 5.4
Kumar and
Thompson
[36]
TORe (Over-
Stitch)
N
9.0 ± 0.2
25.0 ± 1.9
28.8 ± 2.7
24.9 ± 2.6
Jirapinyo [25]
TORe (Over-
Stitch)
N
27 (7–80)
6 (3–10)
11.5**
11.7**
10.8** 1 Surgical Endoscopy (2020) 34:2410–2428 2421 1 3
(
)
Study
Procedure
Combination
therapy (Y/N)
Av. procedure
length (mins)
Av. Complications stoma
diameter at end
of procedure
(mm)
% Excess weight loss
1 week
1 month
2 months
3 months
6 months
12 months
Vargas [23]
TORe (Over-
Stitch)
N
9.31 ± 6.7
20.2 ± 10
Baretta [20]
APC
N
8.40 ± 1.85
Moon [19]
APC
N
(5–10)
14.0 ± 6.3
6.5**
7.7**
Riva [35]
Over-
Stitch + sutur-
ing
Y
91 ± 72.4
9.05
Over-
Stitch + scle-
rotherapy
Study
% Excess weight loss
Recurrence rates
n (%)
Definition of
successful endo-
therapy
Number of suc-
cessful endother-
apy n (%)
18 months
24 months
36 months
48 months
60 months
72 months
Mikami [14]
Ong-Uti [15]
3 (4.7%)
Goyal [38]
4.3 ± 29.8
2 (3.6)–pro-
gressed to fur-
ther procedure
1. Ability to
reduce pouch
and stoma size
2. Weight loss
35 (63.6)
Mullady et al. [29]
1. Ability to
reduce stoma
diameter and
pouch length
2. Weight loss
17 (85)
Horgan et al. [16]
1. Ability to
reduce stoma
diameter and
pouch length
2. Weight loss
112 (97)
Ryou et al. [30]
1. Ability to
reduce stoma
diameter and
pouch length
2. Weight loss
5 (100)
Gallo et al. [17]
6.7
− 10.7
− 13.5
− 5.8
− 4.5
Buttelmann [31]
5.5*** − 4.5 − 13.5 3 Surgical Endoscopy (2020) 34:2410–2428 2422 22
Surgical Endoscopy (2020) 34:2410–242
1 3
Study
% Excess weight loss
Recurrence rates
n (%)
Definition of
successful endo-
therapy
Number of suc-
cessful endother-
apy n (%)
18 months
24 months
36 months
48 months
60 months
72 months
Thompson [18]
1. Ability to
reduce stoma
diameter and
pouch length
2. Weight loss
112 (97)
Patel 2017 [32]
IST 3 (8.8)
PST 0
Tsai [22]
Catalano [33]
19.9
1. Stoma
size < 12 mm
2. Complications Loss of > 75%
of weight
regained after
initial weight
loss
18 (64)
n and
a [34]
2nd session 35
(49), 3rd ses-
sion 10(14),
4th session
1(1.4)
21 (29.6)
yo [24]
Ability to
reduce the GJ
to < 12 mm
pson [21]
Ability to
reduce the GJ
to < 10 mm
89.6%
ura [40]
Weight mainte-
nance/ weight
loss
24 (29.6)
and
mpson [37]
and
mpson [36]
20.0 ± 6.4
19.2 ± 4.6
yo [25]
Ability to
reduce the GJ
to < 12 mm
25 (100)
[23]
8 ± 8.8 (18–
24mths)
11 (8)—repeat
EGD per-
formed
Ability to
reduce the GJ
to < 10 mm Vargas [23] 1 3 Surgical Endoscopy (2020) 34:2410–2428 2423 Study
% Excess weight loss
Recurrence rates
n (%)
Definition of
successful endo-
therapy
Number of suc-
cessful endother-
apy n (%)
18 months
24 months
36 months
48 months
60 months
72 months
Baretta [20]
Moon [19]
8.3**
Riva [35]
36.4
Key: * median value, interquartile range, ** mean weight loss, *** mean BMI loss, **** mean %TBWL
TBWL total body weight loss, EWL excess weight loss, ROSE restorative obesity surgery, endoluminal (ROSE) procedure, EGJR endoscopic gastrojejunal revision, RYGB Roux-en-Y gastric
bypass, OTSC-clip over-the-scope clip, IOP incisionless operating platform, TORe sutured transoral outlet reduction, GJA gastrojejunal anastomosis, APC Argon plasma coagulation Outcomes of combination therapy Only two studies assessed the use of combination therapy
[28, 35]. Riva et al.’s study [35] involved a combination
of mucosal ablation and endoscopic suturing using the
OverStitch device or mucosal ablation, endoscopic sutur-
ing and sclerotherapy, performed following RYGB in 22
patients. Mean pre-revision weight and BMI were 102.3 kg
and 42.4 kg/m2, respectively. Mean post-revision BMI was
reduced by 15.1% at 6 months, and this was increased to
19.8% at 1 year. However, no mean weight or excess weight
loss following the revision procedure was reported. In terms
of complications, only abdominal pain and nausea were
reported (44%).i The other study by Eid [28] consisted of five patients,
in whom a combination of APC and endoscopic suturing
(OverStitch device) was performed. Mean pre-revision
weight was 110.3 kg and mean pre-revision BMI 37 kg/
m2. In this group of patients, greatest weight loss was noted
6 months post-procedure (11.0%) and this reduced slightly
to 10.1% at 1 year. This is consistent with a fall in 9.1% of
mean post-revision BMI measured at 1 year. No complica-
tions were reported. Quality of included studies All studies were evaluated for risk of bias using the New-
castle–Ottawa Scale (NOS), which allows for a maximum of
eight points, and NIH Quality Assessment Tool (Table 3). A score of 6 or more on the NOS is rated “good”, while the
NIH tool judges each study to be of “good”, “fair” or “poor”
quality. Both the NOS and NIH tool assess risk of bias in
the selection of cases, outcome assessment and duration of
follow-up. Additionally, the NOS assesses the representa-
tiveness of the exposed cohort and adequacy of follow-up. Of the 25 observational studies, two studies achieved a score
of 7, seven were scored 6, nine rated 5, five scored 4 and
two scored 3 on the NOS (Table 3, Supplementary Table 1). Twenty-three studies (92%) lacked a comparator group. In all studies, a satisfactory length of follow-up is defined
as 12 months or longer—this criteria was met by 20 (80%)
studies. Four (15.4%) studies were not awarded an “out-
come” score on the Newcastle–Ottawa Scale due to high
attrition rates. Eleven (42.3%) studies failed to report the rate
of patient follow-up. Two studies in particular [14, 16] had
very low follow-up rate (< 20%) at 12 months. The authors
defined 12 months as the ideal duration of follow-up for
post-procedural assessment of weight loss and complications
following StomaphyX [14] and ROSE [16], respectively, in
their selected patients, all of whom were at least 2 years
post-RYGB. However, only 15.4% [14] and 11.2% [16] were
successfully followed up for post-procedural evaluations at All studies were evaluated for risk of bias using the New-
castle–Ottawa Scale (NOS), which allows for a maximum of
eight points, and NIH Quality Assessment Tool (Table 3). A score of 6 or more on the NOS is rated “good”, while the
NIH tool judges each study to be of “good”, “fair” or “poor”
quality. Both the NOS and NIH tool assess risk of bias in
the selection of cases, outcome assessment and duration of
follow-up. Additionally, the NOS assesses the representa-
tiveness of the exposed cohort and adequacy of follow-up. Of the 25 observational studies, two studies achieved a score
of 7, seven were scored 6, nine rated 5, five scored 4 and
two scored 3 on the NOS (Table 3, Supplementary Table 1). Twenty-three studies (92%) lacked a comparator group. Complications Loss of > 75%
of weight
regained after
initial weight
loss
18 (64)
Loewen and
Barba [34]
2nd session 35
(49), 3rd ses-
sion 10(14),
4th session
1(1.4)
21 (29.6)
Jirapinyo [24]
Ability to
reduce the GJ
to < 12 mm
Thompson [21]
Ability to
reduce the GJ
to < 10 mm
89.6%
de Moura [40]
Weight mainte-
nance/ weight
loss
24 (29.6)
Kumar and
Thompson [37]
Kumar and
Thompson [36]
20.0 ± 6.4
19.2 ± 4.6
Jirapinyo [25]
Ability to
reduce the GJ
to < 12 mm
25 (100)
Vargas [23]
8 ± 8.8 (18–
24mths)
11 (8)—repeat
EGD per-
formed
Ability to
reduce the GJ
to < 10 mm 422
Surgical Endoscopy (2020) 34:2410–24
(
)
% Excess weight loss
Recurrence rates
n (%)
Definition of
successful endo-
therapy
Number of suc-
cessful endother-
apy n (%)
18 months
24 months
36 months
48 months
60 months
72 months
pson [18]
1. Ability to
reduce stoma
diameter and
pouch length
2. Weight loss
112 (97)
017 [32]
IST 3 (8.8)
PST 0
2]
no [33]
19.9
1. Stoma
size < 12 mm
2. Quality of included studies In all studies, a satisfactory length of follow-up is defined
as 12 months or longer—this criteria was met by 20 (80%)
studies. Four (15.4%) studies were not awarded an “out-
come” score on the Newcastle–Ottawa Scale due to high
attrition rates. Eleven (42.3%) studies failed to report the rate
of patient follow-up. Two studies in particular [14, 16] had
very low follow-up rate (< 20%) at 12 months. The authors
defined 12 months as the ideal duration of follow-up for
post-procedural assessment of weight loss and complications
following StomaphyX [14] and ROSE [16], respectively, in
their selected patients, all of whom were at least 2 years
post-RYGB. However, only 15.4% [14] and 11.2% [16] were
successfully followed up for post-procedural evaluations at 1 3 2424 Surgical Endoscopy (2020) 34:2410–2428 Table 3 Summary of quality
and risk of bias assessment
using the Newcastle–Ottawa
scale and National Institute of
Health quality assessment tool
for case series studies
Study
Newcastle–Ottawa
scale
National Institute of Health quality
assessment tool
Overall
Mikami et al. [14]
4
Fair
Fair
Manouchehri et al. [26]
6
Good
Good
Ong’Uti et al. [15]
7
Good
Good
Goyal et al. (39)
7
Good
Good
Mullady et al. [29]
5
Fair
Fair
Horgan et al. [16]
6
Good
Good
Ryou et al. [30]
4
Poor
Poor
Gallo et al. [17]
4
Fair
Fair
Buttelmann et al. [31]
6
Good
Good
Thompson et al. [18]
5
Fair
Fair
Heylen et al. [27]
5
Fair
Fair
Patel et al. [32]
5
Fair
Fair
Tsai et al. [22]
6
Good
Good
Catalano et al. [33]
5
Good
Fair
Loewen and Barba [34]
4
Fair
Fair
Jirapinyo et al. [24]
6
Good
Good
de Moura et al. [39]
3
Poor
Poor
Kumar and Thompson [37]
5
Fair
Fair
Kumar and Thompson [36]
6
Good
Good
Jirapinyo et al. [25]
5
Fair
Fair
Vargas et al. [23]
5
Good
Fair
Baretta et al. [20]
4
Fair
Fair
Moon et al. [19]
5
Fair
Fair
Riva et al. [35]
6
Good
Good
Eid [28]
3
Poor
Poor 12 months, and the authors did not elaborate the reason(s)
behind loss to follow-up. to other techniques (i.e. sclerotherapy, APC) post-RYGB. More specifically, analysis of the included studies has shown
successful EWL following the use of endoluminal plication
devices in the first 12 months after revisional procedure. This EWL was, however, not well-sustained past 12 months. Quality of included studies Greater procedural success and lower recurrence rates are
seen in endoluminal plication devices compared to sclero-
therapy and APC. Additionally, this review suggests that
endoluminal plication devices are associated with lower
rates of mild and moderate complications post-procedure
compared to sclerotherapy and APC. With regards to the NIH Quality Assessment Tool
(Table 3, Supplementary Table 2), 12 (46.2%) studies were
subjectively considered to be of “good” quality, while 11
(42.3%) were considered “fair” and three (11.5%) were
rated “poor” in the risk of bias assessment. The studies rated
“poor” lacked a clear definition for cohort selection and out-
come measures, and either failed to describe the results or
statistical methods used. Given the high heterogeneity of the
studies included in this review, the results and conclusions
should be interpreted with caution. Due to the complex nature of weight regain, which
involves an interplay between genetic, anatomical, physi-
ological and behavioural factors [42, 43], there are different
theories as to which factor is most predictive of treatment
response. Excess weight loss (EWL) following revision
surgery using endoluminal plication devices is likely to be
due to anatomical reasons. Horgan et al. describes failure of
maintenance of EWL to be due to loss of restriction attrib-
uted to the enlargement of the gastric pouch, dilatation of
gastrojejunostomy and fistula development between gas-
tric pouch and remnant of the stomach [16]. In this review,
Vargas et al.’s study was focused on stoma size reduction, Table 3 Summary of quality
and risk of bias assessment
using the Newcastle–Ottawa
scale and National Institute of
Health quality assessment tool
for case series studies 3 Discussion Weight regain is estimated to range between 5–7% [2, 4, 5]
with higher failure rates (20–35%) in the superobese patients
(BMI > 50) [6, 40, 41]. This systematic review addresses less
invasive techniques for treatment of post-operative weight
gain and associated short-term outcomes. The use of endo-
luminal plication devices in revisional surgery is associated
with greater initial EWL and fewer complications compared 1 3 3 Surgical Endoscopy (2020) 34:2410–2428 2425 where the revision procedure (TORe) was considered suc-
cessful if the stoma diameter was reduced to < 10 mm [23]. The authors achieved a mean of 70.4% reduction in stoma
diameter post-procedure and a resultant mean EWL of
20.2% at 12 months, and 8.0% at 18 months [23]. Similarly,
Jirapinyo et al. showed a mean reduction of 77.3% in stoma
diameter post-procedure in their cohort of 25 patients post-
procedure (TORe), with a peak mean weight loss of 11.7 kg
at 6 months, which later decreased to 10.8 kg at 12 months
[25]. The greater mean EWL in the initial 12 months fol-
lowing the use of endoluminal plication devices in patients
post-RYGB which decreased in the ensuing months may be
attributed to the lack of durability of endoscopic sutures in
the long term [44]. Follow-up endoscopy after ROSE pro-
cedures showed that superior weight loss is associated with
reduction in stoma size, with good durability of anchors and
tissue fold for up to 12 months post-revision [16, 18]. These
studies provide evidence that stoma size does influence
weight loss post-revision surgery, where EWL is greatest in
the first 12 months. However, long-term data past 18 months
are not recorded for most studies utilising endoluminal plica-
tion devices post-RYGB in this review. Future studies, which
include follow-up evaluations with endoscopy to verify the
link between maintenance of stoma reduction and EWL, are
warranted. 20]. Hence, these studies focused on the anatomical aspect
of causes in weight regain, similar to that in endoluminal
plication devices, although Moon et al. did demonstrate a
sustained mean weight loss up to 24 months, longer than
those noted in revisional procedures utilising endoluminal
plication devices [19]. Furthermore, Manouchehri et al. Discussion has shown that endo-
luminal plication devices in revision surgery, specifically
the StomaphyX™, can effectively contribute to weight loss
in patients following VBG, with only minor complications
experienced by patients [26], although sustained weight
loss is not demonstrated due to limited duration of follow-
up (3 months). However, the role of endoluminal plication
devices is more skewed towards that in RYGB patients
because VBG has largely been supplanted by RYGB as a
primary bariatric surgery technique in recent years. Nev-
ertheless, outcomes following endoluminal revisional tech-
niques in VBG may still be of interest in a small cohort
of patients experiencing weight regain requiring revisional
surgery [49–51]. A previous meta-analysis by Vargas et al. has demon-
strated the safe and efficacious use of TORe (OverStitch
device) in revision surgery performed in RYGB patients
[23]. The present review builds on this finding, and sum-
marises qualitatively the evidence supporting greater long-
term post-procedure weight loss when endoscopic suturing
with OverStitch device is combined with sclerotherapy or
APC, as shown by Riva et al. [35] and Eid [28]. Riva et al.’s
study was aimed at investigating a possible additive effect of
combined sclerotherapy and endoscopic suturing, where the
induced fibrosis could enhance the durability of sutures [35]. Compared with sclerotherapy/APC (EWL 19.9%) or endo-
luminal plication device (EWL 12.9%) alone, combination
therapy is shown to induce the greatest mean EWL of 36.4%
at 18 months in a small study of five patients [28]. Although
combination therapy appears to have some benefit in one
study, this has not translated to a larger study of 22 patients. Another possible predictor of EWL following revision
surgery is ghrelin levels post-procedure. The role of ghre-
lin in obesity in previous work appears to be significant,
however, its exact mechanism requires further investigation
[45, 46]. However, Dayyeh et al. demonstrated a decrease in
ghrelin levels in a group of 33 RYGB patients post-sclero-
therapy, contrary to what was observed following mechani-
cal endoscopic suturing with endoluminal plication devices
[42]. The authors postulated that ghrelin-producing cells
were destructed as a result of sclerosis, hence modulating
the neurohormonal signalling to the brain and other organs,
altering satiety, food intake behaviours insulin secretion and
energy expenditure [42, 47]. This alteration in neurophysi-
ology may account for the greater sustained EWL over a
longer period of time in sclerotherapy compared to endolu-
minal plication devices. Discussion This is because the latter predomi-
nantly depends on the reduction in GJ stoma diameter slow-
ing down the activation of gastric wall mechanoreceptors,
inhibiting the release of orexigenic gastric peptides such as
ghrelin [42, 46–48], which may be less effective compared
to a direct destruction of ghrelin-producing cells in sclero-
therapy in inducing neurophysiological changes contributing
to sustained weight loss. This, compounded by the lack of clear description on
patient selection and specification of outcomes, undermines
the internal validity of the conclusions. This finding may
suggest the potential of combination therapy in managing
weight regain following primary bariatric surgery, however,
there is currently insufficient evidence to support its supe-
riority over endoluminal plication devices, and vice versa. Compliance with ethical standards Disclosures Miss Yan Mei Goh, Nicole Ellen James, En Lin Goh, and
Achal Khanna have no conflicts of interest or financial ties to disclose. Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Additionally, these studies also lacked control of con-
founding factors including patients’ nutritional status, main-
tenance of diet and exercise, as well as important comorbid
conditions such as type 2 diabetes mellitus. Future work may
wish to explore the impact of additional routine follow-up
addressing these behavioural issues and dietary and lifestyle
modifications on maintenance of weight loss. Additionally,
all of the studies were conducted in developed countries. These skewed study populations are unlikely to represent
faithfully the true populations in less developed countries,
thus the generalisability of these findings to the wider pop-
ulation in other parts of the world should be treated with
caution. With the majority of studies being retrospective
in design and the paucity of studies assessing long-term
EWL of greater than 12 months following endoluminal
procedures, the question whether endoluminal techniques
can sustain long-term EWL still remains. Cohort studies or
randomised controlled trials should be performed to not only
clarify the role of endoluminal plication devices, but also
combination therapy in the management of weight regain
following primary bariatric surgery. References 1. English WJ, DeMaria EJ, Brethauer SA, Mattar SG, Rosenthal
RJ, Morton JM (2018) American Society for metabolic and bari-
atric surgery estimation of metabolic and bariatric procedures
performed in the United States in 2016. Surg Obes Relat Dis
14:259–263 2. Brolin RE, Kenler HA, Gorman JH, Cody RP (1992) Long-limb
gastric bypass in the superobese: a prospective randomized study. Ann Surg 215(4):387–395 3. Capella JF, Capella RF (1996) The weight reduction operation of
choice: vertical banded gastroplasty or gastric bypass? Am J Surg
171(1):74–79 4. Fobi MAL, Lee H, Igwe D, Felahy B, James E, Stanczyk M et al
(2001) Revision of failed gastric bypass to distal Roux-en-Y gas-
tric bypass: a review of 65 cases. Obes Surg 11(2):190–195 yp
g
5. Capella JF, Capella RF (1996) The weight reduction operation of
choice: vertical banded gastroplasty or gastric bypass? Am J Surg
1:74–79 6. Biron S, Hould FS, Lebel S, Marceau S, Lescelleur O, Simard S
et al (2004) Twenty years of biliopancreatic diversion: what is the
goal of the surgery? Obes Surg 14(2):160–164 Study limitations The included studies exhibit some limitations, which must
be considered when interpreting the findings of this analy-
sis. Firstly, there is significant variation in primary bariatric
procedure, endoluminal revision techniques, methodology
of reporting, follow-up times, outcomes and complications. Theoretically, the modulation in neurohormonal signal-
ling may presumably be applied to APC, but the APC pro-
cedures that were utilised in two studies included in this
review were aimed at reducing the diameter of the GJ stoma
and, therefore, reinitiate weight loss in RYGB patients [19, 1 3 Surgical Endoscopy (2020) 34:2410–2428 2426 There are limited data on endoscopic revision procedures
following sleeve gastrectomy. Given the rapid increase in
use of sleeve gastrectomy in recent years, future research on
the generalisability and applicability of endoscopic revision
surgery in patients with sleeve gastrectomy will be necessary
to overcome the inherent limitations of the currently avail-
able evidence. Moreover, the heterogeneity of the studies,
especially with regards to the selection criteria of patients
for revisional surgery, limits the statistical analysis of demo-
graphic and procedural variables that appeared to be predic-
tive of maximal weight loss benefit. quality data limits our ability to demonstrate and support
the long-term efficacy of endoluminal techniques in the
management of weight regain following primary bariatric
surgery. However, we suggest that these techniques have an
intermediate role in management of weight regain following
bariatric surgery, delaying surgical revision or conversion to
distal RYGB or biliopancreatic/duodenal switch procedures. Future work is necessary to substantiate the long-term role
of endoluminal bariatric procedures in the management of
this group of patients. i
Most series have small number of patients and some
follow-up data were not available which imposes limits on
our ability to make a meaningful conclusion. These high
attrition rates could be attributed to a poor understanding of
patients’ expressed needs, which is central to the develop-
ment and delivery of effective longer term follow-up care
following revision surgery. Studies have shown that patients
who did not attend regular follow-up commonly described
unmet perceived expectations as well as fear of disappoint-
ing the healthcare professional if they were unable to meet
nutritional or physical activity targets set [52]. Conclusion 7. Zalesin KC, Franklin BA, Miller WM, Nori Janosz KE, Veri S,
Odom J et al (2010) Preventing weight regain after bariatric sur-
gery: an overview of lifestyle and psychosocial modulators. Am J
Lifestyle Med 4:113–120 Our study demonstrates the need for detailed discussion
and tailoring of techniques and resources to the individ-
ual patient. Endoluminal techniques at present affords the
patient an opportunity to alter their lifestyle and delay surgi-
cal revision or conversion to distal RYGB or biliopancreatic/
duodenal switch procedures. However, the paucity of good 8. Odom J, Zalesin KC, Washington TL, Miller WW, Hakmeh B,
Zaremba DL et al (2010) Behavioral predictors of weight regain
after bariatric surgery. Obes Surg 20(3):349–356 1 3 Surgical Endoscopy (2020) 34:2410–2428 2427 26. Manouchehri N, Birch DW, Menzes C, Shi X, Karmali S (2011)
Natural orifice surgery: endoluminal pouch reduction following
failed vertical banded gastroplasty. Obes Surg 21(11):1787–1791 9. Stefanidis D, Malireddy K, Kuwada T, Phillips R, Zoog E, Gersin
KS (2013) Revisional bariatric surgery: perioperative morbidity is
determined by type of procedure. Surg Endosc 26(Suppl):26–32f 10. Schwartz RW, Strodel WE, Simpson WS, Griffen WO (1988)
Gastric bypass revision: lessons learned from 920 cases. Surgery
104(4):806–812 27. Heylen AMF, Jacobs A, Lybeer M, Prosst RL (2011) The
OTSC®-clip in revisional endoscopy against weight gain after
bariatric gastric bypass surgery. Obes Surg 21(10):1629–1633 ( )
11. Spaulding L (2003) Treatment of dilated gastrojejunostomy with
sclerotherapy. Obes Surg 13(2):254–257 28. Eid G (2017) Sleeve gastrectomy revision by endoluminal sleeve
plication gastroplasty: a small pilot case series. Surg Endosc
31:4252–4255 12. Wells G, Shea B, O’Connell D, Peterson J, Welch V, Losos M,
et al. The Newcastle-Ottawa Scale (NOS) for assessing the qual-
ity of nonrandomised studies in meta-analyses [Internet]. 2019;
https://www.ohri.ca/programs/clinical_epidemiology/oxford.asp,
Accessed 23 Aug, 2019 29. Mullady DK, Lautz DB, Thompson CC (2009) Treatment of
weight regain after gastric bypass surgery when using a new
endoscopic platform: initial experience and early outcomes (with
video). Gastrointest Endosc 70(3):440–444 13. National Heart L and BI. Study quality assessment tools [Internet]. 2018; https://www.nhlbi.nih.gov/health-topics/study-quality-asses
sment-tools. Accessed 23 Aug, 2018 30. Ryou M, Mullady DK, Lautz DB, Thompson CC (2009) Pilot
study evaluating technical feasibility and early outcomes of
second-generation endosurgical platform for treatment of
weight regain after gastric bypass surgery. Surg Obes Relat Dis
5(4):450–454 14. Conclusion Mikami D, Needleman B, Narula V, Durant J, Melvin WS (2010)
Natural orifice surgery: initial US experience utilizing the Stoma-
phyXTM device to reduce gastric pouches after Roux-en-Y gastric
bypass. Surg Endosc 24(1):223–228 31. Buttelmann K, Linn JG, Denham W, Ruiz M, Yetasook A, Ujiki
M (2015) Management options for obesity after bariatric surgery. Surg Laparosc Endosc Percutaneous Tech 25(1):15–18 15. OngUti SK, Ortega G, Onwugbufor MT, Ivey GD, Fullum TM,
Tran DD (2013) Effective weight loss management with endo-
scopic gastric plication using StomaphyX device: Is it achievable? Surg Obes Relat Dis. 9(1):113–117 32. Patel LY, Lapin B, Brown CS, Stringer T, Gitelis ME, Linn JG
et al (2017) Outcomes following 50 consecutive endoscopic gas-
trojejunal revisions for weight gain following Roux-en-Y gastric
bypass: a comparison of endoscopic suturing techniques for stoma
reduction. Surg Endosc Other Interv Tech 31(6):2667–2677 16. Horgan S, Jacobsen G, Weiss GD, Oldham JS, Denk PM, Borao F
et al (2010) Incisionless revision of post-Roux-en-Y bypass stomal
and pouch dilation: multicenter registry results. Surg Obes Relat
Dis 6(3):290–295 33. Catalano MF, Rudic G, Anderson AJ, Chua TY (2007) Weight
gain after bariatric surgery as a result of a large gastric stoma:
endotherapy with sodium morrhuate may prevent the need for
surgical revision. Gastrointest Endosc 66(2):240–245 17. Gallo AS, DuCoin CG, Berducci MA, Nino DF, Almadani M,
Sandler BJ et al (2016) Endoscopic revision of gastric bypass:
Holy Grail or Epic fail? Surg Endosc 30(9):3922–3927 34. Loewen M, Barba C (2008) Endoscopic sclerotherapy for dilated
gastrojejunostomy of failed gastric bypass. Surg Obes Relat Dis
4(4):539–542 18. Thompson CC, Jacobsen GR, Schroder GL, Horgan S (2012)
Stoma size critical to 12-month outcomes in endoscopic suturing
for gastric bypass repair. Surg Obes Relat Dis 8(3):282–287 35. Riva P, Perretta S, Swanstrom L (2017) Weight regain following
RYGB can be effectively treated using a combination of endo-
scopic suturing and sclerotherapy. Surg Endosc Other Interv Tech
31(4):1891–1895 19. Moon RC, Teixeira AF, Neto MG, Zundel N, Sander BQ, Ramos
FM et al (2018) Efficacy of utilizing argon plasma coagulation for
weight regain in Roux-en-Y Gastric bypass patients: a multi-center
study. Obes Surg 28(9):2737–2744 36. Kumar N, Thompson CC (2016) Transoral outlet reduction for
weight regain after gastric bypass: Long-term follow-up. Gastro-
intest Endosc 83(4):776–779i 20. Conclusion Baretta GAP, Alhinho HCAW, Matias JEF, Marchesini JB, de
Lima JHF, Empinotti C et al (2015) Argon plasma coagulation of
gastrojejunal anastomosis for weight regain after gastric bypass. Obes Surg 25(1):72–79 37. Kumar N, Thompson CC (2014) Comparison of a superficial
suturing device with a full-thickness suturing device for transoral
outlet reduction (with videos). Gastrointest Endosc 79(6):984–989 21. Thompson CC, Chand B, Chen YK, Demarco DC, Miller L, Sch-
weitzer M et al (2013) Endoscopic suturing for transoral outlet
reduction increases weight loss after Roux-en-Y gastric bypass
surgery. Gastroenterology 145(1):129–137f 38. Goyal V, Holover S, Garber S (2013) Gastric pouch reduction
using StomaphyXTM in post Roux-en-Y gastric bypass patients
does not result in sustained weight loss: a retrospective analysis. Surg Endosc 27(9):3417–3420i 22. Tsai C, Steffen R, Kessler U, Merki H, Zehetner J (2019) Endo-
scopic gastrojejunal revisions following gastric bypass: lessons
learned in more than 100 consecutive patients. J Gastrointest Surg
23(1):58–66 g
39. de Moura DTH, Jirapinyo P, Thompson CC (2019) Modified-ESD
plus APC and suturing for treatment of weight regain after gastric
bypass. Obes Surg 29(6):2001–2002 23. Vargas EJ, Bazerbachi F, Rizk M, Rustagi T, Acosta A, Wilson EB
et al (2018) Transoral outlet reduction with full thickness endo-
scopic suturing for weight regain after gastric bypass: a large mul-
ticenter international experience and meta-analysis. Surg Endosc
32(1):252–259 40. Hsu LK, Benotti PN, Dwyer J, Roberts SB, Saltzman E, Shikora
S et al (1998) Nonsurgical factors that influence the outcome of
bariatric surgery: a review. Psychosom Med 60(3):338–346 41. Nelson DW, Blair KS, Martin MJ (2012) Analysis of obesity-
related outcomes and bariatric failure rates with the duode-
nal switch vs gastric bypass for morbid obesity. Arch Surg
147(9):847–854 24. Jirapinyo P, Dayyeh BKA, Thompson CC (2016) Gastrojejunal
anastomotic reduction for weight regain in roux-en-y gastric
bypass patients: physiological, behavioral, and anatomical effects
of endoscopic suturing and sclerotherapy. Surg Obes Relat Dis
12(10):1810–1816 42. Abu Dayyeh BK, Jirapinyo P, Weitzner Z, Barker C, Flicker MS,
Lautz DB et al (2012) Endoscopic sclerotherapy for the treatment
of weight regain after Roux-en-Y gastric bypass: outcomes, com-
plications, and predictors of response in 575 procedures. Gastro-
intest Endosc 76(2):275–282 25. Jirapinyo P, Slattery J, Ryan MB, Dayyeh BKA, Lautz DB,
Thompson CC (2013) Evaluation of an endoscopic suturing
device for transoral outlet reduction in patients with weight regain
following Roux-en-Y gastric bypass. Endoscopy 45(7):532–536 43. Conclusion Vetter ML, Cardillo S, Rickels MR, Iqbal N (2009) Narrative
review: effect of bariatric surgery on type 2 diabetes mellitus. Ann
Intern Med 150(2):94–103 1 3 2428 Surgical Endoscopy (2020) 34:2410–2428 44. Eid GM, McCloskey CA, Eagleton JK, Lee LB, Courcoulas AP
(2014) StomaphyX vs a sham procedure for revisional surgery
toreduceregainedweightinroux-en-ygastric bypass patients a ran-
domized clinical trial. JAMA Surg 149(4):372–379 50. Cariani S, Agostinelli L, Leuratti L, Giorgini E, Biondi P, Amenta
E (2010) Bariatric revisionary surgery for failed or complicated
vertical banded gastroplasty (VBG): comparison of VBG reopera-
tion (re-VBG) versus Roux-en-Y Gastric bypass-on-VBG (RYGB-
on-VBG). J Obes. https://doi.org/10.1155/2010/206249 g
45. Cummings DE, Shannon MH (2003) Roles for ghrelin in the regu-
lation of appetite and body weight. Arch Surg 138(4):389–396 51. van Gemert W, van Wersch M, Greve J, Soeters P (1998) Revi-
sional surgery after failed vertical banded gastroplasty: restoration
of vertical banded gastroplasty or conversion to gastric bypass. Obes Surg 8(1):21–28 46. Cummings DE, Weigle DS, Scott Frayo R, Breen PA, Ma MK,
Patchen Dellinger E et al (2002) Plasma ghrelin levels after diet-
induced weight loss or gastric bypass surgery. N Engl J Med
346(21):1662–1663 g
52. Parretti HM, Hughes CA, Jones LL (2019) ‘The rollercoaster of
follow-up care’ after bariatric surgery: a rapid review and qualita-
tive synthesis. Obes Rev 20(1):88–107 47. Hansotia T, Maida A, Flock G, Yamada Y, Tsukiyama K, Seino Y
et al (2007) Extrapancreatic incretin receptors modulate glucose
homeostasis, body weight, and energy expenditure. J Clin Invest
117(1):143–152 Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. 48. Ogiso K, Asakawa A, Amitani H, Inui A (2011) Ghrelin: a gut
hormonal basis of motility regulation and functional dyspepsia. J
Gastroenterol Hepatol 26(Suppl 3):67–72 49. Iannelli A, Dainese R, Piche T, Facchiano E, Gugenheim J (2008)
Laparoscopic sleeve gastrectomy for morbid obesity. World J Gas-
troenterol 14(6):821–827 1 3 3
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Influence of breast cancer risk factors and intramammary biotransformation on estrogen homeostasis in the human breast
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Abstract Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in
breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens
present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17β-estradiol, estrone, estrone-3-sulfate, and
2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of
metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on
breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by question-
naire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in
GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and
BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues
as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2,
and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the
exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT
and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tis-
sue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports
biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and
tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the
environment possible. Keywords Estrogens · Human breast · Multiple linear regression * Leane Lehmann
leane.lehmann@uni‑wuerzburg.de Influence of breast cancer risk factors and intramammary
biotransformation on estrogen homeostasis in the human breast Daniela Pemp1 · Leo N. Geppert2 · Claudia Wigmann2 · Carolin Kleider1 · René Hauptstein1 ·
Katja Ickstadt2 · Harald L. Esch1 · Leane Lehmann1 Received: 12 February 2020 / Accepted: 15 June 2020 / Published online: 22 June 2020
© The Author(s) 2020 Archives of Toxicology (2020) 94:3013–3025
https://doi.org/10.1007/s00204-020-02807-1 Archives of Toxicology (2020) 94:3013–3025
https://doi.org/10.1007/s00204-020-02807-1 TOXICOKINETICS AND METABOLISM 2
Mathematical Statistics with Applications in Biometrics, TU
Dortmund University, Vogelpothsweg 87, 44221 Dortmund,
Germany Introduction (E1), and other endogenous steroids in pre- and postmeno-
pausal women (Endogenous Hormones Breast Cancer Col-
laborative Group 2002, 2013) over a prolonged period of
time. Based on these associations as well as an abundance
of experiments in vitro and in animal models, the current
understanding of the molecular etiology of breast cancer
hypothesizes biotransformation of E2/E1 within the breast
tissue to catechols and subsequent oxidation to mutagenic
quinones possibly initiating tumor formation. Tumor promo-
tion is then favored by estrogen receptor (ESR)-mediated
stimulation of proliferation of the initiated cells (Yager and
Davidson 2006). Thus, both tumor initiation and progres-
sion would depend predominately on intramammary levels
of reactive products of estrogen biotransformation, whereas
tumor promotion would depend predominately on levels of
E2. Breast cancer is the most common cancer in women world-
wide. Its development is associated with increased levels
of circulating estrogens, e.g., 17β-estradiol (E2), estrone Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00204-020-02807-1) contains
supplementary material, which is available to authorized users. * Leane Lehmann
leane.lehmann@uni‑wuerzburg.de 1
Institute of Pharmacy and Food Chemistry, University
of Würzburg, Am Hubland, 97074 Würzburg, Germany 1
Institute of Pharmacy and Food Chemistry, University
of Würzburg, Am Hubland, 97074 Würzburg, Germany 2
Mathematical Statistics with Applications in Biometrics, TU
Dortmund University, Vogelpothsweg 87, 44221 Dortmund,
Germany 2
Mathematical Statistics with Applications in Biometrics, TU
Dortmund University, Vogelpothsweg 87, 44221 Dortmund,
Germany :(0123
1 3456789)
3 Archives of Toxicology (2020) 94:3013–3025 3014 Consequently, commonly accepted risk factors such as
early menarche and/or late menopause, late age at first preg-
nancy, small number of pregnancies, and short or no periods
of breastfeeding (Colditz and Bohlke 2014) are supposed
to increase the duration or extent of the local exposure of
the mammary gland to E2 (biotransformation products) by
increasing their systemic production. Concurrently, cur-
rent risk reduction strategies involve the chemical modula-
tion of ESR as well as systemic inhibition of aromatase or
salpingo-oophorectomy (Advani and Moreno-Aspitia 2014)
aimed to reduce levels of circulating estrogens. Recently,
also modifiable risk factors associated with lifestyle such
as (postmenopausal) obesity, alcohol consumption (Colditz
and Bohlke 2014), smoking (Gaudet et al. 2017; Jones et al. 2017; Gram et al. 2019), and intake of estrogen-active drugs
(EADs) for oral contraception (Grosse et al. Origin of biospecimens Breast tissue specimens were obtained from 47 adult
women without breast cancer undergoing reduction mam-
moplasty between 2010 and 2015. All women participat-
ing in the study gave their written informed consent prior
to their inclusion in the study. Women with a personal
and/or family history of breast cancer were not eligible
for participation. Information on age, height, weight, par-
ity (parous/nulliparous), smoking habits (never smoker,
current smoker, current nonsmoker, and the latter two
with daily cigarette consumption) was volunteered by
47 women, and information on the intake of EADs by 45
women. Body mass index (BMI) was calculated in kg/m2. Given the wide range of enzymes present in breast
glandular (GLT) and adipose tissues (ADT; Pemp et al. 2019a), additional (biotrans)formation of estrogens within
the breast tissue can reasonably be assumed (Labrie 2015;
Mueller et al. 2015; Hilborn et al. 2017; Pemp et al. 2019a). Consequently, breast cancer risk factors may also influence
tissue levels of E2 and its biotransformation products by
affecting estrogen homeostasis in the breast. Thus, to better
understand how estrogen homeostasis may affect initiation
and promotion of breast cancer, insight into the influence
of breast cancer risk factors on both levels of estrogen and
estrogen biotransformation in women without breast cancer
is needed. 1 3 Introduction 2009) or hor-
mone replacement therapy (Collaborative Group on Hor-
monal Factors in Breast Cancer 2019) have been associated
with both increased breast cancer risk and higher levels of
circulating E2 and E1 (Endogenous Hormones Breast Can-
cer Collaborative Group 2003, 2011, 2013), suggesting that
these risk factors also act by affecting intramammary levels
of E2/E1 (biotransformation products). (biotrans)formation in breast GLT and ADT of pre- and
postmenopausal women without breast cancer (Pemp et al. 2019a; Fig. 1) providing suitable data to determine vari-
ables affecting intramammary levels of estrogens. Further-
more, it was shown that levels of most estrogens and ratios
thereof as well as levels of transcripts encoding enzymes
involved in their (biotrans)formation differed significantly
between GLT and ADT (Pemp et al. 2019a), demonstrating
that breast GLT and ADT should be considered separately. Thus, the aim of the present study was to identify vari-
ables (reproductive history, lifestyle, and transcript levels
of enzymes involved in intracrine activity) influencing lev-
els of estrogens and ratios thereof in breast GLT and ADT. Sample preparation and characterization Tissue levels of E1 and E2 can be decreased by hydroxylation
catalyzed by CYPs and conjugation, i.e., sulfonation and glucuronida-
tion by sulfotransferases (SULTs) and UDP-glucuronosyltransferases
(UGTs), respectively. However, only E1-G (Pemp et al. 2019a) and E1-S have been detected in breast GLT and ADT up to now. Catecho-
lic hydroxy-estrogens (HO-E) can be oxidized to potentially cancer-
initiating estrogen quinones which can be reduced back to catechols
by NADPH quinone dehydrogenase 1 (NQO1). Detoxification of
catechols is catalyzed by catechol-O-methyl transferase (COMT)
resulting in the formation of methoxy(MeO-)estrogens. Of all possi-
ble MeO-estrogens, only 2-MeO-E1 has been detected mass spectro-
metrically (Fleming et al. 2010; Pemp et al. 2019a), predominately in
ADT (Pemp et al. 2019a). Framed estrogens were quantified recently
in breast tissues (Pemp et al. 2019a) and are used in this study as
dependent variables in multiple linear regression analyses. Gray-
colored estrogens were below the limit of detection in breast tissues
(Pemp et al. 2019a) by Pemp et al (2019a). Data used in statistical analyses are
presented in Online Resource 3. Sample preparation and characterization Biospecimens were prepared as described previously
(Pemp et al. 2019a). Briefly, aliquots of apparently plain
ADT and GLT with less than 15% adhering ADT were
flash-frozen in liquid nitrogen and stored at − 80 °C. From mixed tissues, GLT was isolated from cryosections
(40 µm) at maximum − 20 °C using a scalpel. Biospeci-
mens were characterized by their mass percentages of oil
(oil%), percentage of area covered by intra- and interstro-
mal adipocytes, and lobule type: oil% in GLT and ADT
were determined gravimetrically after extraction with
chloroform. Percentage of area covered by intra- and inter-
stromal adipocytes was estimated microscopically (Leica
LMD6500) in cryosections (10 µm) of GLT stained with
hematoxylin and eosin Y by two different persons and
coded slides (Pemp et al. 2019a). The lobule type of GLT
was determined microscopically according to Russo and
Russo (2004) and Figueroa et al. (2014). However, only two studies in women without breast
cancer have been published in this regard of which only
one has performed statistical analyses (Online Resource 1,
Savolainen-Peltonen et al. 2014). Even considering those
analyzing non-tumor tissue of women with breast cancer
and investigating the association of risk factors with tissue
levels of estrogens, most studies did not provide informa-
tion on parameters statistically compared and whether or
not all positive/negative correlations were reported (Online
Resource 1). Surprisingly, none of the available studies
included reproductive history of the participating women
in their statistical analyses, or collected data on smoking
or analyzed biotransformation products of E2 other than
E1 by recommended methods of specific analysis (Online
Resource 1). Only recently, we described quantitative estrogen
profiles and transcript levels of enzymes involved in E2 1 3 3015 Archives of Toxicology (2020) 94:3013–3025 Fig. 1 Current knowledge on (biotrans)formation of E2 in breast
GLT and ADT of women without breast cancer based on recently
published information on quantitative estrogen profiles and levels of
transcripts encoding enzymes involved in (biotrans)formation of E2
in human breast tissues (Pemp et al 2019a). Intramammary tissue lev-
els of E2 or E1 can be increased by cytochrome P450 (CYP)19A1-
mediated formation form androgenic precursors, interconversion of
E1 and E2 by hydroxysteroid 17-beta dehydrogenases (HSD17Bs),
as well as sulfotransferase (STS)- and glucuronidase beta (GUSB)-
mediated hydrolysis of estrogen sulfates and glucuronides, respec-
tively. Statistical methods E2, E1, and 2-methoxy(MeO)-E1 were determined by
GC–MS/MS (Varian 450-GC, 300-MS; Bruker Daltonics,
Bremen, Germany), whereas E1 sulfate (E1-S) and E1 glu-
curonide was determined by LC–MS/MS (QTrap® 5500;
AB Sciex, Darmstadt, Germany). Tissue levels of E2, E1,
2-MeO-E1, and E1-S were quantified using their respec-
tive deuterated derivatives (Pemp et al. 2019a). Data used
in statistical analyses are presented in Online Resource 2. All statistical analyses were performed with the statistical
programming language R [https://www.R-project.org/], ver-
sion 3.5.2, and all tests of statistical significance were two-
sided. Whenever multiple comparisons were performed, p
values were adjusted using Holm’s method. Instrumental analysis of E2, estrone,
2‑methoxy‑estrone, estrone sulfate,
and glucuronide by Pemp et al (2019a). Data used in statistical analyses are
presented in Online Resource 3. Identification of dependent variables E2, E1, and E1-S were detected in most biospecimens,
whereas 2-MeO-E1 was detected predominately in ADT
(Online Resource 2). Thus, the influence of exVARs on lev-
els of E2, E1, E1-S, and ratios thereof was analyzed in both
GLT and ADT whereas the influence of exVARs on levels
of 2-MeO-E1 could be analyzed in ADT only. Furthermore,
continuous variables significantly influencing estrogen levels
were further analyzed as dependent variables as well. The adjusted coefficients of determination, the num-
bers of conspicuous observations removed, the numbers
of observations contributing to the final models (maxi-
mum of 45 because of two specimens without information
on the intake of EADs). In models considering intracrine
activity, maximum number of observations was further
reduced because of two and one specimens in GLT and
ADT, respectively, without information on transcript lev-
els), and the ratio of observations per exVAR of each final
model is given. To achieve accurate estimation of regres-
sion coefficients, at least two observations per exVAR
(Austin and Steyerberg 2015) were aimed for.fi Identification of possible exVARs related
to the study cohort and to the tissues collected First, the study cohort and the tissues collected were char-
acterized and possible exVARs were defined. Results and discussion To identify exVARs (e.g., breast cancer risk factors) influ-
encing dependent variables (e.g., tissue levels of E2) by
multiple linear regression models, suitable dependent vari-
ables were identified first and potential exVARs were chosen
subsequently. Then, multiple linear regression models using
stepwise forward selection were applied to assess up to 32
exVARs possibly influencing tissue levels of estrogens and
ratios thereof in GLT and ADT. Determination of transcript levels Contingency analyses were performed using Chi-square test. In case of categories following a natural order, Chi-square
test for trend was used. Analysis of transcript levels of genes encoding enzymes
involved in E2 (biotrans)formation and regulation thereof
was performed using customized Taqman® Low Density
Arrays and Taqman® Gene Expression Assays as described 1 3 3016 Archives of Toxicology (2020) 94:3013–3025 interval, as well as the p values of each exVAR selected
are given in Online Resource 4. To test the association of every possible explanatory
variable (exVAR) with the dependent variable, the vari-
able explaining the dependent variable best is chosen by an
automatic procedure. Subsequently, all possible exVARs
are added one after another to the first one, ultimately
choosing the one improving the model most, applying the
Akaike information criterion. This is repeated until the
model cannot be further improved by adding exVARs. Thus, each exVAR selected into the model contributes to
modeling the dependent variable. The significance of the
association is expressed by p values and the magnitude
of impact is expressed by coefficients of regression. The
choice of exVARs is discussed in the results section and
more detailed information is given in Online Resource 4. If
levels of estrogens or transcripts were below limit of quan-
tification (LOQ) in > 40% of samples (Online Resources 2
and 3), they were not included as exVARs. Analyses of independence of variables Spearman’s rank correlation analysis was performed to iden-
tify collinearity between numerical exVARs which might
hinder each other selection and/or influence each other p
values within the models. In the case of variables with > 1
level below LOD or LOQ, correlation was calculated with
randomly distributed ranks for ties 10,000 times and high-
est Spearman correlation coefficients and lowest p values
were used to rather overestimate collinearity. Relationship
between categorical and numerical exVARs was evaluated
by comparison of medians using unpaired Wilcoxon tests. Indications for relationships between variables and possible
consequences for the selection of exVARs are given for each
model in Online Resource 4. For levels of transcripts or estrogens below the respec-
tive limit of detection (LOD) and below the respective
LOQ, LOD and LOQ were set, respectively. When lev-
els of transcripts or estrogens were < LOQ in > 1 sample
and ≤ 40% of samples, the levels of the respective tran-
script/estrogen were included additionally to the con-
tinuous exVAR as the qualitative presence of the exVAR
(binary exVARq, compared to levels < LOQ). If, in the
computed model, observations with Cook’s Distance > 1
appeared, they were removed and the model was com-
puted anew. This process was repeated until no conspicu-
ous observations occurred. To achieve normal distribution,
dependent variables were logarithmized. Data distribu-
tions were evaluated in Quantile–Quantile plots with sim-
ulated confidence bands. Constant standard deviations of
the errors were evaluated using scale-location plots. To
check the model assumption of independent identically
distributed errors, the residual vs. fitted values plot was
used.fi Obesity and related variables According to the WHO BMI classification, 53% and 13% of
women were pre-obese and obese, respectively. The remain-
ing women (34%) were of normal weight (Online Resource
6). Thus, compared to the German adult female population
(29% pre-obese, 24% obese, and 45% of normal weight;
Mensink et al. 2013), a higher percentage was pre-obese, but
lower percentages were obese and of normal weight. BMI
was included as continuous exVAR into the linear regres-
sion models. Regarding reproductive history, 40% of the women par-
ticipating in the study were nulliparous. In the age class
35–54 years old, 27% were nulliparous, compared to 21%
in the respective general population (Online Resource 5). The study population was further classified regarding par-
ity and lobule type of the GLT, the latter reflecting age-
and parity-related histological changes within the breast. These changes are most obvious in parous women, where
lobules type 2 and 3 (Lob2/3), previously induced during Median oil% in GLT and ADT were 16% and 85%,
respectively (Pemp et al. 2019a). In the following lin-
ear regression models, oil% were included as continuous
exVAR. Directly isolated GLT and GLT isolated from mixed
tissue were compared by means of two variables, oil% and
relative areas covered by inter- and intrastromal adipocytes
in GLT, and p values were adjusted for two comparisons. Despite detaching adhering ADT, GLT isolated from mixed
tissues (n = 18) still exhibited significantly higher oil%
(Fig. 3) than GLT which could be isolated directly; indi-
cating a higher number and/or size of adipocytes within
intra- and interlobular stroma. Consistently, microscopic
comparison of GLT isolated directly and GLT isolated from
mixed tissues revealed a significantly larger relative area
covered by inter- and intrastromal adipocytes in GLT derived
from mixed tissues (Fig. 3). Thus, in the following sections,
these specimens will be referred to as large-adipocyte-area
(laa)GLT and small-adipocyte-area GLT. The occurrence of
laaGLT was statistically independent of BMI classification
of the women donating the tissues (p = 0.40, Chi-square test
for trend) and lobule types of GLT specimens (p = 0.28, Chi-
square test, Online Resource 7). The occurrence of laaGLT
was tested as binary exVAR. Fig. 2 Distribution of age and lobule type as well as allocation of
menopausal status (MP) of the women contributing specimens to
the present study. GLT of parous women exhibits lobule type 2/3
(Lob2/3) until age-related regression. Obesity and related variables Lob1 predominates in parous
women after age-related regression as well as in nulliparous (np)
women (see text) Age, reproductive history, and related variables In addition, the regression coefficients (which repre-
sent the mean changes in the dependent variables for one
unit of change in the respective exVAR while holding
other predictors in the models constant), their confidence The age of the 47 women participating in the study ranged
from 18–66 years. Most tissues were derived from women
in the age group of 40–49 years (Online Resource 5). 3 3 3017 Archives of Toxicology (2020) 94:3013–3025 Menopausal status of the study population was allocated
according to the range of age at menopause (46–52 years)
observed in German women participating in the EPIC study
(n > 27,000; Tsilidis et al. 2011) instead of assessment based
on the women’s menstrual cycle characteristics (information
not available). Thus, with a high probability, women > 52
and < 46 years old can be assumed to be post- (19%, Fig. 2)
and premenopausal (55%), respectively. Women between
46–52 years were grouped as perimenopausal (26%). How-
ever, this group is likely to contain pre-, peri-, and post-
menopausal women. To reflect continuous influence of age
as well as abrupt influence of menopause on dependent vari-
ables, both the potential exVARs age and menopausal status
were included into the models. pregnancy, regress back to lobule type 1 (Lob1p; Russo and
Russo 2004). Nulliparous women (exhibiting lobule type 1,
Lob1np) represented the third group of lobule types. 23%,
36%, and 40% of GLT were classified Lob2/3, Lob1p, and
Lob1np, respectively (Fig. 2). In the following linear regres-
sion models, lobule type was tested as categorial exVAR. Relationship of lobule type with menopausal status and age
cannot be completely excluded, but seems to be unlikely
(Fig. 2). Age and menopause Cessation of ovarian estrogen production in menopause
decreases blood levels of estrogens (Endogenous Hormones
Breast Cancer Collaborative Group 2011, 2013) and is thus
considered to affect levels of estrogens in breast tissues. Depypere et al. (2015) observed lower median levels of E2
in GLT derived from postmenopausal than from premeno-
pausal women, yet no statistical analysis was performed. In
the present study, levels of E2 were not directly influenced
by postmenopausal status. However, postmenopausal status
influenced levels of E1 in GLT and levels of E1-S in ADT
negatively (p < 0.05). Furthermore, the ratio of E2 levels
in ADT to E2 levels in GLT (ADT/GLT of E2) was also
influenced negatively by postmenopausal status (p < 0.05). Interestingly, ADT/GLT of E1 was rather positively influ-
enced by postmenopausal status (0.10 > p ≥ 0.05, Fig. 4). Thus, menopause seems not to affect levels of all estrogens
in GLT and ADT and ratios ADT/GLT of estrogens in the
same way which cannot be explained by a mere decrease in
systemic delivery of estrogens via plasma. The continuous Fig. 3 Comparison of oil% and area covered by adipocytes (aa%) in
GLT isolated with and without cryosection. For statistical comparison
of medians, unpaired Wilcoxon test was used. Boxplots depict 25th
percentile, median, and 75th percentile. P values were adjusted for
multiple comparisons (n = 2) using Holm’s method status, lobule type, BMI, intake of estrogen-active drugs, and
smoking are depicted in Online Resource 8. Smoking and intake of EADs Twelve women (26%) declared being current smokers (2–25
cigarettes/day) and EADs were used by eight women. These
information were included as categorial exVARs into the
linear regression models. Fig. 2 Distribution of age and lobule type as well as allocation of
menopausal status (MP) of the women contributing specimens to
the present study. GLT of parous women exhibits lobule type 2/3
(Lob2/3) until age-related regression. Lob1 predominates in parous
women after age-related regression as well as in nulliparous (np)
women (see text) Mosaic plots characterizing the study population used in the
linear regression models regarding the exVARs menopausal 1 Archives of Toxicology (2020) 94:3013–3025 3018 status, lobule type, BMI, intake of estrogen-active drugs, and
smoking are depicted in Online Resource 8. Identification of possible exVARs related to estrogen
biotransformation in tissues
Fig. 3 Comparison of oil% and area covered by adipocytes (aa%) in
GLT isolated with and without cryosection. For statistical comparison
of medians, unpaired Wilcoxon test was used. Boxplots depict 25th
percentile, median, and 75th percentile. P values were adjusted for
multiple comparisons (n = 2) using Holm’s method enzymes directly forming or further metabolizing the
dependent variable according to Fig. 1. In models with
ratios of levels of different estrogens as dependent vari-
ables, further exVAR considered were levels of transcripts
encoding enzymes directly forming or further metabolizing
at least one of the estrogens involved in the ratio. If levels of
transcripts or precursor estrogens were < LOQ in > 1 sam-
ple and ≤ 40% of samples, the qualitative presence of the
respective precursor estrogen or transcript was included as
binary exVARq, as well. Variables influencing tissue levels of estrogens
identified by multiple linear regression models Previous studies investigating variables associated with lev-
els of estrogens in breast tissues either performed no statisti-
cal analysis at all or univariate analysis (i.e., comparisons
of medians in case of categorical variables and correla-
tion analyses in the case of continuous variables, Online
Resource 1). In addition, methods nowadays less recom-
mended for biospecimen analysis (Labrie et al. 2015) were
applied to determine estrogen levels and/or undefined speci-
mens were used without specifying the presence of GLT
or ADT. Because of these differences, the outcome of the
present study is only compared with previous ones if at least
either a specific method or specifically GLT or ADT was
used. Moreover, results observed in previous studies using
tissues derived from women with breast cancer or from both
women with and without breast cancer together for statisti-
cal analyses were included in Online Resource 1, but are not
discussed in the following sections. Identification of possible exVARs related to estrogen
biotransformation in tissues Besides age, lifestyle factors,
and tissue characteristics, oil% may be influenced by
estrogens affecting lipogenesis and adipogenesis (Gao and
Dahlman-Wright 2013), and thus, levels of E2 were also
considered as exVAR. Furthermore, the classification of
GLT regarding adipocyte area laaGLT was considered as
exVAR as well. Oil% in GLT were positively influenced
by laaGLT (p < 0.05), but no other exVAR was selected
(Fig. 4). Interestingly, oil% in ADT were positively influ-
enced by levels of E2, age, and specimens derived from
nulliparous women (compared to parous women prior to Lobule type The impact of lobule type on estrogen levels has not been
investigated yet. Average levels of E1-S in GLT exhibiting
the least developed lobule type 1 (derived from nulliparous
women, Lob1np, or from parous women after age-related
regression, Lob1p) were lower than levels of E1-S in GLT
categorized Lob2/3, yet not significantly (0.10 > p ≥ 0.05
and p > 0.1, respectively; Fig. 4). Furthermore, lobule type
1 influenced levels of E2 and E2/E1 in ADT negatively com-
pared to Lob2/3 (p < 0.05, Fig. 4). Since oil% influenced levels of some estrogens and
ratios thereof in ADT and GLT, variables affecting oil%
were investigated as well. Besides age, lifestyle factors,
and tissue characteristics, oil% may be influenced by
estrogens affecting lipogenesis and adipogenesis (Gao and
Dahlman-Wright 2013), and thus, levels of E2 were also
considered as exVAR. Furthermore, the classification of
GLT regarding adipocyte area laaGLT was considered as
exVAR as well. Oil% in GLT were positively influenced
by laaGLT (p < 0.05), but no other exVAR was selected
(Fig. 4). Interestingly, oil% in ADT were positively influ-
enced by levels of E2, age, and specimens derived from
nulliparous women (compared to parous women prior to Identification of possible exVARs related to estrogen
biotransformation in tissues Further possible continuous exVARs were tissue levels of
the direct precursor estrogen(s) and of transcripts encoding 1 3 Archives of Toxicology (2020) 94:3013–3025 3019 Fig. 4 Influence of various exVARs on levels of estrogens as well as
on ratios thereof and oil% in GLT and ADT (dependent variables)
identified by multiple linear regression models using stepwise for-
ward selection as detailed in Online Resource 4. For each model, the
adjusted coefficient of determination (R2), and ratio of the number of
observations (i.e., biospecimens) to exVAR (O/exVAR) after forward
selection of variables is given adjusted coefficient of determination (R2), and ratio of the number of
observations (i.e., biospecimens) to exVAR (O/exVAR) after forward
selection of variables is given Fig. 4 Influence of various exVARs on levels of estrogens as well as
on ratios thereof and oil% in GLT and ADT (dependent variables)
identified by multiple linear regression models using stepwise for-
ward selection as detailed in Online Resource 4. For each model, the exVAR age did not directly influence levels of any estrogen
or ratio thereof significantly (Fig. 4). influenced levels of E1 in GLT (positively, p < 0.05), levels
of E1-S in GLT (negatively, p < 0.05), and E1-S/E1 in both
GLT and ADT (negatively, p < 0.05; Fig. 4). Interestingly,
levels of 2-MeO-E1 and E2 (which are more and compara-
bly lipophilic than E1) were not influenced by oil% in ADT
(Fig. 4), thereby rendering a mere physicochemical effect
less likely. In line with the influence of oil% on the respec-
tive estrogens, ADT/GLT of both E2 and E1 were positively
influenced by oil% in ADT but negatively in GLT (p < 0.05). %l influenced levels of E1 in GLT (positively, p < 0.05), levels
of E1-S in GLT (negatively, p < 0.05), and E1-S/E1 in both
GLT and ADT (negatively, p < 0.05; Fig. 4). Interestingly,
levels of 2-MeO-E1 and E2 (which are more and compara-
bly lipophilic than E1) were not influenced by oil% in ADT
(Fig. 4), thereby rendering a mere physicochemical effect
less likely. In line with the influence of oil% on the respec-
tive estrogens, ADT/GLT of both E2 and E1 were positively
influenced by oil% in ADT but negatively in GLT (p < 0.05). Since oil% influenced levels of some estrogens and
ratios thereof in ADT and GLT, variables affecting oil%
were investigated as well. Transcripts encoding enzymes involved in estrogen
(biotrans)formation Savolainen-Peltonen et al. (2014) observed no correlation of
levels of CYP19A1, i.e., aromatase, STS, and HSD17B1, with
levels of E2 in ADT and the respective transcript levels did
also not significantly influence levels of E2 in ADT in the
present study. However, in the present study, levels of E2 in
ADT were influenced positively by levels of the transcript
encoding GUSB (Fig. 4, p < 0.05), the enzyme hydrolyzing
estrogen glucuronides (Fig. 1). Estrogen glucuronides are
mostly associated with elimination from tissues and body
but may also contribute to intratissue levels of estrogens,
even though GUSB and substrates only meet in a highly
controlled manner (Naz et al. 2013). Furthermore, levels of
E2 in ADT were influenced negatively (p < 0.05) by levels of
transcripts encoding HSD17B2, the enzyme forming E1 by
oxidation of E2 (Fig. 1). Congruently, levels of E1 in ADT
were positively influenced by levels of HSD17B2. Moreover,
levels of CYP19A1 influenced levels of E1 in ADT posi-
tively (Fig. 4, p < 0.05). The presence of transcripts encoding
the conjugating enzyme UGT1A9 and levels of transcripts
encoding CYP1A1 influenced the levels of the respective
substrates (i.e., E1, 2-MeO-E1, and E2, respectively) in ADT
negatively (0.10 > p ≥ 0.05). Likewise, levels of SULT1A3/4
influenced E1-S in GLT positively (0.10 > p ≥ 0.05). Forward
selection of exVARs into models describing estrogen levels
in ADT and GLT identified levels of further transcripts, yet
the associations exhibited p values ≥ 0.10 (Fig. 4). l
Besides BMI, smoking has also been associated with
higher levels of E2 and E1 in blood of postmenopausal
women (Endogenous Hormones Breast Cancer Collabora-
tive Group 2011) but not of premenopausal women (Endog-
enous Hormones Breast Cancer Collaborative Group 2013). No studies investigating the impact of smoking on levels
of estrogens in breast tissues have been identified. In the
present study, smoking influenced levels of E1 in ADT
positively and, congruently, E2/E1 in ADT negatively (both
p < 0.05, Fig. 4). In all studies analyzing estrogens in breast tissues of
women without breast cancer, the intake of EADs for oral
contraception or hormone replacement therapy was either
an exclusion criterium or not considered in statistical analy-
ses (Online Resource 1). In the present study, the intake of
ethinyl estradiol did not influence the levels of E2 in GLT
or ADT (Fig. 4). Lifestyle‑associated variables A positive association between BMI and estrogens in serum
has been observed in pre- (E2, E1; Endogenous Hormones
Breast Cancer Collaborative Group 2013) and postmeno-
pausal women (E2, E1, E1-S; Endogenous Hormones Breast
Cancer Collaborative Group 2003, 2011). The common
interpretation is that an increase in BMI leads to an increase
in the mass of adipose tissue within the whole body, accom-
panied by a change in intra- and extramammary function of
adipose tissue (Yaghjyan and Colditz 2011; Brown 2014). Consequently, a higher amount of estrogens is produced and
distributed within the body via blood (Lønning et al. 2011),
contributing to estrogen levels in breast tissue. Yet, no stud-
ies investigating associations between BMI and estrogen
levels in breast tissues derived from women without breast
cancer have been identified. In the present study, BMI influ-
enced levels of E2 in GLT (0.10 > p ≥ 0.05) as well as E1 in
GLT and ADT (p < 0.05) positively. In contrast, tissue levels
of E1-S were not influenced by BMI (Fig. 4). Oil% Median oil% in GLT and ADT were 16% and 85%, respec-
tively (Pemp et al. 2019a). Estrogen levels might be affected
by oil% via: (i) physicochemical distribution of the lipophilic
molecules E2 and E1 and the more hydrophilic E1-S and (ii)
cell-specific enzyme expression in stromal adipocytes. Oil% 1 3 3020 Archives of Toxicology (2020) 94:3013–3025 Biotransformation precursors age-related regression, all p < 0.05), and not significantly,
by BMI (p > 0.1, with no apparent statistical reason such
as collinearity interfering with the exVAR BMI, Online
Resource 4). No studies investigating correlations among estrogens in
breast tissues derived from women without breast cancer
have been identified. In the present study, biotransforma-
tion precursors of the respective estrogens influenced tissue
levels of E2, E1, E1-S, and 2-MeO-E1 positively (p < 0.05,
except for E1-S as precursor for E1 in ADT 0.10 > p ≥ 0.05;
Fig. 4). Thus, exVARs affecting levels of, e.g., E1, may
indirectly contribute to levels of E2, E1-S, and 2-MeO-E1. Interestingly, whereas levels of E1 in GLT were positively
influenced by levels of E1-S, levels of E1 in ADT were posi-
tively influenced by the qualitative presence of both E1-G
(p < 0.05) and E1-S (0.10 > p ≥ 0.05). Notably, both linear regression models exhibited low R2
values (Fig. 4) suggesting either important variables miss-
ing in the model (which seems likely in this case) or large
variations within the data set. Transcripts encoding enzymes involved in estrogen
(biotrans)formation Yet, intake of ethinyl estradiol influenced
ADT/GLT of E2, levels of E1 in GLT (p < 0.05), and E1-S/
E1 in ADT negatively (0.10 > p ≥ 0.05). In contrast, intake
of E2-releasing drugs (containing E2 or E2 valerate) used for
hormone replacement therapy positively influenced levels of
E1-S and E1-S/E1 in ADT (p < 0.05), but did not influence
levels of E2 or E1 in GLT or ADT. Regulation of transcription of genes encoding enzymes
by the respective substrates (up-) and products (down-), is
a common biochemical feedback mechanism. Furthermore,
ligand-activated transcription factors are involved in the reg-
ulation of transcription of genes of biotransforming enzyme
families; e.g., CYP (Tralau and Luch 2013), SULT (Runge-
Morris et al. 2013), and UGT (Hu et al. 2014). Positive asso-
ciations of levels of STS as well as presence of UGT1A9 and
UGT1A10 were observed with the respective substrates of 1 3 3 Archives of Toxicology (2020) 94:3013–3025 3021 the encoded enzymes; i.e., E1-S in ADT, E1 in GLT and
E1 in ADT, respectively (p < 0.05; Fig. 4). UGT1A9 (Cho
et al. 2016) and UGT1A10 (Starlard-Davenport et al. 2008)
may be regulated by activated ESR1, whereas STS may be
regulated via G-protein-coupled ESR action (Gilligan et al. 2017). the encoded enzymes; i.e., E1-S in ADT, E1 in GLT and
E1 in ADT, respectively (p < 0.05; Fig. 4). UGT1A9 (Cho
et al. 2016) and UGT1A10 (Starlard-Davenport et al. 2008)
may be regulated by activated ESR1, whereas STS may be
regulated via G-protein-coupled ESR action (Gilligan et al. 2017). prerequisites (Yaghjyan and Colditz 2011; Sherman et al. 2012; Rosner et al. 2013) for data acquisition concerning
sample characterization as well as specificity and reliability
of estrogen analysis (Pemp et al. 2019a). However, mammoplasty specimens raise concern regard-
ing sample bias, in particular (i) “young age”, (ii) “obesity”,
and (iii) “extremely large fatty breasts” (Sherman et al. 2012), thus putatively reflecting a specific subpopulation. Most specimens were derived from women 40–49, which is
also the modal age group of the respective general popula-
tion (Online Resource 5). Yet, the study population indeed
lacked women older than 66 years and the proportion of
pre-obese and obese women was higher and lower than in
the general population, respectively (Online Resource 6). Moreover, 38% of specimens were classified as laaGLT and
exhibited higher oil% than GLT which could be isolated
directly (section “Obesity and related variables”). Transcripts encoding enzymes involved in estrogen
(biotrans)formation Since
no data on the occurrence of laaGLT in the general female
population are available, it is currently unknown whether
women undergoing mammoplasty are predisposed to laa-
GLT. Oil% significantly influenced levels of E1, E1-S in
GLT, E1-S/E1 in GLT and ADT, as well as ADT/GLT of
E1 and E2 and should, therefore, be considered in sample
characterization of human breast biospecimens. Of note,
oil% in GLT were not influenced by any exVAR deducible
by questionnaire. Since levels of CYP19A1, GUSB, and HSD17B2 influ-
enced levels of estrogens in ADT significantly, exVARs
influencing these variables were investigated, as well. Besides exVARs associated with physiology and lifestyle,
levels of transcripts known to be directly or indirectly
involved in regulation of phase I and phase II biotransfor-
mation were included, since little is known about specific
regulation of the transcription of the genes encoding these
enzymes (Naz et al. 2013; Zhao et al. 2016; Hilborn et al. 2017). Levels of CYP19A1 in ADT were exclusively influ-
enced by the intake of ethinyl estradiol (negatively, Fig. 4,
p < 0.05). It is known that the ovarial synthesis of estrogens
is negatively regulated by estrogen-active compounds (Fleis-
chman et al. 2010). Furthermore, CYP19A1 was detected
less frequently in the endometrium of women taking oral
contraceptives containing ethinyl estradiol than in non-users
(Maia et al. 2013). Low R2 value of the model (Fig. 4) sug-
gests at least one other important variable missing in the
model (e.g., transcript levels of glucocorticoid receptor;
Zhao et al. 2016). Intake of exogenous estrogens, menopausal status, BMI,
and smoking were previously shown to be associated with
levels of estrogens in blood (Endogenous Hormones Breast
Cancer Collaborative Group 2003,2011; Fleischman et al. 2010, 2013; Gaudet et al. 2017). The general consensus
is that blood levels of estrogens contribute to breast tissue
levels, yet whether levels of circulating estrogens serve as
surrogate for levels of estrogens in breast tissues, or more
precisely, in GLT or ADT, is a matter of debate (Lønning
et al. 2011; Bulun et al. 2012; Colditz and Bohlke 2014;
Labrie 2015; Stanczyk et al. 2015; Vihma et al. 2016). Levels of GUSB in ADT were significantly influenced by
levels of AHR, ESR2 (positively), and qualitative presence
of NR1I3 (negatively). Binding sites for transcription fac-
tors such as Sp1 and AP-2 (Naz et al. Transcripts encoding enzymes involved in estrogen
(biotrans)formation 2013) in the promo-
tor of the GUSB gene provide a link to estrogen signaling
(Pellikainen and Kosma 2007; Safe and Kim 2008). BMI
influenced levels of GUSB positively (0.10 > p ≥ 0.05). Most
interestingly, levels of HSD17B2 in ADT were significantly
influenced by BMI, smoking, and postmenopausal status as
well as by levels of ARNT (negatively) and levels of ESR2
and N1I2 (positively). Transcription of the HSD17B2 gene
is regulated by retinoic acid via RAR alpha/RXR alpha teth-
ered to transcription factors Sp1 and Sp3 on the HSD17B2
promoter (Cheng et al. 2008), which provides a link to ESR-
mediated signaling. An obvious link between ARNT or N1I2
and expression of HSD17B2 has not been described yet. In the present study, variables known to affect levels of
circulating estrogens indeed influenced estrogen levels in
breast tissues, as well (Fig. 5).f However, these exVARs affecting levels of circulating
estrogens by targeting ovarial and adiposal production of
estrogens did obviously not suffice to explain the variances
in estrogen levels within the breast. In addition, estrogen
levels in both GLT and ADT were further influenced by
levels or the presence of their precursor estrogens and levels
of transcripts encoding enzymes involved in estrogen bio-
transformation. Interestingly, whereas (as expected, Mueller
et al. 2015) E1-S seems to represent a source of E1 in breast
GLT, intratissue levels of E1 in ADT seem additional to be
regulated by glucuronidation via UGT1A9 (Fig. 5). Further-
more, although E2-3-G was not detected in human breast
tissues, in the light of the contribution of levels of GUSB to Concluding this section, it should be emphasized that lack
of influence of transcript levels does not exclude the contri-
bution of the respective enzyme activities to estrogen levels. Relevance Including E2, E1, E1-S, and 2-MeO-E1, the present study
encompasses the major estrogens detectable in breast tis-
sues derived from women without breast cancer. In contrast
to all previous studies, the present study fulfilled published 1 3022 Archives of Toxicology (2020) 94:3013–3025 Fig. 5 ExVARs associated with intramammary biotransformation
pathways, lifestyle, and reproductive history influencing levels of
estrogens (framed) as well as transcript levels of CYP19A1, GUSB,
HSD17B2 in ADT with p < 0.05 (exVARs written in black color) or
0.10 > p ≥ 0.05 (exVARs written in grey color) identified by multi-
ple regression models using stepwise forward selection. Correlations
between estrogen levels in GLT and ADT determined by Spearman
correlation analyses were described in Pemp et al. (2019a). EE ethi-
nyl estradiol, ERD E2-releasing drug, PostMP postmenopausal status Fig. 5 ExVARs associated with intramammary biotransformation
pathways, lifestyle, and reproductive history influencing levels of
estrogens (framed) as well as transcript levels of CYP19A1, GUSB,
HSD17B2 in ADT with p < 0.05 (exVARs written in black color) or
0.10 > p ≥ 0.05 (exVARs written in grey color) identified by multi- ple regression models using stepwise forward selection. Correlations
between estrogen levels in GLT and ADT determined by Spearman
correlation analyses were described in Pemp et al. (2019a). EE ethi-
nyl estradiol, ERD E2-releasing drug, PostMP postmenopausal status E1 in ADT, i.e., levels of CYP19A1, HSD17B2, presence
of UGT1A9, E1-S, and E1-G, as well as BMI and smoking
(Fig. 5). E1 in ADT, i.e., levels of CYP19A1, HSD17B2, presence
of UGT1A9, E1-S, and E1-G, as well as BMI and smoking
(Fig. 5). levels of E2 in ADT and a ten times higher LOD for E2-3-G
compared to E1-G (Pemp et al. 2019a), E2 glucuronides
cannot be excluded to represent a source of estrogens for the
human breast, as well. Inhibition of CYP19A1 suggested in breast cancer pre-
vention (Advani and Moreno-Aspitia 2014; Colditz and
Bohlke 2014) could thus lower intratissue E2 levels not only
by systemic but also by intracrine mechanisms. However,
systemic alteration of E2 biosynthesis harbors the risk of
losing its beneficial biological effects, e.g., in bone health
(Advani and Moreno-Aspitia 2014). Thus, drugs in develop-
ment for the treatment of endocrine disorders by targeting
enzymes involved in more organ-specific E2 homeostasis
(e.g., inhibitors of HSD17B2 and STS; Konings et al. 2018)
may also be successful in breast cancer chemoprevention. 3 Compliance with ethical standards Depypere HT, Bolca S, Bracke M, Delanghe J, Comhaire F, Blondeel P
(2015) The serum estradiol concentration is the main determinant
of the estradiol concentration in normal breast tissue. Maturitas
81:42–45. https://doi.org/10.1016/j.maturitas.2015.01.014 Conflict of interest The authors declare that they have no conflict of
interest. Endogenous Hormones Breast Cancer Collaborative Group (2002)
Endogenous sex hormones and breast cancer in postmenopausal
women: reanalysis of nine prospective studies. J Natl Cancer Inst
94:606–616 Ethics approval This study was performed in line with the principles
of the Declaration of Helsinki and its later amendments. Approval was
granted by the Ethics Committee of the University of Würzburg, Ger-
many (reference numbers 74/10 and 168/10). Endogenous Hormones Breast Cancer Collaborative Group (2003)
Body mass index, serum sex hormones, and breast cancer risk in
postmenopausal women. J Natl Cancer Inst 95:1218–1226 Consent to participate Informed consent was obtained from all indi-
vidual participants included in the study. Endogenous Hormones Breast Cancer Collaborative Group (2011) Cir-
culating sex hormones and breast cancer risk factors in postmeno-
pausal women: reanalysis of 13 studies. Br J Cancer 105:709–722. https://doi.org/10.1038/bjc.2011.254 Consent for publication All individual participants consented to pub-
lish their anonymized data. Endogenous Hormones Breast Cancer Collaborative Group (2013) Sex
hormones and risk of breast cancer in premenopausal women: a
collaborative reanalysis of individual participant data from seven
prospective studies. Lancet Oncol 14:1009–1019. https://doi. org/10.1016/S1470-2045(13)70301-2f Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Relevance Further supporting the role of intratissue biotransforma-
tion, levels of E2 and of E1 in ADT were also significantly
influenced by levels of HSD17B2. Recently, it was shown
that levels of Hsd17b enzymes significantly influenced
intramammary levels of E2 in the ACI rat model of tumori-
genesis (Pemp et al 2019b), as well, supporting the impor-
tant role of HSD17Bs in intramammary estrogen homeosta-
sis (Hilborn et al. 2017) across species. Furthermore, the present study not only supports the
commonly accepted importance of CYP19A1 in estrogen
homeostasis, but suggests its role within the breast tissue
in addition to a systemic effect. Avoiding indirect associa-
tions in linear regression models, the influence of exVARs
present in ADT on levels of E2 in GLT was not tested in the
present study. However, due to correlations between estrogen
levels in GLT and ADT (Pemp et al. 2019a) and the influ-
ence of precursor estrogens on the respective estrogen levels
shown in the present study, levels of E2 in GLT might be
affected indirectly by any variable significantly influencing In conclusion, a thorough characterization of specimens
enabled taking into account variables related to obesity and
“extremely fatty breasts” during statistical analyses. Tissue
characterization of GLT derived from mammoplasty (and
possibly also of biopsy) specimens by oil% as well as by
lobule type seems to be advisable to prevent sample bias. Novel insights in estrogen homeostasis in the normal
human breast GLT and ADT support contribution of varia-
bles affecting both extra- and intratissue (biotrans)formation 1 3 3 Archives of Toxicology (2020) 94:3013–3025 3023 of estrogens and suggest a central role of E1 levels in breast
ADT homeostasis. The susceptibility of estrogen homeosta-
sis to systemic and tissue-specific modulation renders both
beneficial and adverse effects of further variables associated
with lifestyle and the environment possible. Brown KA (2014) Impact of obesity on mammary gland inflammation
and local estrogen production. J Mammary Gland Biol Neoplasia
19:183–189. https://doi.org/10.1007/s10911-014-9321-0 p
g
Bulun SE, Chen D, Moy I, Brooks DC, Zhao H (2012) Aromatase,
breast cancer and obesity: a complex interaction. Trends Endo-
crinol Metab 23:83–89. https://doi.org/10.1016/j.tem.2011.10.003 Cheng YH, Yin P, Xue Q, Yilmaz B, Dawson MI, Bulun SE (2008)
Retinoic acid (RA) regulates 17beta-hydroxysteroid dehydroge-
nase type 2 expression in endometrium: interaction of RA recep-
tors with specificity protein (SP) 1/SP3 for estradiol metabolism. J Clin Endocrinol Metab 93:1915–1923. Relevance https://doi.org/10.1210/
jc.2007-1536 Acknowledgements Open Access funding provided by Projekt DEAL. The authors would like to thank Prof. Peter Eckert, Prof. Rafael Jakubi-
etz, Dr. Iva Neshkova, and Dr. Ulrike Waldhofen (mammoplasty
specimens). Cho SJ, Ning M, Zhang Y, Rubin LH, Jeong H (2016) 17beta-Estra-
diol up-regulates Udp-glucuronosyltransferase 1a9 expression via
estrogen receptor alpha. Acta Pharm Sin B 6:504–509. https://doi. org/10.1016/j.apsb.2016.04.005 Funding This study was funded by the German Research Founda-
tion with a grant to L. Lehmann (DFG LE 1329/10–1) and is part
of the joint research project, “Isoflavones: Cross-species comparison
of metabolism, estrogen sensitivity, epigenetics and carcinogenesis”. Colditz GA, Bohlke K (2014) Priorities for the primary prevention
of breast cancer. CA Cancer J Clin 64:186–194. https://doi. org/10.3322/caac.21225 Availability of data and material The datasets generated during and/or
analyzed during this study are included in this published article [and its
supplementary information files] or are available from the correspond-
ing author on reasonable request. Collaborative Group on Hormonal Factors in Breast Cancer (2019)
Type and timing of menopausal hormone therapy and breast can-
cer risk: individual participant meta-analysis of the worldwide
epidemiological evidence. Lancet. https://doi.org/10.1016/S0140
-6736(19)31709-X Compliance with ethical standards Figueroa JD, Pfeiffer RM, Patel DA, Linville L, Brinton LA, Gierach
GL, Yang XR, Papathomas D, Visscher D, Mies C, Degnim AC,
Anderson WF, Hewitt S, Khodr ZG, Clare SE, Storniolo AM,
Sherman ME (2014) Terminal duct lobular unit involution of the
normal breast: implications for breast cancer etiology. J Natl Can-
cer Inst 106:286. https://doi.org/10.1093/jnci/dju286 Fleischman DS, Navarrete CD, Fessler DM (2010) Oral contraceptives
suppress ovarian hormone production. Psychol Sci 21:750–752. https://doi.org/10.1177/0956797610368062 Fleming JM, Miller TC, Quinones M et al (2010) The normal breast
microenvironment of premenopausal women differentially influ-
ences the behavior of breast cancer cells in vitro and in vivo. BMC
Med 8:27. https://doi.org/10.1186/1741-7015-8-27 References Gao H, Dahlman-Wright K (2013) Implications of estrogen receptor
alpha and estrogen receptor beta for adipose tissue functions and
cardiometabolic complications. Horm Mol Biol Clin Investig
15:81–90. https://doi.org/10.1515/hmbci-2013-0021 Advani P, Moreno-Aspitia A (2014) Current strategies for the pre-
vention of breast cancer. Breast cancer 6:59–71. https://doi. org/10.2147/BCTT.S39114 Gaudet MM, Carter BD, Brinton LA, Falk R, Gram IT, Luo J, Milne
RL, Nyante SJ, Weiderpass E, Beane Freeman LE, Sandler DP,
Robien K, Anderson KE, Giles GG, Chen WY, Feskanich D,
Braaten T, Isaacs C, Butler LM, Koh WP, Wolk A, Adami HO,
White E, Margolis KL, Thun MJ, Gapstur SM (2017) Pooled g
Austin PC, Steyerberg EW (2015) The number of subjects per variable
required in linear regression analyses. J Clin Epidemiol 68:627–
636. https://doi.org/10.1016/j.jclinepi.2014.12.014 1 3 3024 Archives of Toxicology (2020) 94:3013–3025 replacement therapy. Rejuvenation Res 16:352–363. https://doi. org/10.1089/rej.2013.1407 analysis of active cigarette smoking and invasive breast cancer
risk in 14 cohort studies. Int J Epidemiol 46:881–893. https://doi. org/10.1093/ije/dyw288 Pellikainen JM, Kosma VM (2007) Activator protein-2 in carcinogen-
esis with a special reference to breast cancer–a mini review. Int J
Cancer 120:2061–2067. https://doi.org/10.1002/ijc.22648 g
j
y
Gilligan LC, Gondal A, Tang V, Hussain MT, Arvaniti A, Hewitt AM,
Foster PA (2017) Estrone sulfate transport and steroid sulfatase
activity in colorectal cancer: implications for hormone replace-
ment therapy. Front Pharmacol. https://doi.org/10.3389/fphar
.2017.00103 Pemp D, Kleider C, Schmalbach K, Hauptstein R, Geppert LN, Köll-
mann C, Ickstadt K, Eckert P, Neshkova I, Jakubietz R, Esch HL,
Lehmann L (2019a) Qualitative and quantitative differences in
estrogen biotransformation in human breast glandular and adipose
tissues: implications for studies using mammary biospecimens. Arch Toxicol 93:2823–2833. https://doi.org/10.1007/s00204-019-
02564-w Gram IT, Park S-Y, Maskarinec G, Wilkens LR, Haiman CA, Le
Marchand L (2019) Smoking and breast cancer risk by race/eth-
nicity and oestrogen and progesterone receptor status: the Multi-
ethnic Cohort (MEC) study. Int J Epidemiol 48:501–511. https://
doi.org/10.1093/ije/dyy290 Pemp D, Esch HL, Hauptstein R, Möller FJ, Zierau O, Bosland MC,
Geppert LN, Kleider C, Schlereth K, Vollmer G, Lehmann L
(2019b) Novel insight in estrogen homeostasis and bioactivity
in the ACI rat model of estrogen-induced mammary gland car-
cinogenesis. Arch Toxicol 93:1979–1992. https://doi.org/10.1007/
s00204-019-02483-w Grosse Y, Baan R, Straif K, Secretan B, El-Ghissassi F, Bouvard V,
Benbrahim-Tallaa L, Guha N, Galichet L, Cogliano V, W.O Inter-
national Agency for Research on Cancer Monograph Working
Group (2009) A review of human carcinogens—Part A: phar-
maceuticals. References Lancet Oncol 10:13. https://doi.org/10.1016/s1470
-2045(08)70286-9 Rosner W, Hankinson SE, Sluss PM, Vesper HW, Wierman ME (2013)
Challenges to the measurement of estradiol: an endocrine society
position statement. J Clin Endocrinol Metab 98:1376–1387. https
://doi.org/10.1210/jc.2012-3780 Hilborn E, Stal O, Jansson A (2017) Estrogen and androgen-converting
enzymes 17beta-hydroxysteroid dehydrogenase and their involve-
ment in cancer: with a special focus on 17beta-hydroxysteroid
dehydrogenase type 1, 2, and breast cancer. Oncotarget 8:30552–
30562. https://doi.org/10.18632/oncotarget.15547 Runge-Morris M, Kocarek TA, Falany CN (2013) Regulation of the
cytosolic sulfotransferases by nuclear receptors. Drug Metab Rev
45:15–33. https://doi.org/10.3109/03602532.2012.748794 Hu DG, Meech R, McKinnon RA, Mackenzie PI (2014) Transcrip-
tional regulation of human Udp-glucuronosyltransferase genes. Drug Metab Rev 46:421–458. https://doi.org/10.3109/03602
532.2014.973037 Russo J, Russo IH (2004) Development of the human breast. Maturitas
49:2–15. https://doi.org/10.1016/j.maturitas.2004.04.011 Safe S, Kim K (2008) Non-classical genomic estrogen receptor (ER)/
specificity protein and ER/activating protein-1 signaling path-
ways. J Mol Endocrinol 41:263–275. https://doi.org/10.1677/
JME-08-0103 Jones ME, Schoemaker MJ, Wright LB, Ashworth A, Swerdlow AJ
(2017) Smoking and risk of breast cancer in the generations study
cohort. Breast Cancer Res 19:118. https://doi.org/10.1186/s1305
8-017-0908-4 Savolainen-Peltonen H, Vihma V, Leidenius M, Wang F, Turpeinen U,
Hamalainen E, Tikkanen MJ, Mikkola TS (2014) Breast adipose
tissue estrogen metabolism in postmenopausal women with or
without breast cancer. J Clin Endocrinol Metab 99:E2661–2667. https://doi.org/10.1210/jc.2014-2550 Konings G, Brentjens L, Delvoux B, Linnanen T, Cornel K, Koskimies
P, Bongers M, Kruitwagen R, Xanthoulea S, Romano A (2018)
Intracrine regulation of estrogen and other sex steroid levels in
endometrium and non-gynecological tissues; pathology, physi-
ology, and drug discovery. Front Pharmacol 9:940. https://doi. org/10.3389/fphar.2018.00940 Sherman ME, Figueroa JD, Henry JE, Clare SE, Rufenbarger C, Storni-
olo AM (2012) The Susan G. Komen for the Cure Tissue Bank
at the IU Simon Cancer Center: a unique resource for defining
the “Molecular Histology” of the breast. Cancer Prev Res (Phila)
5:528–535. https://doi.org/10.1158/1940-6207.CAPR-11-0234 g
p
Labrie F (2015) All sex steroids are made intracellularly in periph-
eral tissues by the mechanisms of intracrinology after meno-
pause. J Steroid Biochem Mol Biol 145C:133–138. https://doi. org/10.1016/j.jsbmb.2014.06.001 Stanczyk FZ, Mathews BW, Sherman ME (2015) Relationships of sex
steroid hormone levels in benign and cancerous breast tissue and
blood: a critical appraisal of current science. Steroids 99:91–102. https://doi.org/10.1016/j.steroids.2014.12.011 Labrie F, Ke Y, Gonthier R, Belanger A (2015) Why both LC-MS/MS
and FDA-compliant validation are essential for accurate estro-
gen assays? J Steroid Biochem Mol Biol 149:89–91. https://doi. Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast can-
cer. N Engl J Med 354:270–282. https://doi.org/10.1056/NEJMr
a050776 Yager JD, Davidson NE (2006) Estrogen carcinogenesis in breast can-
cer. N Engl J Med 354:270–282. https://doi.org/10.1056/NEJMr
a050776
Yaghjyan L, Colditz GA (2011) Estrogens in the breast tissue: a sys-
tematic review. Cancer Causes Control 22:529–540. https://doi.
org/10.1007/s10552-011-9729-4
Zhao H, Zhou L, Shangguan AJ, Bulun SE (2016) Aromatase expres-
sion and regulation in breast and endometrial cancer. J Mol Endo-
crinol 57:R19–33. https://doi.org/10.1530/JME-15-0310 References org/10.1016/j.jsbmb.2015.02.003 Starlard-Davenport A, Lyn-Cook B, Radominska-Pandya A (2008)
Novel identification of UDP-glucuronosyltransferase 1A10 as an
estrogen-regulated target gene. Steroids 73:139–147. https://doi. org/10.1016/j.steroids.2007.09.007 Lønning PE, Haynes BP, Straume AH, Dunbier A, Helle H, Knapp-
skog S, Dowsett M (2011) Exploring breast cancer estrogen dis-
position: the basis for endocrine manipulation. Clin Cancer Res
17:4948–4958. https://doi.org/10.1158/1078-0432.CCR-11-0043f Tralau T, Luch A (2013) The evolution of our understanding of
endo-xenobiotic crosstalk and cytochrome P450 regulation and
the therapeutic implications. Expert Opin Drug Metab Toxicol
9:1541–1554. https://doi.org/10.1517/17425255.2013.828692 Maia H Jr, Haddad C, Pinheiro N, Casoy J (2013) The effect of oral
contraceptives on aromatase and Cox-2 expression in the endo-
metrium of patients with idiopathic menorrhagia or adenomyosis. Int J Womens Health 5:293–299. https://doi.org/10.2147/IJWH. S45093 Tsilidis KK, Allen NE, Key TJ, Dossus L, Lukanova A, Bakken K et al
(2011) Oral contraceptive use and reproductive factors and risk
of ovarian cancer in the European Prospective Investigation into
Cancer and Nutrition. Br J Cancer 105:1436–1442. https://doi. org/10.1038/bjc.2011.371 Mensink GB, Schienkiewitz A, Haftenberger M, Lampert T, Ziese
T, Scheidt-Nave C (2013) Overweight and obesity in Germany:
results of the German Health Interview and Examination Sur-
vey for Adults (DEGS1). Bundesgesundheitsblatt Gesund-
heitsforschung Gesundheitsschutz 56:786–794. https://doi. org/10.1007/s00103-012-1656-3 Vihma V, Wang F, Savolainen-Peltonen H, Turpeinen U, Hamalainen
E, Leidenius M, Mikkola TS, Tikkanen MJ (2016) Quantitative
determination of estrone by liquid chromatography-tandem mass
spectrometry in subcutaneous adipose tissue from the breast in
postmenopausal women. J Steroid Biochem Mol Biol 155:120–
125. https://doi.org/10.1016/j.jsbmb.2015.10.004 Mueller JW, Gilligan LC, Idkowiak J, Arlt W, Foster PA (2015) The
regulation of steroid action by sulfation and desulfation. Endocr
Rev 36:526–563. https://doi.org/10.1210/er.2015-1036 Naz H, Islam A, Waheed A, Sly WS, Ahmad F, Hassan I (2013) Human
beta-glucuronidase: structure, function, and application in enzyme 1 3 Archives of Toxicology (2020) 94:3013–3025 3025 Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Yaghjyan L, Colditz GA (2011) Estrogens in the breast tissue: a sys-
tematic review. Cancer Causes Control 22:529–540. https://doi. org/10.1007/s10552-011-9729-4 Zhao H, Zhou L, Shangguan AJ, Bulun SE (2016) Aromatase expres-
sion and regulation in breast and endometrial cancer. J Mol Endo-
crinol 57:R19–33. https://doi.org/10.1530/JME-15-0310 1 3
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INTRODUÇÃO Informações sobre a mortalidade desempenham
importante papel no planejamento local, regional e
nacional. Dados sobre mortalidade contribuem na
identificação da situação demográfica do país e per-
mitem fazer inferências sobre o futuro. A par da
perspectiva demográfica, os dados sobre mortalidade
são medidas importantes das condições sócio-econô-
micas e de saúde. Por seu intermédio mostra-se o
progresso numa das áreas de maior preocupação do
homem, qual seja, a do prolongamento da vida e
prevenção da morte prematura. As chamadas esta-
tísticas de mortalidade também se constituem em
indicadores muito sensíveis de diferenças existentes
nas populações, possibilitando a identificação de
grupos de maior risco a fim de implementar progra-
mas especiais de saúde e de desenvolvimento. São
úteis, por outro lado, para a avaliação de resultados
dos programas já realizados. Além disso, os padrões
de mortalidade relacionam-se com outros aspectos
sociais, tais como a capacidade de trabalho e a ferti-
lidade11. morte, ou (b) as circunstâncias do acidente ou vio-
lência que produziram a lesão fatal"6. Para um determinado óbito, a causa básica de
morte deriva das informações dadas pelo médico ao
certificar o óbito, utilizando, para sua codificação, a
estrutura, as regras de seleção e de modificação e as
disposições correlatas da Classificação Internacional
de Doenças. As regras de seleção permitem a identi-
ficação da causa básica de morte tendo em vista a
posição das afecções mencionadas pelo médico no
Modelo Internacional de Atestado Médico da Causa
de Morte e a relação causal entre as mesmas. As re-
gras de modificação têm a finalidade de aprimorar
a informação sobre a causa básica de morte dando
preferência a determinadas afecções em detrimento
de outras ou associando duas ou mais afecções se-
gundo uma rubrica da Classificação Internacional de
Doenças. Tais regras para a codificação da causa
básica de morte vêm sendo apresentadas na Classifi-
cação Internacional de Doenças há décadas, com a
finalidade de padronizar sua identificação, fato que
contribui para a comparabilidade internacional de
estatísticas de mortalidade10. Os estudos estatísticos de mortalidade levam
em consideração numerosas variáveis, tais como
sexo, idade, estado civil, local de residência e ocor-
rência, ocupação, entre outras, utilizando-as para
a elaboração de análises pormenorizadas. Assim,
apenas como exemplo, quanto à variável idade, a
mortalidade infantil é um dos mais sensíveis indica-
dores de níveis de saúde de populações. Além destas
variáveis, a causa de morte constitui-se em outra das
mais importantes formas de sua análise. * Trabalho realizado no Centro Brasileiro de Classificação de Doenças, Departamento de Epidemiologia da Faculdade de
Saúde Pública da Universidade de São Paulo - Av. Dr. Arnaldo, 715 - 01255 - São Paulo, SP - Brasil.
** Do Departamento de Epidemiologia da Faculdade de Saúde Pública da Universidade de São Paulo - Av. Dr. Arnaldo,
715 - 01255 - São Paulo, SP - Brasil. ESTATÍSTICAS DE MORTALIDADE POR CAUSAS MÚLTIPLAS
NOVAS PERSPECTIVAS COM O SISTEMA ACME * Augusto Hasiak Santo **
Ruy Laurenti** INTRODUÇÃO O conheci-
mento preciso de padrões de mortalidade segundo a
causa de morte permite a pesquisa de eventuais fato-
res etiológicos e a tomada de decisões visando a
prevenção desses fatores. O trabalho da identificação e atribuição de um
código da Classificação Internacional de Doenças
à causa básica é realizado pelo codificador de causas
de morte que, para tanto, recebe treinamento espe-
cializado em cursos apropriados. É um trabalho que
está sujeito a falhas que prejudicam a qualidade da
codificação; tais falhas devem-se a vários fatores,
como os ligados ao tipo de treinamento recebido,
diferenças de interpretação de relações causais entre
afecções, esquecimento de considerar afecções ou de
aplicar regras ou disposições de codificação, erros de
registro de códigos e outros9. Tradicionalmente as estatísticas de mortalidade
vem sendo apresentadas e analisadas segundo apenas
uma causa de morte. A Organização Mundial de
Saúde adotou em 1948, para a mesma, a denomina-
ção de causa básica de morte, conceituando-a como
"(a) a doença ou lesão que iniciou a sucessão de
eventos mórbidos que conduziram diretamente a mento de Epidemiologia da Faculdade de Saúde Pública da Universidade de São Paulo - Av. Dr. Arnaldo,
- São Paulo, SP - Brasil. CAUSAS MÚLTIPLAS DE MORTE Tendo-se em vista as limitações existentes nas
estatísticas de mortalidade que levam em conta
somente uma causa e, principalmente, o fato de que
nem sempre uma morte depende de uma só causa,
há décadas vem sendo sentida a necessidade de estu-
dar-se a mortalidade segundo causas múltiplas. Tais
estatísticas são obtidas pela classificação de todas
as causas - básicas e associadas - informadas nos
atestados de óbito, e forneceriam um volume de
dados e informações sensivelmente maior que aquele
oferecido apenas pela causa básica2,5. Dado que apenas uma causa é selecionada, são
desprezadas, em favor da causa básica, as demais
afecções informadas no atestado médico. Por vezes
esta seleção, se bem que com normas uniformes e
padronizadas, é arbitrária e exclui das tabulações es-
tatísticas doenças que desempenham importante pa-
pel no processo mórbido que conduz à morte. Desta
seleção são excluídas as chamadas causas associadas,
que incluem as causas conseqüenciais, importantes
para a compreensão da cadeia mórbida que leva di-
retamente ao óbito, bem como as causas contribuin-
tes, isto é, aquelas afecções presentes no momento
da morte porém não relacionadas com a causa
básica. Deste modo não se capta o quadro global do
processo mórbido. Também, nas mortes violentas,
apenas são apresentados dados sobre as circunstân-
cias de sua ocorrência, as chamadas causas externas,
omitindo-se a natureza da lesão. Para a apresentação de causas múltiplas, dois
tipos de tabulação têm sido propostos3,5,8. O pri-
meiro deles consistiria numa modificação das tabula-
ções tradicionais de mortalidade que descrevem o
número de óbitos por determinada causa classifica-
dos segundo idade e sexo. Nestas tabulações seria
incluída a apresentação da freqüência segundo a
qual as diferentes categorias diagnósticas viessem
a ser informadas como causas associadas no atestado
de óbito. A soma de ambas as freqüências, devidas
à causa básica e às causas associadas, corresponderia
ao total de informações encontradas no atestado
de óbito. A descrição da mortalidade segundo uma só
causa adequava-se aos padrões de mortalidade do
início do século, quando as mortes se deviam a
doenças agudas, infecciosas ou a violências. À medi-
da que tais afecções passaram a ser controladas, a
sobrevida das pessoas numa população aumentou
conseqüentemente. Com isso, a proporção de adul-
tos e idosos aumenta, e nesta parcela da população
aparecem as denominadas doenças crônicas, que se
tornam então as causas de morte mais freqüentes. A CAUSA BÁSICA E SUAS LIMITAÇÕES As tabulações de mortalidade segundo a causa
básica fazem corresponder a cada óbito uma só
causa, constituindo-se em dados estatísticos simples,
unidimensionais e de fácil compreensão, sendo portanto medidas bem aceitas de mortalidade. Apre-
sentam as causas iniciais do processo mórbido e
assim podem ser usadas pelos responsáveis pela saú-
de pública para a prevenção ou controle desse pro-
cesso. Entretanto, apesar das numerosas vantagens
como dado estatístico, algumas limitações vêm sen-
do apontadas em relação ao uso da causa básica de
morte para a descrição da mortalidade. substituí-la pela metodologia das causas múltiplas,
a qual, por outro lado, daria nova dimensão ao estu-
do da mortalidade. portanto medidas bem aceitas de mortalidade. Apre-
sentam as causas iniciais do processo mórbido e
assim podem ser usadas pelos responsáveis pela saú-
de pública para a prevenção ou controle desse pro-
cesso. Entretanto, apesar das numerosas vantagens
como dado estatístico, algumas limitações vêm sen-
do apontadas em relação ao uso da causa básica de
morte para a descrição da mortalidade. CAUSAS MÚLTIPLAS DE MORTE Não se pretende tipo de associação, num estudo sobre mortalidade
de adultos falecidos em hospitais5, pôde-se consta-
tar que, em atestados de óbito nos quais o diabetes
era mencionado, as causas que compareciam associa-
das eram arteriosclerose, doenças cerebrovasculares,
hipertensão arterial e doenças isquêmicas do cora-
ção. nicamente por meio de tabelas de decisão às quais
foram incorporadas toda a estrutura de códigos da
Classificação Internacional de Doenças, as relações
etiológicas entre as afecções representadas por tais
códigos e interpretadas segundo as disposições das
13 regras de classificação da causa básica. Todos
esses elementos foram programados
logicamente
e de tal forma que se relacionassem entre si perfei-
tamente. Instruções especiais foram elaboradas para
atribuir códigos a todas as afecções, doenças, lesões,
circunstâncias de mortes violentas (acidentes, homi-
cídios, suicídios), procedimentos médicos e demais
informações que podem ser mencionadas nos atesta-
dos de óbito. A programação do ACME recebe a
mensagem codificada que transcreve as informações
dos atestados de óbito e, após processamento, atri-
bui um código à causa básica1,4,9. Várias dificuldades têm sido apontadas para o em-
prego generalizado de causas múltiplas. O grande
volume de dados a ser trabalhado é citado como
principal óbice pois, como mencionado anterior-
mente, o trabalho seria, no mínimo, duplicado,
considerando a média de dois diagnósticos por ates-
tado. De modo especial são encontrados problemas
de codificação com o uso da Classificação Interna-
cional de Doenças que está muito mais estruturada
para identificar a causa básica, sendo particular-
mente problemático o uso de rubricas corresponden-
tes a duas ou mais afecções classificadas de modo
conjunto. Além disso, deve ser lembrado que ine-
xiste experiência internacional sobre tabulação e
análise de causas múltiplas. O Centro Brasileiro de Classificação de Doenças
— CBCD (Centro da OMS para a Classificação de
Doenças em Português) da Faculdade de Saúde Pú-
blica da Universidade de São Paulo, em 1976, tomou
conhecimento do sistema ACME e, de imediato,
interessou-se pelo mesmo, tendo em vista seu
imenso potencial de uso para as estatísticas de mor-
talidade. Estabeleceu-se, a seguir, um intercâmbio
entre o CBCD e o NCHS, de Washington, para a
aquisição de informações pormenorizadas sobre
o sistema ACME. Após cuidadosa análise das ques-
tões envolvidas, concluiu-se pela conveniência de
sua introdução no Brasil. CAUSAS MÚLTIPLAS DE MORTE O CBCD consultou a Fun-
dação SEADE (Sistema Estadual de Análise de Da-
dos) que também se interessou pelo sistema e ace-
deu em testar seu uso no Estado de São Paulo. O NCHS enviou ao CBCD cópia do programa, bem
como toda a documentação correlata, que as cedeu
à Fundação SEADE. Teve início um trabalho entre
o CBCD e aquela Fundação para introduzir o siste-
ma ACME na apuração das causas de morte do
Estado de São Paulo, trabalho este que compreen-
deu, dentre outras
atividades, treinamento
de
codificadores, adaptação do programa e análise de
problemas operacionais. A par disso, técnicos do
CBCD e da Fundação SEADE estiveram no NCHS
a fim de observar o funcionamento do sistema
ACME. CAUSAS MÚLTIPLAS DE MORTE Para a descrição destas mortes o conceito de causa
básica já não é tão satisfatório, desde que, mesmo
o médico, por vezes, não é capaz de identificar a
afecção que iniciou uma sucessão de eventos pa-
tológicos, observando, antes, uma associação de
doenças. As mortes devidas a doenças crônicas são
determinadas por diversas afecções presentes no
falecido, as quais nem sempre têm entre si relação
etiológica com vistas à seleção de uma causa básica
claramente determinada. O segundo tipo de tabulação constitui-se em uma
nova forma de apresentação de dados sobre mortali-
dade por meio de associações de causas. A morte
ocorre freqüentemente devido à ação sinérgica de
duas ou mais afecções presentes no falecido e tal
fato não se reflete nas estatísticas segundo causa
básica. Isso pode ser exemplificado para o caso de
sarampo, antes do advento da vacinação, quando as
taxas de mortalidade em países desenvolvidos eram
bem menores que as verificadas nos subdesenvolvi-
dos, nos quais as taxas elevadas eram devidas à pre-
sença de desnutrição atuando como causa associada. Com maior propriedade, a morte devida a doenças
crônicas é melhor descrita considerando-se associa-
ções de causas; por exemplo, freqüentemente as
doenças cerebrovasculares incidem em pessoas com
hipertensão e certas complicações renais em pacien-
tes diabéticos. De modo sintético, considerando-se que estudos
internacionais e em nosso meio verificam média de
cerca de dois diagnósticos informados por atestado
de óbito, pode-se dizer que o uso apenas da causa
básica determina a perda de praticamente a metade
das informações sobre o padrão nosológico da mor-
talidade das populações3,5,8. Vários tipos de associação de causas são possíveis,
tais como a da causa básica com as demais causas
associadas ou de qualquer causa com as demais in-
formadas. Como exemplo do primeiro tipo, num
estudo sobre mortalidade em doentes mentais8,
verificou-se que, nos óbitos em que a cirrose hepática
era selecionada como causa básica, as causas associa-
das mais freqüentes foram desnutrição, alcoolismo e
complicações hepáticas. Relativamente ao segundo Em que pesem as considerações sobre sua limita-
ção, o emprego da causa básica de morte continua
a ser essencial para análise de tendências históricas
e para comparabilidade entre países. O SISTEMA ACME PARA CLASSIFICAÇÃO DE
CAUSA BÁSICA E DE CAUSAS MÚLTIPLAS As citadas dificuldades inerentes ao volume de
dados foram resolvidas com o advento dos compu-
tadores que facultam o seu processamento eletrô-
nico. Tal fato permitiu o desenvolvimento por parte
do "National Center for Health Statistics" (NCHS),
dos Estados Unidos, de um sistema automático para
classificar, além da causa básica de morte, todas as
demais afecções indicadas no atestado médico da
declaração de óbito1,4. Denominado ACME, sigla
para "Automated Classification of Medical Entities",
este sistema apresenta-se, não só como uma alter-
nativa adequada ao trabalho manual para a classifi-
cação da causa básica de morte, como também
permite o registro de todos os códigos das afecções
presentes no atestado médico, tornando disponíveis
dados sobre causas múltiplas. Portanto, o sistema
ACME realiza basicamente dois trabalhos: identifica
a causa básica de morte e arquiva dados sobre as
demais causas associadas. Tradicionalmente, a classificação da causa básica
de morte tem sido feita manualmente pelos chama-
dos codificadores. Esse trabalho é realizado utilizan-
do a estrutura da Classificação Internacional de
Doenças, suas quatro regras de seleção e nove de
modificação. Para tanto, o codificador recebe inten-
sivo treinamento, o qual é internacionalmente nor-
matizado
pela Organização Mundial da Saúde. Entretanto, como foi visto, é um trabalho sujeito a
falhas. O sistema ACME dá margem a maior padro-
nização e controle sobre o processo de codificação
de causa básica de morte, desde que automatiza tais
procedimentos. A causa básica é identificada eletro- Em 1976, foi criado no Brasil, pelo Ministério
da Saúde, o Subsistema Nacional de Informações
sobre Mortalidade integrando todos os serviços
estaduais de apuração de dados das declarações
de óbito, objetivando o aprimoramento das estatís-
ticas de mortalidade. Considerando os trabalhos em
relação ao sistema ACME, estabeleceu-se um convê-
nio entre o Ministério da Saúde e a Fundação
SEADE para instalação preliminar do mesmo no
Estado de São Paulo, convênio esse que previa,
oportunamente, a cessão de todas as rotinas técni-
cas e administrativas ao Ministério, para eventual
aplicação em todo o Brasil7. A partir de 1983, o processamento das declara-
ções de óbito do Estado de São Paulo, em torno
de 180.000, realiza-se ja pelo sistema ACME. O
CBCD, atendendo aos objetivos que desde a sua
criação vem sendo perseguidos, qual seja o de traba-
lhar para a melhoria das informações contidas nas
estatísticas, de mortalidade, vem, continuamente,
prestando colaboração direta à Fundação SEADE. O SISTEMA ACME PARA CLASSIFICAÇÃO DE
CAUSA BÁSICA E DE CAUSAS MÚLTIPLAS Esta colaboração compreende não só treinamento
e supervisão de codificadores como também a elaboração de tabelas de decisão e de validade de
causas de morte que se adequem para o nosso meio,
a promoção de estudos sobre familiarização de pro-
cedimentos e a sugestão das formas de tabulação
final de dados. A implantação definitiva desse sistema amplia,
desse modo, as possibilidades de uso das estatísticas
de mortalidade não somente para os administradores
de saúde como também para epidemiologistas e
demógrafos. REFERÊNCIAS BIBLIOGRÁFICAS 1. CHAMBLEE, R.F. & EVANS, M.C. New dimensions
in cause of death statistics. Amer.J.publ.Hlth, 72:
1265-70, 1982. na Reunião de Diretores de Centros Colaboradores
da OMS para a Classificação de Doenças, São Paulo,
1978 - mimeografado ]. 2. DORN, H.F. Underlying and contributory causes of
death. In: Haenszel, W., ed. Epidemiological appro-
aches to the study of cancer and other chronic
diseases. Bethesda, Md, National Cancer Institute,
1966. p. 421-30. 8. SANTO, A.H. Estudo crítico das estatísticas de causa
de morte em doentes portadores de transtornos
mentais. São Paulo, 1980. [ Dissertação de Mestrado
- Faculdade de Saúde Pública da USP ] . 9. SANTO, A.H. Causas múltiplas de morte: formas de
apresentação e métodos de análise. São Paulo, 1983. [Projeto
de tese de doutoramento apresentado à
Comissão de Pós-Graduação da Faculdade de Saúde
Pública da USP - mimeografado ]. 3. DORN, H.F. & MORIYAMA, I.M. Uses and signifi-
cance of multiple cause tabulations for mortality
statistics. Amer.J.publ. Hlth, 54: 400-6, 1964. 4. ISRAEL, R.A.; ROSENBERG, H.M. &CURTIN, L.R. Analytical potential for multiple cause-of-death
data. [Draft 4/11/84 - National Center for Health
Statistics]. 10. SANTO, A.H. & LAURENTI, R. General review on
mortality coding rules. Geneva. World Health Orga-
nization, 1983. (DES/ICD-10/83.15). [Apresentado
ao Preparatory Meeting on ICD-10, Geneva, 1983 ] 5. LAURENTI, R. Causas múltiplas de morte. São Paulo,
1973. [
Tese de Livre-Docência - Faculdade de
Saúde Pública da USP ]. 11. UNITED NATIONS. Department of International
Economic and Social Affairs. Levels and trends of
mortality since 1950; a joint study by the United
Nations and the World Health Organization. New
York, 1982. Recebido para publicação em 15/08/1986
Aprovado para publicação em 22/08/1986 11. UNITED NATIONS. Department of International
Economic and Social Affairs. Levels and trends of
mortality since 1950; a joint study by the United
Nations and the World Health Organization. New
York, 1982. 6. MANUAL of the international statistical classification
of diseases, injuries and causes of death; 6th revision. Geneva, World Health Organization, 1948. Recebido para publicação em 15/08/1986 7. MARQUES, R.M.; TRONKOS, J.A. & TARDELLI
A.O. Technical report on feasibility testing of
"ACME" system. São Paulo, 1978. [Apresentado
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Pleiotropy of Cancer Susceptibility Variants on the Risk of Non-Hodgkin Lymphoma: The PAGE Consortium
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Abstract doi:10.1371/journal.pone.0089791 Editor: Ludmila Prokunina-Olsson, National Cancer Institute, National Institutes of Health, United States of America Editor: Ludmila Prokunina-Olsson, National Cancer Institute, National Institutes of Health, United States of America Received July 20, 2013; Accepted January 27, 2014; Published March 5, 2014 Received July 20, 2013; Accepted January 27, 2014; Published March 5, 2014 Copyright: 2014 Lim 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. Copyright: 2014 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attrib
use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Lim et al. This is an open-access article distributed under the term
use, distribution, and reproduction in any medium, provided the original author and Funding: The Population Architecture Using Genomics and Epidemiology (PAGE) program is funded by the National Human Genome Research Institute (NHGRI),
supported by U01HG004803 (CALiCo), U01HG004798 (EAGLE), U01HG004802 (MEC), U01HG004790 (WHI), and U01HG004801 (Coordinating Center), and their
respective NHGRI ARRA supplements. The complete list of PAGE members can be found at http://www.pagestudy.org. The data and materials included in this
report result from collaboration between the following studies: The ‘‘Epidemiologic Architecture for Genes Linked to Environment (EAGLE)’’ is funded through the
NHGRI PAGE program (U01HG004798-01 and its NHGRI ARRA supplement). Genotyping services for select NHANES III SNPs presented here were also provided by
the Johns Hopkins University under federal contract number (N01-HV-48195) from NHLBI. The study participants derive from the National Health and Nutrition
Examination Surveys (NHANES), and these studies are supported by the Centers for Disease Control and Prevention. The dataset used for the analyses described
were obtained from Vanderbilt University Medical Center’s BioVU which is supported by institutional funding and by the Vanderbilt CTSA grant UL1 TR000445
from NCATS/NIH. The Multiethnic Cohort study (MEC) characterization of epidemiological architecture is funded through the NHGRI PAGE program
(U01HG004802 and its NHGRI ARRA supplement). The MEC study is funded through the National Cancer Institute (R37CA54281, R01 CA63, P01CA33619,
U01CA136792, and U01CA98758). Funding support for the ‘‘Epidemiology of putative genetic variants: The Women’s Health Initiative’’ study is provided through
the NHGRI PAGE program (U01HG004790 and its NHGRI ARRA supplement). Pleiotropy of Cancer Susceptibility Variants on the Risk
of Non-Hodgkin Lymphoma: The PAGE Consortium Pleiotropy of Cancer Susceptibility Variants on the Risk
of Non-Hodgkin Lymphoma: The PAGE Consortium Unhee Lim1*, Jonathan M. Kocarnik2, William S. Bush3, Tara C. Matise4, Christian Caberto1,
Sungshim Lani Park5, Christopher S. Carlson2, Ewa Deelman4, David Duggan6, Megan Fesinmeyer7,
Christopher A. Haiman5, Brian E. Henderson5, Lucia A. Hindorff8, Laurence N. Kolonel1, Ulrike Peters2,
Daniel O. Stram5, Maarit Tiirikainen1, Lynne R. Wilkens1, Chunyuan Wu2, Charles Kooperberg2,
Loı¨c Le Marchand1 ,
,
,
,
,
Sungshim Lani Park5, Christopher S. Carlson2, Ewa Deelman4, David Duggan6, Megan Fesinmeyer7,
Christopher A. Haiman5, Brian E. Henderson5, Lucia A. Hindorff8, Laurence N. Kolonel1, Ulrike Peters2,
Daniel O. Stram5, Maarit Tiirikainen1, Lynne R. Wilkens1, Chunyuan Wu2, Charles Kooperberg2,
1 1 Epidemiology Program, University of Hawaii Cancer Center, Honolulu, Hawaii, United States of America, 2 Public Health Sciences, Fred Hutchinson Cancer Research
Center, Seattle, Washington, United States of America, 3 Center for Human Genetics Research, Vanderbilt University, Nashville, Tennessee, United States of America,
4 Department of Genetics, Rutgers University, Piscataway, New Jersey, United States of America, 5 Department of Preventive Medicine, Norris Comprehensive Cancer
Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America, 6 Translational Genomics Research Institute, Phoenix,
Arizona, United States of America, 7 Center for Child Health, Behavior and Development, Seattle Children’s Research Institute, Seattle, Washington, United States of
America, 8 Division of Genomic Medicine, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America Abstract Background: Risk of non-Hodgkin lymphoma (NHL) is higher among individuals with a family history or a prior diagnosis of
other cancers. Genome-wide association studies (GWAS) have suggested that some genetic susceptibility variants are
associated with multiple complex traits (pleiotropy). Objective: We investigated whether common risk variants identified in cancer GWAS may also increase the risk of
developing NHL as the first primary cancer. Methods: As part of the Population Architecture using Genomics and Epidemiology (PAGE) consortium, 113 cancer risk
variants were analyzed in 1,441 NHL cases and 24,183 controls from three studies (BioVU, Multiethnic Cohort Study,
Women’s Health Initiative) for their association with the risk of overall NHL and common subtypes [diffuse large B-cell
lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia or small lymphocytic lymphoma (CLL/SLL)]
using an additive genetic model adjusted for age, sex and ethnicity. Study-specific results for each variant were meta-
analyzed across studies. Results: The analysis of NHL subtype-specific GWAS SNPs and overall NHL suggested a shared genetic susceptibility
between FL and DLBCL, particularly involving variants in the major histocompatibility complex region (rs6457327 in
6p21.33: FL OR = 1.29, p = 0.013; DLBCL OR = 1.23, p = 0.013; NHL OR = 1.22, p = 5.96E-05). In the pleiotropy analysis, six risk
variants for other cancers were associated with NHL risk, including variants for lung (rs401681 in TERT: OR per C allele = 0.89,
p = 3.76E-03; rs4975616 in TERT: OR per A allele = 0.90, p = 0.01; rs3131379 in MSH5: OR per T allele = 1.16, p = 0.03), prostate
(rs7679673 in TET2: OR per C allele = 0.89, p = 5.76E-03; rs10993994 in MSMB: OR per T allele = 1.09, p = 0.04), and breast
(rs3817198 in LSP1: OR per C allele = 1.12, p = 0.01) cancers, but none of these associations remained significant after
multiple test correction. Conclusion: This study does not support strong pleiotropic effects of non-NHL cancer risk variants in NHL etiology;
however, larger studies are warranted. Citation: Lim U, Kocarnik JM, Bush WS, Matise TC, Caberto C, et al. (2014) Pleiotropy of Cancer Susceptibility Variants on the Risk of Non-Hodgkin Lymphoma: The
PAGE Consortium. PLoS ONE 9(3): e89791. Abstract The WHI program is funded by the National Heart, Lung, and Blood Institute; NIH; and
U.S. Department of Health and Human Services through contracts N01WH22110, 24152, 32100-2, 32105-6, 32108-9, 32111-13, 32115, 32118-32119, 32122, 42107-
26, 42129-32, and 44221. A full listing of WHI investigators can be found at: http://www.whiscience.org/publications/WHI_investigators_shortlist.pdf. Additional
support for Dr. Kocarnik was provided by R25CA94880 from NCI. Assistance with phenotype harmonization, SNP selection and annotation, data cleaning, data
management, integration and dissemination, and general study coordination was provided by the PAGE Coordinating Center (U01HG004801-01 and its NHGRI March 2014 | Volume 9 | Issue 3 | e89791 1 PLOS ONE | www.plosone.org 1 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma ARRA supplement). The National Institutes of Mental Health also contributes to the support for the Coordinating Center. The funders had no role in study design, data
collection and analysis, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ulim@cc.hawaii.edu * E-mail: ulim@cc.hawaii.edu patients without any prior or prevalent cancer diagnoses (except
non-melanoma skin cancer) and with a similar reported race/
ethnicity and age at clinic visit (within 5 years) as cancer cases. The
MEC is a population-based prospective cohort of over 215,000
men and women in Hawaii and Los Angeles, aged 45–75 years at
recruitment and primarily of five ancestries (white, African
American, Latino, Japanese American or Native Hawaiian)
[19,20]. Incident cancer cases in the MEC were identified by
linkage with Hawaii and California SEER tumor registries, from
1993 through October 2010. The WHI is a prospective cohort
study investigating postmenopausal women’s health in the U.S. [21]. A total of 161,838 women of ages 50–79 and of various race/
ethnic groups (white, African American, Latino, Asian/Pacific
Islander or American Indian) were recruited from 40 clinical
centers throughout the U.S. in 1993–1998 for three clinical trials
and an observational study. Medical history, including cancer
incidence,
is
updated
annually
by
mail
and/or
telephone
questionnaires and confirmed by medical records and pathologic
reports [22]. The current WHI analysis includes NHL cases
identified through August 2009. Selection of Cases and Controls We limited our analysis to NHL cases and controls with no
previous cancer (except non-melanoma skin cancer) in order to
assess genetic pleiotropy without the possibility of confounding by
previous cancers or treatments on the risk of NHL. MEC defined
NHL cases based on the current World Health Organization
classification that considered chronic lymphocytic leukemia (CLL)
as a different presentation of the same disease as small lymphocytic
lymphoma (SLL) [23,24]. BioVU and WHI defined NHL based
on the SEER classification and did not include CLL. Histology
information based on the International Classification of Disease-
Oncology (ICD-O3) was available in BioVU [25] and MEC [19]
through linkage with tumor registries and in WHI [26] through
systematic morphology coding of medical record information for
the classification of the three most common NHL subtypes: diffuse
large B-cell lymphoma (DLBCL; 9678–9680, 9684, 9689, 9699),
follicular lymphoma (FL; 9690–9691, 9695, 9698) and CLL
(9823)/SLL (9670) [24]. DLBCL was not ascertained in BioVU
due to a prioritization for more common cancers in their PAGE
analyses. For the current NHL analysis, all three studies included
controls that were matched to cases of common cancers being
investigated in the PAGE consortium (breast, colorectal, ovarian
and prostate cancers and melanoma in all studies, and endometrial
and lung cancers, and NHL in MEC and WHI). The matching
was performed using frequency matching based on age at Abstract All studies were approved by
Institutional Review Boards at their respective study sites: the
Vanderbilt Institutional Review Board for BioVU, the Human
Studies Program at the University of Hawaii and Office for the
Protection of Research Subjects at the University of Southern
California for MEC, and the Fred Hutchinson Cancer Research
Center Institutional Review Board for WHI. All participants of
MEC and WHI provided written informed consent. All BioVU
participants signed a ‘‘consent-to-treatment’’ form, informing
them that anonymized genetic information from their discarded
blood, along with de-identified EMR information, will be used for
research and were given the choice to check an ‘‘opt-out’’ box if
declining to participate [17]. Introduction Non-Hodgkin lymphoma (NHL) is the sixth most common
incident cancer in the U.S. [1]. Although immune suppression,
autoimmune disorders and certain infectious agents have been
identified as strong risk factors for NHL, common host charac-
teristics are also likely to be involved in the etiology of NHL [2]. Risk of NHL has been reported to be greater among individuals
with a first-degree family history of hematopoietic cancers [3]. NHL is also a common second primary cancer among survivors of
adult leukemia, laryngeal/pharyngeal cancer, renal cell carcinoma
and melanoma, suggesting common genetic and/or environmen-
tal etiology, although it is difficult to rule out a treatment effect
from the first cancer [4–6]. In searching for the shared genetic
basis of disease, genome-wide association studies (GWAS) have
discovered a number of risk variants that demonstrate associations
with two or more complex traits (pleiotropy) [7]. A systematic
review of the U.S. National Human Genome Research Institute
(NHGRI) Catalog of Published GWAS reported that 16.9% of
genes and 4.6% of single nucleotide polymorphisms (SNPs) in the
catalog have shown such pleiotropic associations [8,9]. The
proportion of pleiotropic variants was higher than expected by
chance and was particularly high among cancer risk variants, as
well as among the variants associated with altered immunity and
metabolic syndrome. Thus, genetic variations involved in cancer-
related pathways may increase the risk of cancer of multiple types
[10], including NHL. A good example is the multiple cancer site
associations reported for variants at 8q24 [10,11], a region where
some lymphoid malignancies also exhibit translocations and a
common susceptibility SNP [12–15]. In this study, we examined whether established risk variants
identified in published GWAS of 17 common cancers present
pleiotropic associations with the risk of NHL and its histologic
subtypes in three well-characterized studies, as part of the
Population
Architecture
using
Genomics
and
Epidemiology
(PAGE) consortium [16]. We also explored whether variants
identified for specific NHL subtypes are also associated with
overall NHL risk, and whether any such associations differ across
ethnic groups. Biospecimen Collection, SNP Selection, and Genotyping Biospecimen Collection, SNP Selection, and Genotyping Biospecimen Collection, SNP Selection, and Genotyping
BioVU extracted DNA from discarded whole blood samples for
patients drawn as part of routine clinical testing [17]. In the MEC,
DNA of NHL cases was extracted from pre-diagnostic samples
included in its prospective blood repository of over 67,000 cohort
participants assembled in 2001–2006. DNA samples for controls
were from either the prospective blood repository or from case-
control studies of breast, colorectal and prostate cancers [20,27]. The distribution of established cancer risk factors in the biospeci-
men sub-cohort was similar to that in the entire MEC cohort. DNA samples in WHI were extracted from pre-diagnostic blood
collected at time of enrollment. A total of 113 SNPs were selected and genotyped by one or
more of the three PAGE studies based on genome-wide significant
associations (p,5.06E-08) [28] in the cancer GWAS literature at
the time of the study design (March 2010). These non-NHL cancer
SNPs included risk variants for bladder, brain (glioma), breast,
colorectal, esophageal, lung, nasopharyngeal, neuroblastoma,
ovarian, pancreatic, acute lymphoblastic leukemia, prostate, skin
(basal cell carcinoma, melanoma), testicular germ cell, and thyroid
cancers. The NHL SNPs, which included one risk variant for
follicular lymphoma (FL) [29,30] and 8 variants for chronic
lymphocytic leukemia (CLL) [15,30,31], were only considered in
associations with NHL and were excluded from the pleiotropy
analysis. All samples were additionally genotyped for the ancestry
informative markers (AIMs) described by Kosoy et al. [32] BioVU
used Sequenom’s iPLEX Gold coupled with MassARRAY
MALDI-TOF MS detection and Illumina’s BeadXpress with a
custom
GoldenGate
genotyping
assay. MEC
used
Applied
Biosystems Taqman SNP genotyping assays on the OpenArray
and the 7900HT Real-Time PCR platforms. WHI used Illumina
BeadXpress with the Veracode GoldenGate genotyping assay. All
sites used blind duplicate controls. Samples with low overall call
rates (,90% of SNPs) were excluded. SNPs were excluded based
on deviation from ethnicity-specific Hardy-Weinberg equilibrium
(p,0.01), low call rates (,95%) or low concordance rates – range
of minimum varied between 96.5 and 99% in the studies. In
addition to site-specific quality control as above, all PAGE study
sites genotyped the same 360 DNA samples from the International
HapMap Project with excellent concordance rates with the
published genotype data [16]. Results Characteristics of the NHL cases and controls in the BioVU,
MEC and WHI studies are shown in Table 1. Median age of
NHL cases and controls was the highest in MEC, followed by
WHI and BioVU, and both BioVU and MEC had a slightly
higher representation of men over women. Cases in BioVU and
WHI were mostly whites, whereas MEC had more even
distribution of four ethnic groups. We first investigated nine previously published GWAS risk
variants for specific NHL subtypes (one for FL and 8 for CLL) for
an association with overall NHL risk to test for a shared genetic
susceptibility. The association reported for FL with rs6457327, in
6p21.33, the major histocompatibility complex region (MHC),
replicated in our data for FL [summary OR per allele C vs. A = 1.29 (1.05–1.57), p = 0.013; Figure 1(a), Table S1 in File
S1] and was also observed for DLBCL [OR = 1.23 (1.04–1.44),
p = 0.013] and overall NHL [OR = 1.22 (1.11–1.34), p = 5.92E-05;
Figure 1(b)], but not for CLL/SLL [OR = 1.05 (0.81–1.36),
p = 0.73]. When the meta-analysis on overall NHL was limited to
MEC and WHI, considering that BioVU did not include DLBCL,
the association with the published FL risk variant remained the
same [OR = 1.22 (1.11–1.33), p = 5.92E-05]. In the MEC, where
all main subtypes were examined, including CLL, the OR for
the association of allele C of rs6457327 with FL did not differ from
the corresponding OR for DLBCL (p-het. from polytomous
regression = 0.61), CLL/SLL (p-het. = 0.23) or other subtypes
(p-het. = 0.73). We did observe heterogeneity in the association
for the FL risk variant (rs6457327) and overall NHL across study
sites (Cochran Q = 6.58; p-het. = 0.01), which was eliminated when
the analysis was limited to whites only [Figure 1(c); OR for allele
C
of
rs6457327 = 1.30
(1.16–1.47),
p = 8.83E-06;
Cochran Biospecimen Collection, SNP Selection, and Genotyping After these stringent quality control
procedures, 1,441 NHL cases (BioVU, n = 293; MEC, n = 372;
WHI, n = 776) and 24,183 controls (BioVU, n = 9,002; MEC,
n = 9,091; WHI, n = 6,090) were included in the current analysis. Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma for number of risk alleles). The unconditional logistic regression
model was adjusted for age, sex and race/ethnicity. Residual
confounding by race/ethnicity was examined by additionally
adjusting for principal components of genetic ancestry (top three in
BioVU and WHI and top four in MEC). Effect modification by
sex was assessed by a Wald test of the cross-product terms of sex
and the continuous SNP variable in BioVU and MEC (WHI
includes only women). Heterogeneity across race/ethnic groups
was tested similarly using a Wald test in the MEC, where cases of
non-white ethnic groups were available in substantial numbers. Also, heterogeneity in the SNP-cancer associations across common
NHL subtypes (DLBCL, FL, CLL/SLL) was examined in the
MEC, where all subtypes were ascertained, by performing
polytomous logistic regression using common controls. A risk
score was computed to examine the combined effect of 53 cancer
variants that were genotyped in all three studies, by summing up
the number of risk alleles (0, 1 or 2 for each SNP) in individuals
across SNPs. For subjects with missing genotypes for any of the 53
variants, missing genotypes were estimated using the allele
frequencies among controls of the same ethnicity in each study. The risk score was examined as both a continuous and a
categorical variable (using quartile cut points based on the
distribution among controls). To summarize results from the three
studies, we carried out a meta-analysis for each variant and for the
risk score variable in fixed-effects models using METAL [33]. Heterogeneity across studies was evaluated using Cochran’s Q
statistic. Analyses were conducted initially with significance
considered at p,0.05 (two-sided). To control for the potentially
inflated Type 1 error due to multiple comparisons, we used
Bonferroni correction (p = 0.05/113 = 4.42E-04) to determine the
statistical significance threshold for results. diagnosis or clinic visit (+/25 yrs), sex and race/ethnicity in
BioVU; and individual matching for each case based on age at
cohort entry (+/25 yrs), sex and race/ethnicity in MEC; and age
at enrollment (+/23 yrs), enrollment date (+/2365 days), race/
ethnicity, and randomization arms (observational study or clinical
trial
assignment
to
hormone
replacement
therapy,
dietary
modification, or calcium/vitamin D supplement) in WHI. WHI
also included additional controls selected from other genetic
studies based on the availability of biomarkers. Study Populations The PAGE consortium was established in 2008 by the U.S. National Human Genome Research Institute to investigate
well-replicated genetic variants for complex diseases in several
large, ethnically diverse studies (https://www.pagestudy.org) [16]. Three PAGE studies participated in this analysis: biorepository of
the Vanderbilt University (BioVU), the Multiethnic Cohort Study
(MEC) and the Women’s Health Initiative (WHI). BioVU is a study at Vanderbilt University Medical Center that
links de-identified electronic medical records (EMR) to a DNA
biobank [17,18]. Out of ,130,000 BioVU participants, over 6,098
cancer cases were identified from 2009–2011 through linkage with
the hospital tumor registry or search of diagnostic codes in the
EMR. Race/ethnicity was recorded by hospital staff in the EMR
(white, African American, Latino or Asian American) and
confirmed using principal components analysis of ancestry-
informative markers (AIMs). Controls included 9,152 BioVU March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 2 Statistical Analysis Unconditional logistic regression analysis was used in each study
to estimate the association of cancer risk variants and NHL risk as
odds ratios (ORs) and 95% confidence intervals (CIs). For each
cancer risk variant, the allele that increased the risk of cancer in
the original report was modeled against the low risk allele. Thus,
ORs for NHL would be expected to be .1 if the association was in
the same direction as the one found in the cancer GWAS study. Each biallelic SNP was coded as a continuous variable (0, 1 or 2 March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 3 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Table 1. Characteristics of non-Hodgkin lymphoma (NHL) cases and controls in the PAGE studies. Statistical Analysis Among the eight GWAS risk variants for CLL, one
variant replicated in our data for CLL/SLL [OR per allele G of
rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027]
and remained significant after Bonferroni correction (p,0.0063;
Table S1 in File S1). None of the CLL variants were associated
with overall NHL risk (p.0.05; data not shown). For example, in
the
MEC,
the
association
of
rs17483466
with
CLL/SLL
significantly differed from that with FL (p-het. from polytomous
regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). Of the 113 GWAS risk variants for cancers other than NHL
that were examined in PAGE, 53 SNPs were genotyped in all
three studies, and the other 60 variants were typed in one or two
studies (Table S2 in File S1). Six of the 53 SNPs showed nominal
associations with the risk of overall NHL, including three risk
variants originally identified for lung cancer, two risk variants
identified for prostate cancer and one risk variant for breast cancer
(Table 2). None of these associations remained significant after
multiple test correction (i.e., p.4.4E-04 for 113 SNPs). Two lung
cancer risk variants in the TERT region (rs401681, OR per
allele C = 0.89 (0.82–0.96), p = 0.0037; rs4975616, OR per allele
A = 0.90 (0.83–0.97), p = 0.010), as well as the prostate cancer risk
variant (rs7679673 in TET2; OR per allele C = 0.89 (0.82–0.97), p = 0.0057), were associated with a decreased risk of overall NHL. The two TERT variants were in linkage disequilibrium (LD)
among whites (R2 = 0.84) and Native Hawaiians (R2 = 0.79) but
less so in other ethnic groups (R2 = 0.53 for African Americans;
0.47 for Latinos, 0.30 for Japanese Americans). The breast cancer
susceptibility variant rs3817198 in LSP1 [OR per allele C = 1.12
(1.03–1.22), p = 0.011], the lung cancer SNP rs3131379 in the
MHC region in chromosome 6 (6p21.33) [OR per allele T = 1.16
(1.01–1.33), p = 0.030] and the prostate cancer variant rs10993994
in MSMB [OR per allele T = 1.09 (1.01–1.18), p = 0.036] were
each associated with an increased risk of overall NHL. The
associations for the six variants above did not differ significantly
across study sites, except for rs401681 (TERT), which showed a
stronger inverse association in BioVU and MEC than in WHI
(Cochran Q = 6.26, p-het. = 0.04; Table 2). Statistical Analysis These 6 variants
showed the same or similar summary ORs when the analysis was
limited to MEC and WHI, where overall NHL included DLBCL,
with 4 variants showing nominal significance (unadjusted p,0.05;
data not shown). Of the other 60 variants genotyped in only two
studies or a single study, 7 SNPs showed moderate associations
(unadjusted p,0.05; data not shown). In particular, an esophageal
cancer variant (rs1229984 in ADH1B) available in MEC and WHI
showed an inverse association with NHL risk [OR per allele
C = 0.77 (0.66–0.90), p = 4.4E-04]. Q = 0.05, p-het. = 0.82], although the interaction between the
variant and race/ethnicity was not significant in the MEC (p-
int. = 0.10). Among the eight GWAS risk variants for CLL, one
variant replicated in our data for CLL/SLL [OR per allele G of
rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027]
and remained significant after Bonferroni correction (p,0.0063;
Table S1 in File S1). None of the CLL variants were associated
with overall NHL risk (p.0.05; data not shown). For example, in
the
MEC,
the
association
of
rs17483466
with
CLL/SLL
significantly differed from that with FL (p-het. from polytomous
regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). Q = 0.05, p-het. = 0.82], although the interaction between the
variant and race/ethnicity was not significant in the MEC (p-
int. = 0.10). Among the eight GWAS risk variants for CLL, one
variant replicated in our data for CLL/SLL [OR per allele G of
rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027]
and remained significant after Bonferroni correction (p,0.0063;
Table S1 in File S1). None of the CLL variants were associated
with overall NHL risk (p.0.05; data not shown). For example, in
the
MEC,
the
association
of
rs17483466
with
CLL/SLL
significantly differed from that with FL (p-het. from polytomous
regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). Of the 113 GWAS risk variants for cancers other than NHL
that were examined in PAGE, 53 SNPs were genotyped in all
three studies, and the other 60 variants were typed in one or two
studies (Table S2 in File S1). Six of the 53 SNPs showed nominal
associations with the risk of overall NHL, including three risk
variants originally identified for lung cancer, two risk variants
identified for prostate cancer and one risk variant for breast cancer
(Table 2). Statistical Analysis BioVU
MEC
WHI
Type of Study
Cross-sectional [17,18]
Nested Case-Control in Cohort [19,20]
Nested Case-Control in Cohort [21,22]
Focus of Study
Cancer
Cancer
Women’s health
Years of Data
Collection
Enrollment and Blood Draw 2007–2011;
Diagnoses between 2009–2011
Enrollment 1993–1996; Blood draw
1995–2006; Diagnoses between 1993
and October 2010
Enrollment and Blood Draw 1993–
1998; Diagnoses between 1993 and
August 2009
NHL Cases
Controls
NHL Cases
Controls
NHL Cases
Controls
Selection
First primary incident
NHL diagnoses from
hospital tumor
registry and
electronic medical
records (EMR)
Combined controls
for multiple cancer
sites; matched on
age, sex, ethnicity
First primary incident
NHL diagnoses from
linkage of cohort
with SEER* registries
Combined controls
for multiple cancer
sites; matched on
age, sex, ethnicity
First primary incident
NHL diagnoses from
active follow-up
(semi/annual) and
EMR verification
Combined controls
for multiple cancer
sites; matched on
age, enrollment date,
ethnicity,
randomization
Total, n*
293
9,002
372
9,091
776
6,090
Age, median (range)
57 (18–102)
63 (19–110)
71 (45–92)
71 (45–88)
65 (50–79)
65 (50–79)
Sex, n (%) women
139 (47%)
3,711 (41%)
166 (45%)
4,321 (48%)
776 (100%)
6,090 (100%)
Race/Ethnicity, n (%)
White
275 (94%)
8,061 (90%)
102 (27%)
1844 (20%)
718 (93%)
4,763 (78%)
African American
16 (5%)
804 (9%)
68 (18%)
2228 (25%)
27 (3%)
714 (12%)
Latino
0
56 (0.6%)
80 (22%)
1864 (21%)
16 (2%)
332 (5%)
Asian American/
Pacific Islander
2/0 (0.7%)
81/0 (0.9%)
104/18 (33%)
2513/642 (35%)
15/0 (2%)
281/0 (5%)
NHL Subtypes, n (%)
293
N/A
372
N/A
776
N/A
DLBCL
-
102 (27%)
258 (33%)
FL
72 (25%)
68 (18%)
178 (23%)
CLL/SLL
42 (SLL only; 14%)
71 (19%)
66 (SLL only; 9%)
Others
179 (61%)
131 (35%)
274 (35%)
* Any prior cancer cases were excluded from the NHL cases and controls for the current analysis, based on self-report (BioVU, MEC, WHI), the SEER registry linkage
(BioVU, MEC), and medical record reviews (BioVU, WHI). Abbreviations: BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative); CLL/SLL (chronic
lymphocytic leukemia/small lymphocytic lymphoma), DLBCL (diffuse large B-cell lymphoma), FL (follicular lymphoma), SEER (Surveillance, Epidemiology and End
Results). doi:10 1371/journal pone 0089791 t001 Table 1. Characteristics of non-Hodgkin lymphoma (NHL) cases and controls in the PAGE studies. Q = 0.05, p-het. = 0.82], although the interaction between the
variant and race/ethnicity was not significant in the MEC (p-
int. = 0.10). Statistical Analysis (a) follicular lymphoma, (b) overall NHL, and (c) overall NHL among whites only. doi:10.1371/journal.pone.0089791.g001 Figure 1. Forest plots for the association between a published follicular lymphoma risk variant (rs6457327) and the risk of follicular
lymphoma or overall non-Hodgkin lymphoma (NHL) in the Multiethnic Cohort (MEC) and the Women’s Health Initiative (WHI) in
the PAGE consortium. (a) follicular lymphoma, (b) overall NHL, and (c) overall NHL among whites only. doi:10.1371/journal.pone.0089791.g001 The
pleiotropy
analysis
for
specific
NHL
subtypes
was
conducted on all non-NHL GWAS cancer SNPs (n = 113) (Table
S3 in File S1). None of the subtype-specific associations were
significant after Bonferroni correction (i.e., p.0.05/113 = 4.4E-04
for 113 tests on each subtype). The most significant association for
follicular lymphoma was with a breast cancer risk variant
[rs11249433 in EMBP1: summary OR per allele C = 1.29 (1.08–
1.54), p = 0.0095]. For CLL/SLL, the most significant association
was with a prostate cancer risk variant [rs2735839 in KLK3-KLK2:
OR per allele G = 1.51 (1.10–2.07), p = 0.0099]. The associations
of overall NHL described above (Table 2) with the risk variants for
lung cancer (rs3131379 in MSH5) and prostate cancer (rs7679673
in TET2) appeared to be due to their associations with the risk of
DLBCL subtype [OR per allele T in rs3131379 = 1.41 (1.10–
1.80), p = 0.0061; OR per allele C in rs7679673 = 0.83 (0.71–0.98),
p = 0.030]. subtype specifically, as indicated in previous studies for shared
etiology [34–36]. The CLL risk variants did not extend to other
subtypes or NHL overall, indicating the subtype-specificity of the
CLL variants. For non-NHL cancer variants, we found no
convincing evidence of pleiotropy, with only weak suggestions that
specific risk variants for lung, prostate and breast cancers may also
be associated with the risk of developing first primary incident
NHL among those without a history of other cancers or prior
cancer treatments. The effects of three of the six non-NHL GWAS variants
nominally associated with NHL (rs401681, rs7679673, rs4975616)
were in the opposite direction compared to the original reports. These variants showed an association with increased risks for lung
and prostate cancers in the original reports but a lower risk of
NHL in our study. Statistical Analysis None of these associations remained significant after
multiple test correction (i.e., p.4.4E-04 for 113 SNPs). Two lung
cancer risk variants in the TERT region (rs401681, OR per
allele C = 0.89 (0.82–0.96), p = 0.0037; rs4975616, OR per allele
A = 0.90 (0.83–0.97), p = 0.010), as well as the prostate cancer risk
variant (rs7679673 in TET2; OR per allele C = 0.89 (0.82–0.97), Q = 0.05, p-het. = 0.82], although the interaction between the
variant and race/ethnicity was not significant in the MEC (p-
int. = 0.10). Among the eight GWAS risk variants for CLL, one
variant replicated in our data for CLL/SLL [OR per allele G of
rs17483466 in ACOXL/BCL2L11 = 1.57 (1.17–2.11), p = 0.0027]
and remained significant after Bonferroni correction (p,0.0063;
Table S1 in File S1). None of the CLL variants were associated
with overall NHL risk (p.0.05; data not shown). For example, in
the
MEC,
the
association
of
rs17483466
with
CLL/SLL
significantly differed from that with FL (p-het. from polytomous
regression = 0.03), DLBCL (p-het. = 0.02) and others (p-het. = 0.01). regression
0.03), DLBCL (p het. 0.02) and others (p het. 0.01). Of the 113 GWAS risk variants for cancers other than NHL
that were examined in PAGE, 53 SNPs were genotyped in all
three studies, and the other 60 variants were typed in one or two
studies (Table S2 in File S1). Six of the 53 SNPs showed nominal
associations with the risk of overall NHL, including three risk
variants originally identified for lung cancer, two risk variants
identified for prostate cancer and one risk variant for breast cancer
(Table 2). None of these associations remained significant after
multiple test correction (i.e., p.4.4E-04 for 113 SNPs). Two lung
cancer risk variants in the TERT region (rs401681, OR per
allele C = 0.89 (0.82–0.96), p = 0.0037; rs4975616, OR per allele
A = 0.90 (0.83–0.97), p = 0.010), as well as the prostate cancer risk
variant (rs7679673 in TET2; OR per allele C = 0.89 (0.82–0.97), March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 4 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Figure 1. Forest plots for the association between a published follicular lymphoma risk variant (rs6457327) and the risk of follicular
lymphoma or overall non-Hodgkin lymphoma (NHL) in the Multiethnic Cohort (MEC) and the Women’s Health Initiative (WHI) in
the PAGE consortium. Statistical Analysis This may be a chance finding given that none
of the associations remained significant after correcting for
multiple tests, though such effects in opposite directions for
different cancer types have been previously demonstrated in the
TERT region and SNP rs401681 in particular [37]. A prostate
cancer variant (rs7679673 in TET2) was specifically associated
with a lower risk of DLBCL. The risk score based on the 53 non-NHL cancer SNPs was not
significantly associated with the risk of overall NHL or subtypes,
either as a continuous variable (Table 3) or categorized in
quartiles (p.0.05; data not shown). There was no significant
heterogeneity in any of the associations for individual SNPs or the
risk score by sex, or by ethnic group (p.0.05; data not shown). One of the other three nominally positive associations for
overall NHL was found with a breast cancer SNP in the coding
region for a lymphocyte-specific protein (rs3817198 in LSP1). This
gene encodes an intracellular F-actin binding protein that is
expressed in endothelium and various hematopoietic cells (lym-
phocytes, neutrophils, macrophages) [38]. As such, this protein
may be involved in lymphomagenesis through the regulation of
lymphocyte motility and migration, as evidenced by an association
of another variant in LSP1 (rs2089910) with NHL in a study of an
immune and inflammation SNP panel [39]. Discussion BioVU
MEC
WHI
Summary
Mean RS, case/
control
OR (95% CI)*
Mean RS, case/
control
OR (95% CI)*
Mean RS, case/
control
OR (95% CI)*
N, case/control
OR (95% CI)*
p-value
Cochran Q
(p-het)
Overall NHL
47.4/46.9
0.98 (0.96, 1.01)
44.1/44.2
0.98 (0.94, 1.02)
42.4/42.4
1.00 (0.98, 1.01)
1,414/23,469
1.00 (0.98, 1.01)
0.80
3.27 (0.20)
DLBCL
-
-
43.5/44.2
0.98 (0.94, 1.02)
42.8/42.4
1.01 (0.99, 1.04)
360/23,469
1.00 (0.98, 1.03)
0.79
2.00 (0.16)
FL
47.0/46.9
1.00 (0.96, 1.05)
44.7/44.2
1.03 (0.98, 1.09)
42.9/42.4
1.02 (0.98, 1.05)
318/23,469
1.02 (0.99, 1.04)
0.17
0.85 (0.65)
CLL/SLL
48.1/46.9
1.05 (0.98, 1.12)
46.2/44.2
1.04 (0.99, 1.09)
42.2/42.4
0.98 (0.93, 1.04)
179/23,469
1.02 (0.99, 1.05)
0.19
2.75 (0.25)
* ORs and 95% CIs in individual studies were estimated per risk allele in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity. Summary odds ratios (ORs) and 95% confidence
intervals (CIs) were estimated in a meta-analysis of fixed effects models. Abbreviations: p-het. (p-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). doi:10.1371/journal.pone.0089791.t003 Table 2. Pleiotropic association of selected cancer susceptibility variants with the risk of overall non-Hodgkin lymphoma (NHL). Discussion BioVU
MEC
WHI
Summary
SNP
Gene
GWAS
Risk (Ref.)
Allele
Cases/
Controls
OR (95% CI)*
Cases/
Controls
OR (95% CI)*
Cases/
Controls
OR (95% CI)*
OR (95% CI)*
p-value
(0.00044){
Cochran Q
(p-het)
rs401681
TERT
Lung
C (T)
292/8984
0.88 (0.74, 1.04)
372/9053
0.76 (0.66, 0.89)
733/5939
0.97 (0.87, 1.09)
0.89 (0.82, 0.96)
0.0037
6.26 (0.04)
rs7679673
TET2
Prostate
C (A)
290/8899
1.02 (0.86, 1.22)
372/8946
0.88 (0.75, 1.04)
733/5936
0.84 (0.75, 0.94)
0.89 (0.82, 0.97)
0.0057
3.42 (0.18)
rs4975616
TERT
Lung
A (G)
293/9000
0.85 (0.72, 1.00)
368/9058
0.80 (0.68, 0.95)
732/5938
0.97 (0.87, 1.09)
0.90 (0.83, 0.97)
0.0103
4.06 (0.13)
rs3817198
LSP1
Breast
C (T)
293/8995
1.06 (0.89, 1.27)
371/9042
1.11 (0.93, 1.32)
733/5942
1.15 (1.02, 1.30)
1.12 (1.03, 1.22)
0.0112
0.56 (0.76)
rs3131379
MSH5
Lung
T (C)
290/8916
1.08 (0.83, 1.41)
334/9053
1.11 (0.78, 1.59)
733/5946
1.21 (1.01, 1.43)
1.16 (1.01, 1.33)
0.0302
0.51 (0.78)
rs10993994
MSMB
Prostate
T (C)
292/9001
1.20 (1.01, 1.42)
369/9054
1.08 (0.93, 1.26)
732/5944
1.05 (0.94, 1.17)
1.09 (1.01, 1.18)
0.0356
1.68 (0.43)
* ORs and 95% CIs in individual studies were estimated in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity (ancestry informative markers). Summary ORs and 95% CIs were
estimated in a meta-analysis of fixed-effects models. {The Bonferroni corrected p-value for 53 SNPs/tests is 4.4E-04. Abbreviations: p-het. (P-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). doi:10.1371/journal.pone.0089791.t002
Table 3. Associations between a risk score (RS) for 53 GWAS-identified cancer risk variants and the overall and subtype-specific risks of NHL. Discussion Increasing evidence supports the pleiotropic involvement of
common genetic risk variants in multiple diseases or complex
traits. Thus, we examined a substantial number of risk variants
identified in GWAS of common cancers in relation to overall and
subtype-specific risk of NHL. Our analysis extended the associ-
ation of the FL risk variant to overall NHL and the DLBCL March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org 5 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma able 2. Pleiotropic association of selected cancer susceptibility variants with the risk of overall non-Hodgkin lymphoma (NHL). BioVU
MEC
WHI
Summary
P
Gene
GWAS
Risk (Ref.)
Allele
Cases/
Controls
OR (95% CI)*
Cases/
Controls
OR (95% CI)*
Cases/
Controls
OR (95% CI)*
OR (95% CI)*
p-value
(0.00044){
Cochran Q
(p-het)
01681
TERT
Lung
C (T)
292/8984
0.88 (0.74, 1.04)
372/9053
0.76 (0.66, 0.89)
733/5939
0.97 (0.87, 1.09)
0.89 (0.82, 0.96)
0.0037
6.26 (0.04)
679673
TET2
Prostate
C (A)
290/8899
1.02 (0.86, 1.22)
372/8946
0.88 (0.75, 1.04)
733/5936
0.84 (0.75, 0.94)
0.89 (0.82, 0.97)
0.0057
3.42 (0.18)
975616
TERT
Lung
A (G)
293/9000
0.85 (0.72, 1.00)
368/9058
0.80 (0.68, 0.95)
732/5938
0.97 (0.87, 1.09)
0.90 (0.83, 0.97)
0.0103
4.06 (0.13)
817198
LSP1
Breast
C (T)
293/8995
1.06 (0.89, 1.27)
371/9042
1.11 (0.93, 1.32)
733/5942
1.15 (1.02, 1.30)
1.12 (1.03, 1.22)
0.0112
0.56 (0.76)
131379
MSH5
Lung
T (C)
290/8916
1.08 (0.83, 1.41)
334/9053
1.11 (0.78, 1.59)
733/5946
1.21 (1.01, 1.43)
1.16 (1.01, 1.33)
0.0302
0.51 (0.78)
0993994
MSMB
Prostate
T (C)
292/9001
1.20 (1.01, 1.42)
369/9054
1.08 (0.93, 1.26)
732/5944
1.05 (0.94, 1.17)
1.09 (1.01, 1.18)
0.0356
1.68 (0.43)
Rs and 95% CIs in individual studies were estimated in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity (ancestry informative markers). Summary ORs and 95% CIs were
imated in a meta-analysis of fixed-effects models. he Bonferroni corrected p-value for 53 SNPs/tests is 4.4E-04. breviations: p-het. (P-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). :10.1371/journal.pone.0089791.t002
able 3. Associations between a risk score (RS) for 53 GWAS-identified cancer risk variants and the overall and subtype-specific risks of NHL. Table 3. Associations between a risk score (RS) for 53 GWAS-identified cancer risk variants and the overall and subtype-specific risks of NHL. Discussion BioVU
MEC
WHI
Summary
Mean RS, case/
control
OR (95% CI)*
Mean RS, case/
control
OR (95% CI)*
Mean RS, case/
control
OR (95% CI)*
N, case/control
OR (95% CI)*
p-value
Cochran Q
(p-het)
Overall NHL
47.4/46.9
0.98 (0.96, 1.01)
44.1/44.2
0.98 (0.94, 1.02)
42.4/42.4
1.00 (0.98, 1.01)
1,414/23,469
1.00 (0.98, 1.01)
0.80
3.27 (0.20)
DLBCL
-
-
43.5/44.2
0.98 (0.94, 1.02)
42.8/42.4
1.01 (0.99, 1.04)
360/23,469
1.00 (0.98, 1.03)
0.79
2.00 (0.16)
FL
47.0/46.9
1.00 (0.96, 1.05)
44.7/44.2
1.03 (0.98, 1.09)
42.9/42.4
1.02 (0.98, 1.05)
318/23,469
1.02 (0.99, 1.04)
0.17
0.85 (0.65)
CLL/SLL
48.1/46.9
1.05 (0.98, 1.12)
46.2/44.2
1.04 (0.99, 1.09)
42.2/42.4
0.98 (0.93, 1.04)
179/23,469
1.02 (0.99, 1.05)
0.19
2.75 (0.25)
* ORs and 95% CIs in individual studies were estimated per risk allele in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity. Summary odds ratios (ORs) and 95% confidence
intervals (CIs) were estimated in a meta-analysis of fixed effects models. Abbreviations: p-het. (p-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiative). Table 2. Pleiotropic association of selected cancer susceptibility variants with the risk of overall non-Hodgkin lymphoma (NHL). Discussion BioVU
MEC
WHI
Summary
SNP
Gene
GWAS
Risk (Ref.)
Allele
Cases/
Controls
OR (95% CI)*
Cases/
Controls
OR (95% CI)*
Cases/
Controls
OR (95% CI)*
OR (95% CI)*
p-value
(0.00044){
Cochran Q
(p-het)
rs401681
TERT
Lung
C (T)
292/8984
0.88 (0.74, 1.04)
372/9053
0.76 (0.66, 0.89)
733/5939
0.97 (0.87, 1.09)
0.89 (0.82, 0.96)
0.0037
6.26 (0.04)
rs7679673
TET2
Prostate
C (A)
290/8899
1.02 (0.86, 1.22)
372/8946
0.88 (0.75, 1.04)
733/5936
0.84 (0.75, 0.94)
0.89 (0.82, 0.97)
0.0057
3.42 (0.18)
rs4975616
TERT
Lung
A (G)
293/9000
0.85 (0.72, 1.00)
368/9058
0.80 (0.68, 0.95)
732/5938
0.97 (0.87, 1.09)
0.90 (0.83, 0.97)
0.0103
4.06 (0.13)
rs3817198
LSP1
Breast
C (T)
293/8995
1.06 (0.89, 1.27)
371/9042
1.11 (0.93, 1.32)
733/5942
1.15 (1.02, 1.30)
1.12 (1.03, 1.22)
0.0112
0.56 (0.76)
rs3131379
MSH5
Lung
T (C)
290/8916
1.08 (0.83, 1.41)
334/9053
1.11 (0.78, 1.59)
733/5946
1.21 (1.01, 1.43)
1.16 (1.01, 1.33)
0.0302
0.51 (0.78)
rs10993994
MSMB
Prostate
T (C)
292/9001
1.20 (1.01, 1.42)
369/9054
1.08 (0.93, 1.26)
732/5944
1.05 (0.94, 1.17)
1.09 (1.01, 1.18)
0.0356
1.68 (0.43)
* ORs and 95% CIs in individual studies were estimated in unconditional logistic regression models that were adjusted for age, sex (in BioVU and MEC) and ethnicity (ancestry informative markers). Summary ORs and 95% CIs we
estimated in a meta-analysis of fixed-effects models. {The Bonferroni corrected p-value for 53 SNPs/tests is 4.4E-04. Abbreviations: p-het. (P-values for heterogeneity across studies measured in Cochran’s Q statistic); BioVU (the biorepository of the Vanderbilt University), MEC (the Multiethnic Cohort Study), WHI (the Women’s Health Initiativ
doi:10.1371/journal.pone.0089791.t002 PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e89791 6 Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma subtypes of non-Hodgkin lymphoma (NHL). Table S2. List of
113 GWAS-based cancer risk variants examined for pleiotropy on
NHL in PAGE; the 53 SNPs listed as genotyped in all three studies
were included in the risk score analysis. Table S3. Pleiotropic
association of selected cancer susceptibility variants with the risk of
common subtypes of non-Hodgkin lymphoma (NHL). (DOC) Another non-significant positive association for NHL, especially
with DLBCL, was with a lung cancer susceptibility variant,
rs3131379, in MSH5 or mutS homolog 5, a gene involved in the
DNA mismatch repair pathway [40]. References susceptibility loci at 2p16.1 (REL), 8q24.21 and 10p14 (GATA3). Nat Genet
42: 1126–1130. susceptibility loci at 2p16.1 (REL), 8q24.21 and 10p14 (GATA3). Nat Genet
42: 1126–1130. 1. Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA
Cancer J Clin 63: 11–30. 15. Crowther-Swanepoel D, Broderick P, Di Bernardo MC, Dobbins SE, Torres M,
et al. (2010) Common variants at 2q37.3, 8q24.21, 15q21.3 and 16q24.1
influence chronic lymphocytic leukemia risk. Nat Genet 42: 132–136. 2. Smedby KE, Hjalgrim H (2011) Epidemiology and etiology of mantle cell
lymphoma and other non-Hodgkin lymphoma subtypes. Semin Cancer Biol 21:
293–298. 3. Wang SS, Slager SL, Brennan P, Holly EA, De Sanjose S, et al. (2007) Family
history of hematopoietic malignancies and risk of non-Hodgkin lymphoma
(NHL): a pooled analysis of 10 211 cases and 11 905 controls from the
International Lymphoma Epidemiology Consortium (InterLymph). Blood 109:
3479–3488. 16. Matise TC, Ambite JL, Buyske S, Carlson CS, Cole SA, et al. (2011) The Next
PAGE in understanding complex traits: design for the analysis of Population
Architecture Using Genetics and Epidemiology (PAGE) Study. Am J Epidemiol
174: 849–859. 17. Roden DM, Pulley JM, Basford MA, Bernard GR, Clayton EW, et al. (2008)
Development of a large-scale de-identified DNA biobank to enable personalized
medicine. Clin Pharmacol Ther 84: 362–369. 4. Nielsen SF, Bojesen SE, Birgens HS, Nordestgaard BG (2011) Risk of thyroid
cancer, brain cancer, and non-Hodgkin lymphoma after adult leukemia: a
nationwide study. Blood 118: 4062–4069. 18. Ritchie MD, Denny JC, Crawford DC, Ramirez AH, Weiner JB, et al. (2010)
Robust replication of genotype-phenotype associations across multiple diseases
in an electronic medical record. Am J Hum Genet 86: 560–572. 5. Chakraborty S, Tarantolo SR, Batra SK, Hauke RJ (2013) Incidence and
prognostic significance of second primary cancers in renal cell carcinoma. Am J Clin Oncol 36: 132–142. 19. Kolonel LN, Henderson BE, Hankin JH, Nomura AM, Wilkens LR, et al. (2000)
A multiethnic cohort in Hawaii and Los Angeles: baseline characteristics. AmJEpidemiol 151: 346–357. 6. Nielsen SF, Nordestgaard BG, Bojesen SE (2012) Associations between first and
second primary cancers: a population-based study. CMAJ 184: E57–69. p
20. Kolonel LN, Altshuler D, Henderson BE (2004) The Multiethnic Cohort study:
exploring genes, lifestyle and cancer risk. NatRevCancer 4: 519–527. 7. Hindorff LA, MacArthur J, Wise A, Junkins HA, Klemm AK, et al. A catalog of
published genome-wide association studies. U.S. National Human Genome
Research Institute. References Available: www.genome.gov/gwastudies. Accessed 2013 Nov
15. 21. The Women’s Health Initiative Study Group (1998) Design of the Women’s
Health Initiative clinical trial and observational study. The Women’s Health
Initiative Study Group. Control Clin Trials 19: 61–109. 8. Sivakumaran S, Agakov F, Theodoratou E, Prendergast JG, Zgaga L, et al. (2011) Abundant pleiotropy in human complex diseases and traits. Am J Hum
Genet 89: 607–618. 22. Curb JD, McTiernan A, Heckbert SR, Kooperberg C, Stanford J, et al. (2003)
Outcomes ascertainment and adjudication methods in the Women’s Health
Initiative. Ann Epidemiol 13: S122–128. 9. Hindorff LA, Sethupathy P, Junkins HA, Ramos EM, Mehta JP, et al. (2009)
Potential etiologic and functional implications of genome-wide association loci
for human diseases and traits. Proc Natl Acad Sci U S A 106: 9362–9367. 23. Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, et al. (2000)
The World Health Organization classification of neoplasms of the hematopoietic
and lymphoid tissues: report of the Clinical Advisory Committee meeting–Airlie
House, Virginia, November, 1997. HematolJ 1: 53–66. 10. Hindorff LA, Gillanders EM, Manolio TA (2011) Genetic architecture of cancer
and other complex diseases: lessons learned and future directions. Carcinogen-
esis 32: 945–954. 24. Morton LM, Turner JJ, Cerhan JR, Linet MS, Treseler PA, et al. (2007)
Proposed classification of lymphoid neoplasms for epidemiologic research from
the Pathology Working Group of the International Lymphoma Epidemiology
Consortium (InterLymph). Blood 110: 695–708. 11. Haiman CA, Patterson N, Freedman ML, Myers SR, Pike MC, et al. (2007)
Multiple regions within 8q24 independently affect risk for prostate cancer. Nat
Genet 39: 638–644. 12. Au WY, Horsman DE, Viswanatha DS, Connors JM, Klasa RJ, et al. (2000)
8q24 translocations in blastic transformation of mantle cell lymphoma. Haematologica 85: 1225–1227. 25. Bush WS, Boston J, Pendergrass SA, Dumitrescu L, Goodloe R, et al. (2013)
Enabling high-throughput genotype-phenotype associations in the Epidemio-
logic Architecture for Genes Linked to Environment (EAGLE) project as part of
the Population Architecture using Genomics and Epidemiology (PAGE) study. Pac Symp Biocomput: 373–384. 13. Okhowat R, Dorner S, Hinterberger W, Fonatsch C (2003) Unusual karyotype
aberrations involving 2p12, 3q27, 18q21, 8q24, and 14q32 in a patient with
non-Hodgkin lymphoma/acute lymphoblastic leukemia. Cancer Genet Cyto-
genet 142: 60–64. 26. De Roos AJ, Martinez-Maza O, Jerome KR, Mirick DK, Kopecky KJ, et al. (2013) Investigation of epstein-barr virus as a potential cause of B-cell non-
hodgkin lymphoma in a prospective cohort. Author Contributions Conceived and designed the experiments: UL UP LLM LAH CAH CK. Performed the experiments: UL JMK WSB TCM CC SLP CSC ED DD
MF CAH BEH LAH LNK UP DOS MT LRW CW CK LLM. Analyzed
the data: UL JMK WSB CC. Contributed reagents/materials/analysis
tools: WSB TCM CC CAH BEH LAH LNK UP DOS MT LRW CW CK
LLM. Wrote the paper: UL JMK WSB LLM. Discussion This variant is also located
near the major histocompatibility complex (MHC or human
leukocyte antigen, HLA) region in chromosome 6 (6p21.33), as is
the GWAS variant for FL (rs6457327), and has been associated
with the risk of systemic lupus erythematosus in a GWAS [41]. Our findings on MSH5 and MHC variants indicate possible
involvement of variants in or near this highly-conserved immune-
regulatory region in the etiology of NHL (including FL and
DLBCL), in addition to that of lung cancer. Acknowledgments The authors thank the WHI investigators and staff for their dedication, and
the study participants for making the program possible. The PAGE
consortium thanks the staff and participants of all PAGE studies for their
important contributions. The authors also gratefully acknowledge the
contribution of Julia Higashio and Rasheeda Williams at the PAGE
Coordinating Center and of Dr. Kylee Spencer at Heidelberg University. Disclaimers: The contents of this paper are solely the responsibility of the
authors and do not necessarily represent the official views of the NIH. The
findings and conclusions in this report are those of the authors and do not
necessarily represent the views of the Centers for Disease Control and
Prevention. This study was nested in three large studies with well-
characterized phenotypes and pathology-confirmed histologic
information for subtype classification. However, despite the
sizeable number of NHL cases included, we had limited power,
in part likely due to the heterogeneous nature of NHL. Also, only a
subset of total cancer variants was genotyped in all three PAGE
studies for the NHL analysis. Our analyses do not provide clear
evidence that these common cancer genetic susceptibility loci may
play a role in the etiologies of NHL. A more systematic approach
in larger pooled analyses of specific subtypes, with larger SNP
panels, is warranted in future research. Supporting Information Files S1
Supporting tables. Table S1. Association between
established GWAS risk variants for follicular lymphoma (FL) and
for chronic lymphocytic leukemia (CLL) with the risk of these Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma 27. Lim U, Wilkens LR, Monroe KR, Caberto C, Tiirikainen M, et al. (2012)
Susceptibility variants for obesity and colorectal cancer risk: the multiethnic
cohort and PAGE studies. Int J Cancer 131: E1038–1043. 35. Cerhan JR, Fredericksen ZS, Novak AJ, Ansell SM, Kay NE, et al. (2012) A two-
stage evaluation of genetic variation in immune and inflammation genes with
risk of non-Hodgkin lymphoma identifies new susceptibility locus in 6p21.3
region. Cancer Epidemiol Biomarkers Prev 21: 1799–1806. J
28. Hoggart CJ, Clark TG, De Iorio M, Whittaker JC, Balding DJ (2008) Genome-
wide significance for dense SNP and resequencing data. Genet Epidemiol 32:
179–185. 36. Slager SL, Camp NJ, Conde L, Shanafelt TD, Achenbach SJ, et al. (2012)
Common variants within 6p21.31 locus are associated with chronic lymphocytic
leukaemia and, potentially, other non-Hodgkin lymphoma subtypes. Br J Haematol 159: 572–576. 29. Skibola CF, Bracci PM, Halperin E, Conde L, Craig DW, et al. (2009) Genetic
variants at 6p21.33 are associated with susceptibility to follicular lymphoma. Nat
Genet 41: 873–875. 37. Mocellin S, Verdi D, Pooley KA, Landi MT, Egan KM, et al. (2012)
Telomerase reverse transcriptase locus polymorphisms and cancer risk: a field
synopsis and meta-analysis. J Natl Cancer Inst 104: 840–854. 30. Conde L, Halperin E, Akers NK, Brown KM, Smedby KE, et al. (2010)
Genome-wide association study of follicular lymphoma identifies a risk locus at
6p21.32. Nat Genet 42: 661–664. 38. Easton DF, Pooley KA, Dunning AM, Pharoah PD, Thompson D, et al. (2007)
Genome-wide association study identifies novel breast cancer susceptibility loci. Nature 447: 1087–1093. 31. Di Bernardo MC, Crowther-Swanepoel D, Broderick P, Webb E, Sellick G,
et al. (2008) A genome-wide association study identifies six susceptibility loci for
chronic lymphocytic leukemia. Nat Genet 40: 1204–1210. 39. Cerhan JR, Ansell SM, Fredericksen ZS, Kay NE, Liebow M, et al. (2007)
Genetic variation in 1253 immune and inflammation genes and risk of non-
Hodgkin lymphoma. Blood 110: 4455–4463. 32. Kosoy R, Nassir R, Tian C, White PA, Butler LM, et al. (2009) Ancestry
informative marker sets for determining continental origin and admixture
proportions in common populations in America. Hum Mutat 30: 69–78. 40. Kazma R, Babron MC, Gaborieau V, Genin E, Brennan P, et al. (2012) Lung
cancer and DNA repair genes: multilevel association analysis from the
International Lung Cancer Consortium. Carcinogenesis 33: 1059–1064. p
p
p p
33. References Cancer Epidemiol Biomarkers Prev
22: 1747–1755. 14. Enciso-Mora V, Broderick P, Ma Y, Jarrett RF, Hjalgrim H, et al. (2010) A
genome-wide association study of Hodgkin’s lymphoma identifies new March 2014 | Volume 9 | Issue 3 | e89791 March 2014 | Volume 9 | Issue 3 | e89791 7 PLOS ONE | www.plosone.org Pleiotropy of Cancer SNPs on Non-Hodgkin Lymphoma Willer CJ, Li Y, Abecasis GR (2010) METAL: fast and efficient meta-analysis of
genomewide association scans. Bioinformatics 26: 2190–2191. genomewide association scans. Bioinformatics 26: 2190–2191 g
g
41. Harley JB, Alarcon-Riquelme ME, Criswell LA, Jacob CO, Kimberly RP, et al. (2008) Genome-wide association scan in women with systemic lupus erythema-
tosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other
loci. Nat Genet 40: 204–210. 34. Smedby KE, Foo JN, Skibola CF, Darabi H, Conde L, et al. (2011) GWAS of
follicular lymphoma reveals allelic heterogeneity at 6p21.32 and suggests shared
genetic susceptibility with diffuse large B-cell lymphoma. PLoS Genet 7:
e1001378. March 2014 | Volume 9 | Issue 3 | e89791 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
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Targeting STAT3 in Cancer Immunotherapy
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© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
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data made available in this article, unless otherwise stated in a credit line to the data. Targeting STAT3 in Cancer Immunotherapy Sailan Zou1†, Qiyu Tong1†, Bowen Liu2, Wei Huang3, Yan Tian1* and Xianghui Fu1* Abstract As a point of convergence for numerous oncogenic signaling pathways, signal transducer and activator of transcription
3 (STAT3) is central in regulating the anti-tumor immune response. STAT3 is broadly hyperactivated both in cancer and
non-cancerous cells within the tumor ecosystem and plays important roles in inhibiting the expression of crucial
immune activation regulators and promoting the production of immunosuppressive factors. Therefore, targeting the
STAT3 signaling pathway has emerged as a promising therapeutic strategy for numerous cancers. In this review, we
outline the importance of STAT3 signaling pathway in tumorigenesis and its immune regulation, and highlight the
current status for the development of STAT3-targeting therapeutic approaches. We also summarize and discuss recent
advances in STAT3-based combination immunotherapy in detail. These endeavors provide new insights into the
translational application of STAT3 in cancer and may contribute to the promotion of more effective treatments toward
malignancies. Keywords: STAT3, Cancer, Immunosuppression, Immunotherapy, Immune checkpoint blockade, CAR-T identified and developed for immunotherapy appropriate
for the clinical use. REVIEW Open Access Zou et al. Molecular Cancer (2020) 19:145
https://doi.org/10.1186/s12943-020-01258-7 Zou et al. Molecular Cancer (2020) 19:145
https://doi.org/10.1186/s12943-020-01258-7 Introduction Dysregulation of immune checkpoints is a protective
mechanism used by a number of malignancies to es-
cape from the immune surveillance allowing for can-
cer development [1]. This has inspired the idea of
boosting the host immune response as an anti-cancer
therapy. Indeed, the blockage of immune checkpoints,
including programmed cell death protein 1 (PD-1),
programmed cell death 1 ligand 1 (PD-L1) and cyto-
toxic
T-lymphocyte-associated protein 4 (CTLA-4),
improves clinical outcomes in subsets of patients with
cancers previously considered to be essentially un-
treatable [2–4]. In order to expand the array of treat-
able
cancers
as
well
as
increase
the
number
of
patients that respond to the therapy, novel therapeutic
targets and new molecules/strategies should be urgently The signal transducer and activator of transcription
(STAT) proteins are a family of cytoplasmic transcription
factors which share an overall general structure, organized
into functional modular domains. The mammalian STAT
family
comprises
STAT1,
STAT2,
STAT3,
STAT4,
STAT5a, STAT5b and STAT6 that mediate multiple intra-
cellular signaling pathways [5]. Among them, STAT3 is in-
volved in numerous biological processes including cell
proliferation, survival, differentiation, and angiogenesis [6,
7]. In normal cells, transient activation of STAT3 (predom-
inantly by phosphorylation) transmits transcriptional sig-
nals from cytokines and growth factor receptors at the
plasma membrane to the nucleus [5]. In contrast, STAT3
becomes hyperactivated in the majority of human cancers
and is generally associated with poor clinical prognosis [8]. Therefore, it is not surprising that STAT3 signaling path-
way has long been recognized as a potential therapeutic tar-
get for cancer therapy owing to their roles in tumor
formation, metastasis and drug resistance [9–12]. More-
over, accumulating evidence reveals that STAT3 hyperacti-
vation can mediate tumor-induced immunosuppression at * Correspondence: tyfxh@163.com; xfu@scu.edu.cn
†Sailan Zou and Qiyu Tong are co-first author.
1Division of Endocrinology and Metabolism, National Clinical Research Center
for Geriatrics, State Key Laboratory of Biotherapy and Cancer Center, West
China Hospital, Sichuan University and Collaborative Innovation Center of
Biotherapy, Chengdu 610041, Sichuan, China
Full list of author information is available at the end of the article The STAT3 signaling pathway g
g p
y
STAT3 is a protein consisting of 770 amino acids and
characterized by the presence of 6 functionally con-
served domains, including the amino-terminal domain
(NH2), the coiled-coil domain (CCD), the DNA-binding
domain (DBD), the linker domain, the SRC homology 2
(SH2) domain, and the carboxyl-terminal transactivation
domain (TAD) (Fig. 1a). Among them, SH2 is the most
highly conserved STAT domain and plays a crucial role
in signaling via binding to specific phosphotyrosine mo-
tifs [15]. In an unstimulated cell, STAT3 is tightly regu-
lated by negative modulators to maintain an inactive
state in the cytoplasm. These modulators include mem-
bers of the protein inhibitor of activated STAT (PIAS),
suppressor of cytokine signaling (SOCS) families, protein
tyrosine phosphatases (SHP1, SHP2, PTPN1, PTPN2
PTPRD, PTPRT and DUSP22), and ubiquitin enzymes
[8]. In response to stimuli, STAT3 becomes activated
mainly by direct phosphorylation at tyrosine (705) and
serine (727) residues induced by its upstream ligands in-
cluding Janus kinases (JAKs), tyrosine kinases, cytokines
and several non-receptor tyrosine kinases such as SRC
and ABL; the phosphorylation induces dimerization of
STAT3 proteins followed by nuclear translocation, DNA
binding, and eventually execution of their nuclear func-
tions [15]. y
Additionally, increasing evidence suggests that non-
coding RNAs (ncRNAs) can directly or indirectly modu-
late STAT3 activity (Fig. 1c). As the most extensively
studied ncRNAs, numerous microRNAs (miRNAs) have
been shown to target STAT3 directly and certain com-
ponents
of
STAT3
signaling
pathway
(IL-6,
JAK2,
SOCS1, PIAS3, etc.), thereby modulating STAT3 expres-
sion and activation [8, 25–29]. For instance, miR-125b-
5p can directly target STAT3 and inhibit its expression
[27], while miR-218 indirectly suppresses STAT3 activa-
tion by targeting IL-6 receptor and JAK3 [28]. More re-
cently, it has shown that exosome-mediated transfer of
certain miRNAs, such as miR-193a-3p, miR-210-3p and
miR-5100, can promote metastasis of lung cancer by en-
hancing STAT3 activity [29], although the molecular
mechanisms await further investigation. Likewise, long
non-coding RNAs (lncRNAs) can modulate the expres-
sion and activation of STAT3 directly or indirectly
through multiple mechanisms [30–33]. For instance,
lincRNA-p21 can inhibit the STAT3 transcriptional ac-
tivity via directly binding to STAT3 [30]. Lnc-BM can
bind to the JH2 domain of JAK2, which increases JAK2
activation, and thus indirectly enhances activity of
STAT3 [32]. Zou et al. Molecular Cancer (2020) 19:145 Page 2 of 19 important regulatory mechanisms for STAT3 activity. SMYD2-dependent methylation of STAT3 contributes
to the hyperphosphorylation of STAT3, whereas EZH2-
and SET9-dependent dimethylation of STAT3 inhibits
the activity of DNA-bound STAT3 dimers [20–22]. Sumoylation at the lysine 451 of STAT3 by SUMO2/3
can promote its interaction to the nuclear phosphatase
TC45, thereby restraining phosphorylated STAT3 in the
nucleus, while de-sumoylation by SENP3 leads to the
hyperphosphorylation of STAT3 [23]. However, andro-
gen receptor degradation enhancer ASC-J9 can inhibit
the STAT3 phosphorylation via inducing the sumoyla-
tion of STAT3 at lysine 679 [24]. many levels [13, 14]. Given the similarities between tumori-
genesis and STAT3-dependent immunity, new therapeutic
strategies that target STAT3 signaling pathway may open
up new avenues for long-lasting and multilayered tumor
control. This review outlines the role of the STAT3 pathway in
tumor immunity, summarizes the recent progress in
STAT3-centered anti-cancer approaches, and highlights
future directions for the clinical immunotherapy. The STAT3 signaling pathway STAT3 is characterized by the presence of six different
functional domains, including an amino-terminal domain (NTD) for cooperative binding of STAT proteins to multiple consensus DNA sites, a coiled-coil domain
(CCD) for recruitment of STAT3 to the receptor as well as subsequent phosphorylation, dimerization and nuclear translocation, a DNA-binding domain (DBD) for
recognizing and binding to a specific consensus DNA sequence, a linker domain for connecting the DBD with the SRC homology 2 (SH2) domain, a SH2
domain for recruitment and activation as well as dimerization of the STAT3 molecule by interacting with phosphorylated tyrosine residues in the opposing
subunit, and a carboxyl-terminal transactivation domain (TAD). b STAT3 signaling pathway. STAT3 is activated by upstream growth factor kinases and cytokine
receptors. Non-receptor tyrosine kinases such as SRC and ABL can also lead to constitutive activation of STAT3. Phosphorylated STAT3 dimerizes and
translocates to nucleus, which causes the transcription of target genes including immunosuppression, angiogenesis, metastasis, proliferation and survival. The
signaling pathway can be inhibited by SOCS proteins, PIAS proteins, and protein tyrosine phosphatases (PTPases), etc. c Interplay between noncoding RNAs
and STAT3 signaling pathway. On the one hand, miRNAs and lncRNAs can regulate STAT3 activation through not only directly targeting STAT3, but also
targeting the components of the STAT3 signaling pathway, such as IL-6, JAK2, SOCS1 and PIAS3; CircRNAs usually regulate STAT3 by acting as sponges for
miRNAs. On the other hand, STAT3 is able to regulate miRNAs and lncRNAs expression in many ways. [53]. For instance, STAT3 can suppress the secretion
of type 1 IFNs (IFN-Is) and IFN-I-responsive genes
via multiple actions, such as attenuating the activation
of IFN-I signaling, reducing the expression of ISGF3
components, and impairing the potential of ISGF3
transactivation [54, 55]. other hand, STAT3 can upregulate miR-21 through in-
creasing IL-6 expression [40]. The regulatory effect of
STAT3 in lncRNAs is also emerging. STAT3 can dir-
ectly bind to the promoter region of certain lncRNAs,
such as SNHG17, DUXAP8 and HAGLROS, and thus
contributes to their overexpression in cancers [41–43]. In tumor cells, STAT3 often interacts with other
signaling pathways, such as NF-κB, to confer robust-
ness for tumor progression [44, 56, 57]. NF-κB signal-
ing is of importance for both inflammation-induced
carcinogenesis and anti-tumor immunity [57]. NF-κB
(especially RELA) can upregulate a spectrum of tar-
gets involved in chronic inflammation and cancer ini-
tiation such as cyclooxygenase 2, IL-6, IL-23, and IL-
1β [44]. The STAT3 signaling pathway FLANC, a novel primate-specific lncRNA,
has shown to upregulate and prolong the half-life of
phosphorylated STAT3, but not total STAT3, albeit the
underlying mechanism remains unknown [34]. Intri-
guingly,
a
recent
study
revealed
that
LINC00908-
encoded polypeptide ASRPS can directly bind to the
CCD domain of STAT3, and thus reduce STAT3 phos-
phorylation [35]. In general, circular RNAs (circRNAs)
can modulate gene expression by acting as sponges of
endogenous miRNAs. It has shown that circ-HIPK3,
circ_0076305
and
circ-STAT3
positively
modulate
STAT3 signaling by sponging miR-124-3p, miR-296-5p,
and miR-29a/b/c-3p, respectively [36–38]. In parallel,
STAT3 has a capacity of regulating ncRNAs directly or
indirectly. The regulation of miR-21 by STAT3 has been
extensively studied. On the one hand, STAT3 can dir-
ectly regulate miR-21 transcription in myeloma cells by
binding to its upstream enhancer region [39]. On the Beyond phosphorylation, other posttranslational modi-
fications (i.e. acetylation, methylation, and sumoylation)
can also regulate STAT3 transcriptional activity through
altering STAT3 phosphorylation, and thus add another
layer of complexity for STAT3 hyperphosphorylation in
cancers (Fig. 1b). For instance, acetylation at several ly-
sine residues within both the NH2 and SH2 domains,
primarily mediated by the CBP/p300 acetyltransferase,
can enhance STAT3 transactivating potential, which is
associated with increased dimer stabilization, tyrosine
705 phosphorylation, nuclear translocation, and localized
histone hyperacetylation of target promoters [16]. In
contrast, deacetylation by several deacetylases, such as
HADC1-3, SIRT1 and Loxl3, inhibits transcription of
STAT3 targets [17–19]. The dynamic balance of acetyl-
ation and deacetylation plays a role in STAT3 activation
and is involved in various cellular events. Similarly,
methylation and sumoylation are also emerging as Page 3 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer Fig. 1 (See legend on next page.) Fi
1 (S
l
d
t
) Fig. 1 (See legend on next page.) Page 4 of 19 Zou et al. Molecular Cancer (2020) 19:145 (See figure on previous page.)
Fig. 1 The domain structure and signaling pathway of STAT3. a Schematic domain structure of STAT3. STAT3-driven tumor immunosuppression STAT3-driven tumor immunosuppression
The tumor microenvironment (TME) is a highly com-
plex and heterogenous ecosystem consisting tumor-
infiltrating immune cells, cancer-associated fibroblasts
(CAFs), smooth muscle cells, endothelial cells, and the
tumor cells [45, 46]. It is becoming increasingly evident
that TME can promote the progression of cancer and
mediate therapeutic resistance, particularly against can-
cer immunotherapy [47, 48]. Gathered evidence suggests
that STAT3 becomes hyperactivated not only in cancer
cells themselves but also in immune cells and CAFs
within the TME [13, 49–52]. The hyperactivation of
STAT3 in TME compartments might have a significant
impact on anti-tumor immunity through various mecha-
nisms (described below in more detail). The STAT3 signaling pathway Several layers of STAT3-NF-κB crosstalk
have been identified thus far: (1) both NF-κB and
STAT3 are frequently activated in the same tumor
cells and TME-associated infiltrating immune cells,
and share a wide range of common targets that par-
ticipate in cell proliferation, metastasis, anti-apoptosis,
and angiogenesis [56]; (2) STAT3 can prolong nuclear
retention of RELA through p300-mediated acetylation,
leading to the persistent activation of NF-κB [58]; (3)
many cytokines (i.e. IL-6) can in turn simultaneously
activate STAT3 and NF-κB [57]; (4) it has recently
been demonstrated that NF-κB activity in pancreatic
CAFs shielded cancer cells from immune attack by
increasing CXCL12 expression [59]. Given the well-
known
feedforward
loop
between
CXCL12
and
STAT3 [60, 61], it is possible that STAT3 contributes
to NF-κB-mediated immune evasion by this vicious
cycle. As described above, STAT3 activity can be influenced
by many factors such as numerous post-translational
modifications and multiple ncRNAs regulation. These
complexities, together with the fact that the STAT3 sig-
naling pathway is responsive to a great variety of cellular
stresses and stimuli [44], pose difficulties in our under-
standing of abnormal hyperactivated STAT3 in cancers. Future investigations delineating the regulatory network
of STAT3 will likely facilitate the clinical translation of
STAT3-based therapies for human malignancy. STAT3-mediated crosstalk between cancer cells and
diverse cell subsets in the TME Hyperactivation of STAT3 in tumor-infiltrating im-
mune cells causes immunosuppression by inhibiting
both innate and adaptive immune responses. In brief,
excessive STAT3 activity in innate immune cell sub-
sets may impair the production of pro-inflammatory
mediators such as IFNγ, dampen antigen presenta-
tion, and inhibit the tumor-killing activities of ef-
fector cells. In adaptive immune subsets, elevated
STAT3 activity has the ability to inhibit the accumu-
lation of effector T cells, thereby restraining their
anti-tumor effects [62–64]. Interestingly, some recent
studies
suggest
previously
unknown
functions
of
STAT3 in tumor immunity. For instance, placental
growth factor (PlGF) [65] and Cxxc finger protein 1
(Cxxc1) [66] can act as key upstream regulators of
STAT3 signaling, which subsequently contributes to
the
differentiation
and
function
of
Th17
cells. STAT3 mediates the major impact of β2 adrenergic
receptor
on
the
immunosuppressive
potential
of
myeloid-derived
suppressor
cells
(MDSCs)
in
the
TME
[67]. In
glioblastoma-infiltrating
tumor-
associated macrophages, STAT3 acts as a positive
regulator of aryl hydrocarbon receptor (AHR) and
thus increases the recruitment of tumor-associated
macrophages and tumor growth [68]. It has been
shown that STAT3 modulates the abundance and
function of regulatory T (Treg) cells in response to
radiation therapy in head and neck cancer, suggest-
ing that STAT3 inhibition may be beneficial for pa-
tients receiving radiation [69]. Aberrantly activated STAT3 can lead to tumor-induced
immunosuppression via propagating the crosstalk between
cancer cells and their immunological microenvironment. In tumor cells, hyperactivated STAT3 promotes the ex-
pression of immunosuppressive factors such as VEGF, IL-
6, and IL-10 [53]. Meanwhile, these tumor-derived factors
that also happen to be STAT3 activators could be trans-
ited to the TME, and thus enhance STAT3 signaling in
various immune cell subsets and CAFs (Fig. 2). ( g
)
In particular, STAT3 hyperactivation in tumor cells
has a vital role in dendritic cells (DCs) maturation. DCs
essentially are monocytes at a differentiated stage and
the key antigen presenting cells of the immune system. As immune sentinels, DCs play an important role in the
initiation of T-cell response against tumors, while imma-
ture DCs generally induce immune tolerance [75]. Hyperactivated STAT3 in tumor cells can suppress the
expression of IL-12 and TNF-α, leading to a decrease in
Bcl-2 expression in DCs [53]. STAT3 also represses the
expression of major histocompatibility complex (MHC)
class II complexes and co-stimulatory signals (CD80 and
CD86), which are essential to the antigen presenting
function of DCs [13]. STAT3-mediated crosstalk between cancer cells and
diverse cell subsets in the TME Meanwhile, STAT3 inhibits DC
maturation and innate immunity through negatively
regulating the expression of CXCL10 and CCL5 [53]. Furthermore, the immunosuppressive factors such as IL-
6, IL-10, and VEGF induced by STAT3 can inhibit DC
generation through reducing protein kinase C beta II
(PKCβII) expression [76]. Given that immature DCs can-
not activate antigen-specific CD8+ T cells, activated
STAT3 signaling in tumor cells reduces the anti-
tumorigenic effector functions of CD8+ T cells. g
What’s more, certain factors released by CAFs can
modulate STAT3 signaling in other cell types in the
tumor milieu. TGFβ, an evolutionarily conserved regula-
tor of tumorigenesis, is a crucial driver of the activity of
CAFs. Accumulating
evidence
suggests
that
TGFβ-
stimulated CAFs increase the secretion of IL-6 and IL-
11, which trigger GP130/STAT3 signaling in cancer cells
and thus promote cancer metastasis and progression
[77–80]. STAT3 is also involved in the crosstalk between
CAFs and immune cells. For example, CCL2 secreted
from CAFs with STAT3 hyperactivation can promote
the recruitment of immunosuppressive MDSCs and
hepatocarcinogenesis [72]. Moreover, the differentiation
of these recruited MDSCs has been shown to be con-
trolled in an IL-6/STAT3-dependent manner [81]. In
addition, IL-6 derived from CAFs can activate STAT3 in
DCs, which subsequently induce liver cancer immune
escape through impairing T-cell proliferation and pro-
moting Treg cells expansion [82]. STAT3 signaling in
CAFs and other cells orchestrates stromal remodeling of STAT3 in immune cells In tumor cells per se, hyperactivated STAT3 decreases
the expression of immune-stimulating factors includ-
ing
interferons
(IFNs),
pro-inflammatory
cytokines
(IL-12, TNF-α) and chemokines (CCL5, CXCL10),
while increases the expression of certain cytokines
and growth factors (IL-6, IL-10, TGFβ, and VEGF),
thereby exerting profound immune effects (Fig. 1b) STAT3 also plays a pivotal role in a plethora of tumor-
infiltrating immune cells that predominantly comprise
the TME and recent comprehensive reviews have cov-
ered this topic [62–64]. Here we would only like to
briefly mention the diverse functions of STAT3 in im-
mune cell milieu, together with some recent advances Page 5 of 19 Zou et al. Molecular Cancer (2020) 19:145 Page 5 of 19 STAT3-mediated crosstalk between cancer cells and
diverse cell subsets in the TME that throw new lights on our understanding of its ex-
tremely sophisticated regulation. STAT3 in CAFs CAFs are the key component of the tumor stroma
and contribute to cancer progression and treatment
failure by modifying the extracellular matrix, secreting
soluble factors, supporting angiogenesis and metasta-
sis, and inhibiting anti-tumor immune responses [70]. There is a growing body of evidence to support that
STAT3 can be activated in CAFs by numerous cyto-
kines including leukemia inhibitory factor (LIF) [71]. This STAT3 hyperactivation enables CAFs to produce
various
immunosuppressive
factors
such
as
IL-6,
TGFβ, EGF, VEGF, and CCL2, thereby contributing
to the pro-oncogenic phenotype of these fibroblasts
[72, 73]. Moreover, a recent study revealed that in-
creased phosphorylation of STAT3 in CAFs is associ-
ated with reduced overall survival in colorectal cancer
patients, and STAT3 activation in CAFs enhances in-
testinal tumor growth in vivo [74], exemplifying the
importance of STAT3 activation in CAFs for cancer
initiation and progression. Zou et al. Molecular Cancer (2020) 19:145 Page 6 of 19 Fig. 2 STAT3 induces the immunosuppression in the TME. STAT3 activity in tumor cells supports multiple hallmarks of cancer, including increased
secretion of immunosuppressive factors such as IL-6, IL-10 and EGFR, which can activate STAT3 in the innate and adaptive immune cell subsets as
well as CAFs in the TME. Likewise, immune cells and CAFs within the TME can release certain factors including IL-6, which subsequently enhance
STAT3 signaling in tumor cells. Elevated STAT3 in the TME has dual effects. On the one hand, STAT3 favors the accumulation and enrichment of
immunosuppressive Treg cells and B cells, as well as the polarization of M2-like macrophages, which instigate immune evasion. Particularly, STAT3
is a major driver for increased expression of immune checkpoint molecules (such as PD-L1, PD-L2 and CTLA-4) in these cells. On the other hand,
STAT3 in CD8+ T cells, NK cells and neutrophils evokes restrained anti-tumor cytolytic activities. STAT3 can also inhibit the anti-tumor ability of
DCs through dampening their maturation, activation and antigen presentation. Besides, STAT3 in CAFs can promote their proliferation, survival
and migration, and drive the remodeling of tumor stroma for tumor progression. Collectively, STAT3 induces the immunosuppression in the TME,
thereby promoting tumor progression with diminishing the anti-tumor immunity. Fig. 2 STAT3 induces the immunosuppression in the TME. STAT3 in CAFs STAT3 activity in tumor cells supports multiple hallmarks of cancer, including increased
secretion of immunosuppressive factors such as IL-6, IL-10 and EGFR, which can activate STAT3 in the innate and adaptive immune cell subsets as
well as CAFs in the TME. Likewise, immune cells and CAFs within the TME can release certain factors including IL-6, which subsequently enhance
STAT3 signaling in tumor cells. Elevated STAT3 in the TME has dual effects. On the one hand, STAT3 favors the accumulation and enrichment of
immunosuppressive Treg cells and B cells, as well as the polarization of M2-like macrophages, which instigate immune evasion. Particularly, STAT3
is a major driver for increased expression of immune checkpoint molecules (such as PD-L1, PD-L2 and CTLA-4) in these cells. On the other hand,
STAT3 in CD8+ T cells, NK cells and neutrophils evokes restrained anti-tumor cytolytic activities. STAT3 can also inhibit the anti-tumor ability of
DCs through dampening their maturation, activation and antigen presentation. Besides, STAT3 in CAFs can promote their proliferation, survival
and migration, and drive the remodeling of tumor stroma for tumor progression. Collectively, STAT3 induces the immunosuppression in the TME,
thereby promoting tumor progression with diminishing the anti-tumor immunity. Overall, the outcome of STAT3-mediated crosstalk
between
cancer
cells
and
tumor-infiltrating
cells
within the TME is to promote tumor growth and de-
velopment, along with diminished anti-tumor immun-
ity (Fig. 2). the TME characterized by collagen fibrogenesis, collagen
disorganization and fibroblast contractility; the remodel-
ing of the TME is not only important for cancer cell mi-
gration and invasion, but also plays a critical role in
resistance to therapeutic intervention [83, 84]. Zou et al. argeting STAT3 for cancer immunotherapy recently emerged to overcome this dilemma, and show
great promise to yield therapeutic agents to targeting
transcription factors, including STAT3. For instance, the
small-molecule
proteolysis-targeting
chimera
(PRO-
TAC)-based strategy has attracted a lot of attention
because it can inhibit target protein function as well
as
counteract
increased
target
protein
expression
[128]. The studies on PROTAC-mediated degradation
of oncogenic proteins such as BRD4 [129], BCR-ABL
[130], receptor tyrosine kinase (RTK) [131], and BCL-
XL [132] have shown encouraging results, suggesting
the potential clinical applicability of this ingenious ap-
proach. SD-36, a novel inhibitor identified by the
PROTAC-based strategy, exhibits high selectivity for
STAT3 and high cell permeability [98]. Moreover,
SD-36 treatment can cause a profound and long-
lasting suppression of tumor in mouse models of
leukemia
and
lymphoma
[98],
suggesting
that
PROTAC-based strategy may be a promising and reli-
able avenue for searching small molecule inhibitors
against STAT3. Further, the outstanding performance
of SD-36 in cancer treatment suggests that the strat-
egy of targeting STAT3 protein degradation may be
superior to suppress STAT3 expression. Although targeting STAT3 has been extensively investi-
gated for decades, this field still remains largely unex-
plored. The most common approach in targeting STAT3
directly is to prevent the formation of functional STAT3
dimers through disrupting the domains of SH2, DBD, or
NTD [85, 86]. In general, direct inhibitors of STAT3 can
be classified into three categories: peptides, small mole-
cules and oligonucleotides. Studies of these inhibitors on
pre-clinical cancer models are summarized in Table 1
[87–112] and relevant ongoing clinical trials are intro-
duced in Table 2 [117–119]. Peptides are usually designed based on the structure of
amino acid residues in STAT3 protein and can be di-
rected towards different domains. Phosphopeptide in-
hibitor (PY*LKTK), derived from the binding peptide
sequence of the STAT3-SH2 domain, represents the first
successful attempt to disrupt STAT3 dimerization [89]. However, the further development of peptide for the
clinical use is currently limited due to their poor cellular
permeability and lack of stability in vivo, and even the
second-generation peptidomimetics are largely suffering
from similar limitations [127]. Oligonucleotides represent a new treatment strategy
for
‘undruggable’
cancer
targets
such
as
STAT3. STAT3-binding decoy oligodeoxynucleotides, can se-
quester STAT3 and thus decrease its binding to cog-
nate DNA sites within target genes [133]. Antisense
oligonucleotides (ASOs) are designed to block STAT3
activity by targeting STAT3 mRNA. STAT3 in CAFs Molecular Cancer (2020) 19:145 Page 8 of 19 Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued)
Therapy
Type
Agent
Cell line tested
Mouse model
Functional outcome
Ref
VEGFR2
antibody
DC101
n/d
Xenograft: LLC, CT26
↓Proliferation; ↑Anti-tumor
immunity, Vascular normalization
[116]
STING agonist
cGAMP,
RR-CDA
AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC
Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung
cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued)
Therapy
Type
Agent
Cell line tested
Mouse model
Functional outcome
Ref
VEGFR2
antibody
DC101
n/d
Xenograft: LLC, CT26
↓Proliferation; ↑Anti-tumor
immunity, Vascular normalization
[116]
STING agonist
cGAMP,
RR-CDA
AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC
Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung
cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued)
Therapy
Type
Agent
Cell line tested
Mouse model
Functional outcome
Ref
VEGFR2
antibody
DC101
n/d
Xenograft: LLC, CT26
↓Proliferation; ↑Anti-tumor
immunity, Vascular normalization
[116
STING agonist
cGAMP,
RR-CDA
AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC
Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung
cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models (Continued) AOM/DSS Azoxymethane/dextran sodium sulfate, BC Breast cancer, CML Chronic myelogenous leukemia, CRC Colorectal cancer, GC Gastric cancer, HCC
Hepatocellular carcinoma, LLC Lewis lung carcinoma, MPLW515L Somatic mutations at codon 515 of the thrombopoietin receptor, NSCLC Non-small cell lung
cancer, PC Pancreatic cancer, PCa Prostate cancer, n/d Not determined, hpdODN hairpin decoy oligodeoxynucleotide STAT3 in CAFs Molecular Cancer (2020) 19:145 Page 7 of 19 Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models Table 1 Studies of STAT3 inhibitors on pre-clinical cancer models
Therapy
Type
Agent
Cell line tested
Mouse model
Functional outcome
Ref
Direct
inhibitors
Peptides
DBD-1
Melanoma, Myeloma
n/d
↑Apoptosis; ↓Proliferation
[87]
ISS-610
prodrugs
BC
n/d
↑Apoptosis
[88]
PY*LKTK
NIH3T3/v-Src or v-Ras
n/d
↓Transformation
[89]
Small molecules
6o
BC, PC, PCa, NSCLC
n/d
↑Apoptosis; ↓Proliferation
[90]
FLLL32
BC, PC
Xenograft: MDA-MB-231,
PANC-1
↑Apoptosis; ↓Proliferation,
Vascularization
[91]
HJC0152
Glioblastoma
Xenograft: U87
↑Apoptosis; ↓Metastasis,
Proliferation
[92]
LL1
CRC
Xenograft: HCT116
↑Apoptosis; ↓Metastasis,
Proliferation
[93]
LLL-3
BC, Glioblastoma
Xenograft: U87
↑Apoptosis; ↓Metastasis,
Proliferation
[94]
LLL12
HCC
Xenograft: SNU398
↑Apoptosis; ↓Proliferation
[95]
LYW-6
CRC
AOM/DSS induced CRC
model; Xenograft: HCT116
↑Apoptosis; ↓Metastasis,
Proliferation
[96]
Nitidine
chloride
Oral cancer
Xenograft: HSC3
↑Apoptosis; ↓Proliferation
[97]
SD-36
BC, CRC, Leukemia,
Lymphoma
Xenograft: MOLM-16, SUP-
M2, SU-DHL-1
↑Apoptosis; ↓Proliferation
[98]
Stattic
BC
n/d
↑Apoptosis
[99]
STX-0119
n/d
Humanized NOG-dKO model
↑Anti-tumor immunity;
↓Proliferation
[100]
S3I-1757
Melanoma
Xenograft: B16-F10
↓Proliferation
[101]
S3I-201
BC
Xenograft: MDA-MB-231
↑Apoptosis; ↓Proliferation
[102]
CPA-7
BC, CRC, Melanoma, PCa,
NSCLC
Xenograft: CT26
↑Apoptosis; ↓Proliferation
[103]
C48
BC, CML, Melanoma, PCa
Xenograft: MDA-MB-468,
C3L5
↓Proliferation
[104]
GPA512
PCa
Xenograft: DU145
↓Proliferation
[105]
MMPP
BC, CRC, PCa, HCC, Lung,
Ovary and Skin cancer
Xenograft: Patient-derived
NSCLC, NCI-H460
↑Apoptosis; ↓Proliferation
[106]
Oligonucleotides InS3-
54A18
BC, NSCLC
Xenograft: A549
↓Metastasis, Proliferation
[107]
STAT3
hpdODN
CRC
n/d
↓Proliferation
[108]
Indirect
inhibitors
JAK2
INCB16562 Leukemia
MPLW515L model
↓Proliferation
[109]
TG101209
Leukemia
AML1-ETO9a leukemia model ↑Apoptosis; ↓Proliferation
[110]
EGFR
JND3229
BaF3
Xenograft: BaF3-EGFR
↓Proliferation
[111]
FGFR, VEGFR
ODM-203
Bladder cancer, NSCLC, GC
Xenograft: H1581, KMS11,
RT4, SNU16
↑Anti-tumor immunity;
↓Metastasis, Proliferation
[112]
Combination Direct inhibitor
HJC0152
BC, THP1
Xenograft: 4T1
↑Anti-tumor immunity;
↓Proliferation
[113]
STING agonist
c-diAM
(PS)2
JAK1/2 inhibitor
Ruxolitinib
PC
Xenograft: PANC02-H7
↑Anti-tumor immunity;
↓Proliferation
[114]
Anti-PD-1
antibody
RMP1-14
SRC, ABL
inhibitor
Dasatinib
n/d
Tgfbr1/Pten 2cKO model
↑Anti-tumor immunity;
↓Proliferation
[115]
Anti-CTLA-4
antibody
9D9 Zou et al. argeting STAT3 for cancer immunotherapy For example,
AZD9150, a second-generation STAT3 ASO, targets
the 3'-untranslated region (3'-UTR) of the STAT3
gene [134]. Preclinical testing and clinical evaluation
have revealed the high efficacy and low toxicity of
AZD9150 in oncotherapy [135, 136]. Although oligo-
deoxynucleotides inhibitors of STAT3 provide exquis-
ite specificity and potency, their poor cell membrane
penetrance, rapid degradation, and the lack of effect-
ive targeted delivery carriers, remain the major obsta-
cles that impede their use in solid tumors. Aptamers
have also emerged as useful targeted delivery agents
for conventional drugs and small RNAs including siR-
NAs and miRNAs due to several advantages, such as small
physical size, high stability and low immunogenicity [137]. Non-peptide small molecules capable of disrupting phos-
phorylation of STAT3 or STAT3-STAT3 dimerization have
recently emerged as an attractive alternative approach to
the above. These small molecule inhibitors usually select-
ively bind to the SH2, the DBD, or the NTD domain of
STAT3 to block transcription of target genes [85]. BBI608
(Napabucasin), a small molecule inhibitor that selectively
binds to the DBD domain of STAT3, is the only direct
STAT3 inhibitor that has advanced into phase III trials thus
far. The excellent outcome of a recent phase III monother-
apy trial suggested that BBI608 has potential implication in
advanced colorectal cancer [117]. Moreover, FDA has ap-
proved BBI608 as an orphan drug for treatment of gastric
and pancreatic cancer based on the promising results in
phase I/II clinical trials. Numerous small molecule inhibitors of STAT3 have
been identified by virtual screening. Of note, although
these inhibitors exhibit excellent physicochemical prop-
erties in vitro, most of them show poor clinical efficacy,
which might be due to low aqueous solubility and low
cell permeability [86]. Several novel approaches have Page 9 of 19 Zou et al. argeting STAT3 for cancer immunotherapy Molecular Cancer (2020) 19:145 Table 2 STAT3 inhibitors in currently on-going clinical trials
Therapy
Type
Agent
Indication
Phase NCT number Ref
Direct
inhibitors
Small molecules
BBI608 (FDA approved)
Advanced malignancies
I/II
NCT01775423 NA
CRC
III
NCT01830621 [117]
Celecoxib* (FDA approved)
CRC
III
NCT00087256 NA
C188-9
BC, CRC, HNSCC, HCC, NSCLC, GAC, Melanoma,
Advanced cancer
I
NCT03195699 NA
OPB-111077
Acute myeloid leukemia
I
NCT03197714 NA
Advanced HCC
I
NCT01942083 NA
OPB-31121
Advanced cancer, Solid tumors
I
NCT00955812 NA
HCC
I/II
NCT01406574 NA
OPB-51602
Malignant solid tumors
I
NCT01184807 NA
Hematological malignancies
I
NCT01344876 NA
Nasopharyngeal carcinoma
I
NCT02058017 NA
Pyrimethamine* (FDA
approved)
CLL, Small lymphocytic lymphoma
I/II
NCT01066663 NA
Oligonucleotides
AZD9150
Lymphoma
I/II
NCT01563302 [118]
STAT3 decoy
Head and neck cancer
0
NCT00696176 [119]
Indirect
inhibitors
JAK1/2
AZD-1480
Solid tumors
I
NCT01112397 NA
CYT 387
Myelofibrosis
I/II
NCT01423058 [120]
PMF, Post-PV, Post-ET MF
III
NCT02101268 NA
Ruxolitinib (FDA approved)
Myelofibrosis
II
NCT03427866 NA
JAK2
LY2784544
Myeloproliferative neoplasms
II
NCT01594723 [121]
SB1518
Myelofibrosis
III
NCT02055781 [122]
EGFR
Cetuximab (FDA approved)
Metastatic CRC
I/II
NCT02117466 NA
Panitumumab (FDA
approved)
Advanced CRC
II
NCT03311750 NA
Metastatic CRC
IV
NCT02301962 NA
FGFR
Ponatinib (FDA approved)
CML
II
NCT04043676 NA
CML, ALL
II
NCT04233346 NA
IL-6R
Siltuximab (FDA approved)
Multiple myeloma
II
NCT03315026 NA
Tocilizumab (FDA approved)
HCC
I/II
NCT02997956 NA
VEGF
Bevacizumab (FDA approved)
Metastatic CRC
II
NCT02226289 NA
VEGFR
Apatinib
Lung carcinoma
II
NCT03709953 NA
VEGFR, PDGFR
Sorafenib (FDA approved)
Advanced HCC
IV
NCT02733809 NA
VEGFR, PDGFR, c-KIT
Sunitinib (FDA approved)
Clear cell renal carcinoma
II
NCT03066427 NA
Pancreatic neuroendocrine tumor metastatic
II
NCT02713763 NA
SRC, ABL
Dasatinib (FDA approved)
Chronic-phase CML
IV
NCT01660906 [123]
SRC
Bosutinib (FDA approved)
CML
II
NCT02810990 NA
KX2-391
Bone-metastatic, Castration-resistant PCa
II
NCT01074138 [124]
Combination
Direct inhibitors and
ICB
AZD9150, Durvalumab (anti-
PD-L1)
NSCLC
II
NCT03334617 NA
PC, CRC, NSCLC
II
NCT02983578 NA
Advanced solid tumors, Metastatic HNSCC
I/II
NCT02499328 NA
Diffuse large B-cell lymphoma
I
NCT02549651 NA
BBI608, Nivolumab (anti-PD-
L1)
Metastatic CRC
II
NCT03647839 NA
BBI608, Pembrolizumab (anti-
PD-1)
Metastatic CRC
I/II
NCT02851004 NA Phase NCT number Ref Page 10 of 19 Zou et al. argeting STAT3 for cancer immunotherapy Molecular Cancer (2020) 19:145 Table 2 STAT3 inhibitors in currently on-going clinical trials (Continued)
Therapy
Type
Agent
Indication
Phase NCT number Ref
Indirect inhibitors
and ICB
Apatinib, SHR-1210 (anti-PD-1)
Melanoma
II
NCT03955354 NA
Bevacizumab, Atezolizumab
(anti-PD-L1)
Unresectable HCC
III
NCT03434379 [125]
Cetuximab, Pembrolizumab
(anti-PD-1)
Recurrent or metastatic HNSCC
II
NCT03082534 NA
Dasatinib, Ipilimumab (anti-
CTLA-4)
GIST, Stage III /IV soft tissue sarcoma
I
NCT01643278 [126]
Dasatinib, Nivolumab (anti-
PD-L1)
Philadelphia chromosome positive ALL
I
NCT02819804 NA
Ruxolitinib, Pembrolizumab
(anti-PD-1)
Hematological malignancies
II
NCT04016116 NA
Metastatic stage IV TNBC
I
NCT03012230 NA
Sorafenib, BGB-A317(anti-PD-
1)
HCC
III
NCT03412773 NA
Sorafenib, Nivolumab (anti-
PD-L1)
Advanced or metastatic HCC
II
NCT03439891 NA
Indirect inhibitor and
CAR-T
Tocilizumab, CAR-T 19
Lymphoblastic leukemia
NA
NCT02906371 NA
ALL Acute lymphoblastic leukemia, BC Breast cancer, Celecoxib* An FDA approved nonsteroidal anti-inflammatory drug, CML Chronic myelogenous leukemia, CLL
Chronic lymphocytic leukemia, CRC Colorectal cancer, HNSCC Head and neck squamous cell carcinoma, NA Not available, NSCLC Non-small cell lung cancer, HCC
Hepatocellular carcinoma, GAC Gastric adenocarcinoma, Pyrimethamine* An FDA approved anti-parasitic drug, PMF Primary myelofibrosis, Post-PV Post-
polycythemia vera, Post-ET MF Post-essential thrombocythemia myelofibrosis, PC Pancreatic cancer, PCa Prostate cancer, GIST Gastrointestinal stromal tumor, TNBC
Triple negative breast cancer ALL Acute lymphoblastic leukemia, BC Breast cancer, Celecoxib* An FDA approved nonsteroidal anti-inflammatory drug, CML Chronic myelogenous leukemia, CLL
Chronic lymphocytic leukemia, CRC Colorectal cancer, HNSCC Head and neck squamous cell carcinoma, NA Not available, NSCLC Non-small cell lung cancer, HCC
Hepatocellular carcinoma, GAC Gastric adenocarcinoma, Pyrimethamine* An FDA approved anti-parasitic drug, PMF Primary myelofibrosis, Post-PV Post-
polycythemia vera, Post-ET MF Post-essential thrombocythemia myelofibrosis, PC Pancreatic cancer, PCa Prostate cancer, GIST Gastrointestinal stromal tumor, TNBC
Triple negative breast cancer ALL Acute lymphoblastic leukemia, BC Breast cancer, Celecoxib* An FDA approved nonsteroidal anti-inflammatory drug, CML Chronic myelogenous leukemia, CLL
Chronic lymphocytic leukemia, CRC Colorectal cancer, HNSCC Head and neck squamous cell carcinoma, NA Not available, NSCLC Non-small cell lung cancer, HCC
Hepatocellular carcinoma, GAC Gastric adenocarcinoma, Pyrimethamine* An FDA approved anti-parasitic drug, PMF Primary myelofibrosis, Post-PV Post-
polycythemia vera, Post-ET MF Post-essential thrombocythemia myelofibrosis, PC Pancreatic cancer, PCa Prostate cancer, GIST Gastrointestinal stromal tumor, TNBC
Triple negative breast cancer Recently, STAT3 silencing by aptamer-siRNA chimera ob-
tained excellent inhibition in the therapy of glioblastoma
[138, 139], suggesting that the improved oligonucleotides
might offer translational potential for the treatment of
solid tumors. argeting STAT3 for cancer immunotherapy downstream components of the STAT3 signaling path-
way, and hundreds of leading compounds have been
identified [144–148]. Out of those, Ruxolitinib, Dasatinib
and Siltuximab that target JAK, SRC/ABL, and IL-6 re-
spectively, have been approved by FDA for cancer ther-
apy. Indirect STAT3 inhibitors in currently on-going
clinical trials are summarized in Table 2 [120–124]. Of
note, indirect STAT3 inhibitors lack specificity for
STAT3 and may cause fairly extensive kinase inhibition
because the targeted molecules are often involved in in-
tricate signaling pathways. Since STAT3 is a transcription factor, it is traditionally
regarded as an undruggable target. Direct targeting of
STAT3 has proven to be considerably challenging, owing
in part to high sequence similarity with the other STAT
members [86, 140]. Moreover, several issues such as
high toxicity and poor bioavailability have become sig-
nificant impediments to the clinical development of dir-
ect STAT3 inhibitors [86]. Interestingly, some FDA-
approved compounds, such as Pyrimethamine and Cele-
coxib, have been identified as STAT3 inhibitors through
drug-repositioning screening [141, 142]. These findings
not only provide another source for searching STAT3
inhibitors, but also suggest potential applications of
these drugs in cancer therapy. In addition, similar to
combined therapy, certain bifunctional compounds are
emerging and may represent a new generation of highly
efficacious STAT3 inhibitors for cancer therapy in the
future. For example, the compound 8u has dual immu-
notherapeutic and anticancer efficacy through simultan-
eously inhibiting indoleamine-2,3-dioxygenase 1 (IDO1)
and STAT3 [143]. Intriguingly, it has recently been shown that phosphory-
lated STAT3 is present in exosomes from 5-fluorouracil
(5-FU) resistant colorectal cancer cells, which contributes
to acquired 5-FU resistance [149]. Given the importance
and efficiency of exosomes in intercellular and interorgan
communication [150, 151], these findings not only add an-
other complexity to STAT3 regulation, but also pave a
new way to inhibit the oncogenic function of STAT3, as
well as to delivery STAT3 inhibitors via exosomes. Integrating STAT3 in combination cancer
immunotherapy Immunotherapy is currently among the most promising
approaches for cancer treatment. This therapeutic strat-
egy, represented mainly by immune checkpoint blockade
(ICB) and chimeric antigen receptor T cells (CAR-T),
has obtained unprecedented results in patients with pre-
viously incurable cancers [3, 152]. However, there are
some key challenges that need to be resolved urgently, Indirect targeting of STAT3 Preclinical
and clinical data suggest that combination cancer im-
munotherapies have enhanced therapeutic efficacy and
reduced drug resistance compared with monotherapy
[155, 156]. These encouraging data has triggered many
investigations of combination strategies, and the com-
bination of STAT3 inhibitors with other immunotherapy
agents are also emerging (Fig. 3). p
Combined blockade of STAT3 and immune check-
point has shown encouraging results, whereby the
addition of STAT3 inhibitors can enhance therapeutic
efficacy, and reduce resistance to ICB immunotherapy in
parallel. Dasatinib, an indirect STAT3 inhibitor against
SRC/ABL, significantly facilitated anti-CTLA-4 immuno-
therapy in head and neck squamous cell carcinoma
[115], while the combined blockade of IL-6 and PD-L1
remarkably inhibited the growth of pancreatic ductal
adenocarcinoma and hepatocellular carcinoma (HCC)
[167, 168]. The resistance to anti-PD-1 antibodies can
be overcome by treatment with JAK inhibitor in mice
with pancreatic orthotopic tumors [114]. Niclosamide
blocked STAT3-induced PD-L1 transcription, and thus
enhanced the efficacy of anti-PD-1/PD-L1 antibodies in
non-small cell lung cancer [169]. More recently, a phase
III trial reported exciting results of STAT3-based com-
bination therapy in treatment of advanced HCC. Com-
pared to the first-line drug sorafenib, the combination of
bevacizumab (a monoclonal antibody targeting VEGF)
and atezolizumab (a PD-L1 inhibitor) can significantly
prolong the overall survival and progression-free survival
of patients with unresectable HCC, along with compar-
able adverse effects [125]. In addition, certain STAT3 in-
hibitors, such as BBI608 and AZD9150, combining with
immune checkpoint inhibitors, are currently being tested
in pre-clinical (Table 1) and clinical trials (Table 2)
[126]. The promising early phase clinical trials encour-
age further clinical development of this combination
strategy. Combined blockade of STAT3 and immune checkpoint
Up-regulated expression of the immune checkpoint mol-
ecules, including CTLA-4, PD-1, and PD-L1, has been
documented to facilitate tumor immune escape. A sub-
stantial amount of evidence has shown that STAT3 is
able to directly or indirectly regulate these immune
checkpoint molecules. As a transcription factor, STAT3
can increase expression of PD-1, PD-L1, and PD-L2 by
direct binding to their promoters [157–160]. Meanwhile,
STAT3 has been identified to indirectly induce expres-
sion of immune checkpoint molecules through modulat-
ing diverse signaling pathways. For example, STAT3
increased CTLA-4 expression in tumor-associated B
cells in a JAK-dependent manner [161] and enhanced
CTLA-4 expression in Treg cells through IL-10 [162]. In
addition, STAT3 mediated HDAC6-induced PD-L1 ex-
pression in osteosarcoma cells [163]. Indirect targeting of STAT3 In parallel with direct inhibitors, indirect inhibitors of
STAT3 have been pursued by targeting the upstream or Zou et al. Molecular Cancer (2020) 19:145 Page 11 of 19 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Page 12 of 19 Zou et al. Molecular Cancer (2020) 19:145 (See figure on previous page.)
Fig. 3 Targeting STAT3 in combination cancer immunotherapy. a Summary of the key steps in the development of STAT3-targeting therapeutics. The first step in the development of STAT3-targeting therapeutics involves the systematic selection of STAT3 inhibitors (including direct or
indirect inhibitors) and STAT3 inhibitors-based combined immunotherapy, and then elucidating the biology and effects of these candidates to
cancer using tumor cell lines and patient samples. The next major challenge involves the in vivo model-based validation that these therapeutic
candidates must undergo rigorous disease-specific in vivo testing using rodents and non-human primate models. Key challenges in translating
STAT3 inhibitors into the clinic are low bioavailability and the lack of specific targeting of the tumor site. b Targeting STAT3 in combination
cancer immunotherapy. Targeting STAT3 in combination cancer immunotherapy can not only enhance the anti-tumor effects, but also reduce
drug resistance. Besides, combined STAT3 inhibitors with CAR-T cells can reduce excessive expansion of CAR-T cells and alleviate cytokine release
syndrome (CRS), resulting in lower occurrence of immune-related adverse effects. CD4+ T cells [165]. Given PI3K/AKT as a known repres-
sor of STAT3 transcription [166], it is likely that PD-1
indirectly enhances STAT3 expression through inhib-
ition of PI3K. The reciprocal regulation of STAT3 and
immune checkpoint molecules not only suggests an in-
volvement of STAT3 in anti-tumor immunity, but also
provides a promising strategy to improve the efficacy of
current immune checkpoint inhibitors. including limited clinical response rates and significant
autoimmune-related side effects [3, 153]. For instance,
ICB has shown remarkable effectiveness in solid tumors
including melanoma, non-small cell lung cancers and
renal cancer, however, even in these cancers, the major-
ity of patients still do not respond to the treatment [3]. Furthermore, certain types of cancer such as pancreatic
cancer and prostate cancer show resistance to immune
checkpoint inhibition therapy [3, 154]. Thus, combin-
ation therapy is considered to be a promising direction
for improving outcomes for cancer treatment. Indirect targeting of STAT3 Conversely, recent
evidence also suggests a role of immune checkpoint
molecules in STAT3 expression. Celada et al. reported
that PD-1 upregulation in CD4+ T cells leads to an in-
crease in STAT3 mRNA expression by undescribed
mechanism, and the latter is required for IL-17 and
TGFβ1 production [164]. Interestingly, an early study
from the same research group showed PD-1 can attenu-
ate TCR-dependent activation of PI3K/AKT pathway in Combined STAT3 inhibitors and CAR-T For example, CAR-T cells targeting B7-H3, a
transmembrane protein belonging to the B7 immune fam-
ily, inhibited the growth of neuroblastoma, pancreatic and
ovarian cancer in vitro and in xenograft mouse models
without evident toxicity [171, 172]. The involvement of STAT3 signaling in CAR-T ther-
apy is emerging. Transcriptomic profiling showed that
anti-CD19 CAR-T cells from responsive patients with
chronic lymphocytic leukemia had an increased IL-6/
STAT3 signature, which promoted the expansion of
CAR-T cells [173]. In line with this, a novel anti-CD19
CAR-T cells with constitutive activation of STAT3
showed increased proliferation and reduced terminal dif-
ferentiation of CAR-T cells, and conferred superior anti-
tumor effects [174]. Similarly, CAR-T cells expressing
the ectodomain of the IL-4 receptor and the end domain
of the IL-21 receptor activated the STAT3 pathway and
enhanced Th17-like polarization, representing a poten-
tial clinical CAR-T therapy for solid tumors enriching
IL-4 [175]. These studies suggest a beneficial effect of
STAT3 activation in CAR-T cells. Several previous studies have suggested potential inter-
actions between STING signaling and STAT3-driven
oncogenic pathways [181–183]. It has been observed
that rapid colorectal cancer progression in STING-
deficient mice is associated with STAT3 hyperactivation
[181]. Further research found that STING plays a vital
role in regulation of MDSC differentiation and anti-
tumor immunity in nasopharyngeal carcinoma by in-
creasing the expression of SOCS1, a classic repressor of
STAT3 [182]. Besides, TBK1, which is activated by cyto-
solic DNA in a STING-dependent manner, can restrain
activation of STAT3 through direct phosphorylation of
STAT3 at serine 754 in the TAD [183]. As mentioned above, STAT3 hyperactivation in tumor
stroma is immunosuppressive and can increase the ex-
pression of certain cytokines and growth factors. Ac-
cordingly,
constitutive
expression
of
an
array
of
cytokines such as IL-6 and IL-10 potentially could in-
crease the risk of serious adverse events of CAR-T ther-
apy including cytokine release syndrome [176]. Thus,
there are some attempts to combine CAR-T therapy
with STAT3 inhibitors for improving the persistence
and anti-tumor effects, as well as negating toxicities of
CRA-T cells in vivo. For instance, the JAK2/STAT3 axis
is a crucial driver of liver-associated MDSCs and inhib-
ition of STAT3 increased the efficacy of CAR-T cells in
liver cancer metastasis [177]. In addition, a clinical study
is currently on-going, which tests the efficacy and ad-
ministration of the anti-IL-6 therapy (tocilizumab) on
anti-CD19 CAR-T cells associated cytokine release syn-
drome (NCT02906371). Combined STAT3 inhibitors and CAR-T Combined STAT3 inhibitors and CAR-T
CAR-T cell therapy, a rapidly emerging and effective im-
munotherapeutic approach, has revolutionized anti-cancer
therapies for hematologic malignancies, especially acute
lymphoblastic leukemia and lymphoma [152]. Two Anti- Page 13 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 [178]. Activated STING can propagate interferon recep-
tor signaling in tumor-infiltrating DCs and elicit CD8+ T
cells against tumor-associated antigens in vivo [179]. Therefore, STING agonists are of continuing research
interest as novel adjuvants to boost cancer immunother-
apy. A recent study showed that STING-activating nano-
particles (STING-NPs) can convert immunosuppressive
tumors to immunogenic microenvironments and then
induce anti-tumor immune responses and immuno-
logical memory in mice with melanoma [180]. In an-
other study, Ramanjulu et al. reported that STING
agonist can lead to complete and lasting regression of
tumors
in
mice
with
colon
tumors
[179]. These
encouraging results of preclinical studies point towards
the potential for improving clinical outcomes of im-
munotherapy, and some STING agonists such as c-
diAM (PS)2 and cGAMP are currently being evaluated
in clinical trials (NCT03937141, NCT02986867). [178]. Activated STING can propagate interferon recep-
tor signaling in tumor-infiltrating DCs and elicit CD8+ T
cells against tumor-associated antigens in vivo [179]. Therefore, STING agonists are of continuing research
interest as novel adjuvants to boost cancer immunother-
apy. A recent study showed that STING-activating nano-
particles (STING-NPs) can convert immunosuppressive
tumors to immunogenic microenvironments and then
induce anti-tumor immune responses and immuno-
logical memory in mice with melanoma [180]. In an-
other study, Ramanjulu et al. reported that STING
agonist can lead to complete and lasting regression of
tumors
in
mice
with
colon
tumors
[179]. These
encouraging results of preclinical studies point towards
the potential for improving clinical outcomes of im-
munotherapy, and some STING agonists such as c-
diAM (PS)2 and cGAMP are currently being evaluated
in clinical trials (NCT03937141, NCT02986867). CD19 CAR-T therapies are currently approved by FDA
for the treatment of CD19-positive leukemia or lymph-
oma. Although the efficacy of CAR-T therapy in solid tu-
mors has lagged far behind, a great number of CAR-T
trials are ongoing and positive outcomes are increasingly
being reported for multiple solid tumors, including glio-
blastoma, gastrointestinal, genitourinary, breast, and lung
cancer [170]. Combined STAT3 inhibitors and CAR-T Recent evidence indicates that the combination of STING
agonists with STAT3 inhibitors can enhance tumor im-
munogenicity and optimize the immunotherapeutic effects
[113, 116]. For example, combined STAT3 direct inhibitor
HJC0152 with STING agonist c-diAM (PS)2 increased CD8+
T cells, reduced Treg cells and MDSCs in the TME, and thus
effectively enhanced anti-tumor immunity in mice with
breast cancer [113]. A preclinical study demonstrated the
combination of STING agonist (cGAMP or RR-CDA) with
the indirect STAT3 inhibitor VEGFR2 was maximally effect-
ive for immunotherapy-resistant tumors in breast and lung
cancer [116]. STAT3 blockade can also markedly improve other ef-
fective immunotherapeutic approaches including cancer
vaccines
and
immunostimulatory
Toll-like
receptor
(TLR) agonists (such as CpG oligodeoxynucleotides). For
example, a novel strategy combining STAT3 ASO with
TLR9 stimulation (CpG oligonucleotide) has been shown
to enhance the anti-tumor immunity and overcome
tumor immune tolerance in prostate cancer [184]. The
combination of STAT3 inhibitor and DC-based vaccine
led to improved therapeutic outcomes in mouse colon
cancer
[185]. The
beneficial
outcomes
of
these Combined STAT3 inhibitors and STING agonists
Stimulator of interferon genes (STING) is a major
adaptor protein that plays an important role in anti-viral
and anti-tumor immunity [178]. When stimulated by
cytosolic DNA, STING activates TANK-binding kinase 1
(TBK1), which subsequently phosphorylates interferon
regulatory factor 3 (IRF3) to promote IFN expression Page 14 of 19 Page 14 of 19 Page 14 of 19 Zou et al. Molecular Cancer (2020) 19:145 immunotherapy combinations about STAT3 inhibitors
warrant further clinical validation. Notably, STAT3 an-
tagonists, either direct or indirect STAT3 inhibitors, are
generally less likely to completely block STAT3 signaling
and might not trigger severe autoimmune disorders. However, it cannot be ignored that the use of STAT3 in-
hibitors and other immunotherapy agents in combin-
ation may result in more frequent and severe immune-
related adverse events (irAEs) compared to monother-
apy. Accordingly, risk evaluations for irAEs should be
part of the decision criteria for determining immuno-
therapy combinations. Likewise, early recognition and
adequate management for irAEs are indispensable to
minimize treatment-related serious complications. predictive biomarkers that can provide a basis for im-
proved precision medicine, though the related studies
are currently not explored in depth. Abbreviations
AHR A l h d AHR: Aryl hydrocarbon receptor; ALL: Acute lymphoblastic leukemia;
AOM: Azoxymethane; ASOs: Antisense oligonucleotides; BC: Breast cancer;
Bcl-2: B-cell lymphoma-2; BRD4: Bromodomain-containing protein 4;
CAFs: Cancer-associated fibroblasts; CAR-T: Chimeric antigen receptor T cells;
CCD: Coiled-coil domain; CCL: C-C motif ligand; CD80/86: Cluster of
differentiation 80/86; circRNAs: Circular RNAs; CLL: Chronic lymphocytic
leukemia; CML: Chronic myelogenous leukemia; CRS: Cytokine release
syndrome; CTLA-4: Cytotoxic T-lymphocyte-associated protein 4; CXCL: C-X-C
motif chemokine ligand; Cxxc1: Cxxc finger protein 1; DBD: DNA-binding
domain; DCs: Dendritic cells; DSS: Dextran sodium sulfate; DUSP22: Dual
specificity phosphatase 22; EGF: Epidermal growth factor; EGFR: Epidermal
growth factor receptor; EZH2: Enhancer of zeste homolog 2; FGFR: Fibroblast
growth factor receptor; GAC: Gastric adenocarcinoma; GC: Gastric cancer;
GIST: Gastrointestinal stromal tumor; GP130: Glycoprotein 130; HADC: Histone
deacetylase; HCC: Hepatocellular carcinoma; HIF1α: Hypoxia-inducible factor
1α; HNSCC: Head and neck squamous cell carcinoma; hpdODN: Hairpin
decoy oligodeoxynucleotide; ICB: Immune checkpoint blockade;
IDO1: Indoleamine-2,3-dioxygenase 1; IFNs: Interferons; IFN-Is: Type I
interferons; IL: Interleukin; Loxl3: Lysyl oxidase-like 3; irAEs: Immune-related
adverse events; IRF3: Interferon regulatory factor 3; ISGF3: Interferon-
stimulated gene factor 3; JAKs: Janus kinases; LLC: Lewis lung carcinoma;
lncRNAs: Long non-coding RNAs; LIF: Leukemia inhibitory factor;
MDSCs: Myeloid-derived suppressor cells; MHC class II: Major
histocompatibility complex class II; MMPs: Matrix metalloproteinases;
MPLW515L: Somatic mutations at codon 515 of the thrombopoietin
receptor; ncRNAs: Non-coding RNAs; NH2: Amino-terminal domain; NSCL
C: Non-small cell lung cancer; NF-κB: Nuclear factor-κB; NTD: N-terminal
domain; PC: Pancreatic cancer; PCa: Prostate cancer; PD-1: Programmed cell
death protein 1; PDGFR: Platelet-derived growth factor receptor; PD-
L1: Programmed cell death 1 ligand 1; PKCβII: Protein kinase C β II;
PMF: Primary myelofibrosis; PIAS: Protein inhibitor of activated STAT;
PlGF: Placenta growth factor; PI3K: Phosphatidylinositol 3 kinase; Post-ET
MF: Post-essential thrombocythemia myelofibrosis; Post-PV: Post-
polycythemia vera; PROTAC: Proteolysis-targeting chimera; PTPases: Protein
tyrosine phosphatases; PTPN1: Protein tyrosine phosphatase non-receptor
type 1; PTPRD: Protein tyrosine phosphatase receptor type D; PTPRT: Protein
tyrosine phosphatase receptor type T; RTK: Receptor tyrosine kinase; SH2: Src
homology 2 domain; SHP: Src homology 2 domain containing protein
tyrosine phosphatase; SIRT1: Sirtuin 1; SMYD2: SET (Suppressor of variegation,
Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domain
containing 2; SOCS: Suppressor of cytokine signaling; STAT: Signal transducer
and activator of transcription; STING: Stimulator of interferon genes; STING-
NPs: STING-activating nanoparticles; SUMO2/3: Small ubiquitin-like modifier
2/3; TAD: Transactivation domain; TBK-1: TANK-binding kinase 1; TCR: T cell
receptor; TGFβ: Transforming growth factor β; Th1: T helper 1; TLR: Toll-like
receptor; TME: Tumor microenvironment; TNFα: Tumor necrosis factor α;
Treg: Regulatory T-cell; VEGF: Vascular endothelial growth factor; VEGF
R: Vascular endothelial growth factor receptor; 3ʹ-UTR: 3ʹ-untranslated region;
5-FU: 5-fluorouracil Combined STAT3 inhibitors and CAR-T In summary, therapeutically targeting mediators of the
STAT3 signaling, which has already been shown to be
beneficial in the restoration of anti-tumor immunity,
provides attractive avenues that are currently being ex-
plored for the immunotherapy of cancers both as mono-
therapy and in combination therapies. Y.T. and X.F. conceived the idea; S.Z., Q.T. and B.L. performed the literature
search and draft the manuscript; W.H. revised and edited the manuscript; X.F.
and Y.T. supervised and revised the manuscript. The author(s) read and
approved the final manuscript. Conclusions and perspectives STAT3 becomes excessively activated in multiple human
cancers, and acts as a crucial signaling node for tumor
cells
and
TME
comprising
cells,
especially
tumor-
infiltrating immune cells. Therefore, targeting STAT3 is
expected to offer multiple benefits, including reduced
tumor cell intrinsic proliferation, enhanced anti-tumor
effects of tumor-infiltrating immune cells, and improved
the immunosuppressive crosstalk within the TME. These
effects have positioned STAT3 as an arisen potential
promising target for cancer therapy. To date, many endeavors have been made to target
STAT3 for the development and application of new
drugs. These approaches are devised to inhibit STAT3
directly by peptides, small molecules and decoy oligonu-
cleotides, or indirectly by blocking upstream signaling
pathways such as IL-6 and JAK2 pathways. Currently,
the core idea of direct targeting STAT3 is to prevent the
formation of functional STAT3 dimers through disrupt-
ing phosphorylation of STAT3. Beyond phosphorylation,
other posttranslational modifications, such as acetyl-
ation, methylation and sumoylation, are emerging to
modulate STAT3 activation through diverse mecha-
nisms, providing a broadened list of candidate regulatory
targets for the STAT3 inhibitors. To improve the response rate and the number of
responding cancer types, combined immunotherapies
are now being undertaken. Combination therapies of
STAT3 inhibitors with currently therapeutic anti-tumor
drugs including the immune-checkpoint inhibitors may
open up new possibilities for long-lasting and multi-
layered tumor control. Although preclinical studies and
early clinical trials on combined blockade of STAT3 and
immune checkpoint have shown encouraging results,
their
clinical
outcomes
await
further
investigation. Moreover, predictive biomarkers are urgently required
to rationally incorporate STAT3 inhibitors into the com-
bination
immunotherapy. The
ncRNAs,
particularly
miRNAs,
might
prove
to
be
potentially
promising References Jak-STAT pathways and transcriptional
activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–21. 31. Luo Z, Cao P. Long noncoding RNA PVT1 promotes hepatoblastoma cell
proliferation through activating STAT3. Cancer Manag Res. 2019;11:8517–27. 6. Hanlon MM, Rakovich T, Cunningham CC, Ansboro S, Veale DJ, Fearon U,
et al. STAT3 Mediates the Differential Effects of Oncostatin M and TNFalpha
on RA Synovial Fibroblast and Endothelial Cell Function. Front Immunol. 2019;10:2056. 32. Wang S, Liang K, Hu Q, Li P, Song J, Yang Y, et al. JAK2-binding long
noncoding RNA promotes breast cancer brain metastasis. J Clin Invest. 2017;127:4498–515. 7. Rawlings JS, Rosler KM, Harrison DA. The JAK/STAT signaling pathway. J Cell
Sci. 2004;117:1281–3. 33. Liang C, Zhao T, Li H, He F, Zhao X, Zhang Y, et al. Long Non-coding
RNA ITIH4-AS1 Accelerates the Proliferation and Metastasis of Colorectal
Cancer by Activating JAK/STAT3 Signaling. Mol Ther Nucleic Acids. 2019;18:183–93. 8. Johnson DE, O'Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling
axis in cancer. Nat Rev Clin Oncol. 2018;15:234–48. 9. Ishibashi K, Koguchi T, Matsuoka K, Onagi A, Tanji R, Takinami-Honda R,
et al. Interleukin-6 induces drug resistance in renal cell carcinoma. Fukushima J Med Sci. 2018;64:103–10. 34. Pichler M, Rodriguez-Aguayo C, Nam SY, Dragomir MP, Bayraktar R, Anfossi
S, et al. Therapeutic potential of FLANC, a novel primate-specific long non-
coding RNA in colorectal cancer. Gut. 2020. Fukushima J Med Sci. 2018;64:103–10. 10. Priego N, Zhu L, Monteiro C, Mulders M, Wasilewski D, Bindeman W, et al. STAT3 labels a subpopulation of reactive astrocytes required for brain
metastasis. Nat Med. 2018;24:1024–35. 35. Wang Y, Wu S, Zhu X, Zhang L, Deng J, Li F, et al. LncRNA-encoded
polypeptide ASRPS inhibits triple-negative breast cancer angiogenesis. J Exp
Med. 2020;217. metastasis. Nat Med. 2018;24:1024–35. 11. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C,
et al. Stat3 as an oncogene. Cell. 1999;98:295–303. 36. Hu D, Zhang Y. Circular RNA HIPK3 promotes glioma progression by
binding to miR-124-3p. Gene. 2019;690:81–9. 12. Wang T, Fahrmann JF, Lee H, Li YJ, Tripathi SC, Yue C, et al. JAK/STAT3-
Regulated Fatty Acid beta-Oxidation Is Critical for Breast Cancer Stem Cell
Self-Renewal and Chemoresistance. Cell Metab. 2018;27:136–50 e5. 37. Dong Y, Xu T, Zhong S, Wang B, Zhang H, Wang X, et al. Circ_0076305
regulates cisplatin resistance of non-small cell lung cancer via positively
modulating STAT3 by sponging miR-296-5p. Life Sci. 2019;239:116984. Received: 20 May 2020 Accepted: 4 September 2020 Received: 20 May 2020 Accepted: 4 September 2020 25. Zhang L, Li J, Wang Q, Meng G, Lv X, Zhou H, et al. The relationship
between microRNAs and the STAT3-related signaling pathway in cancer. Tumour Biol. 2017;39:1010428317719869. Competing interests The authors declare that they have no competing interests. 21. Yang J, Huang J, Dasgupta M, Sears N, Miyagi M, Wang B, et al. Reversible
methylation of promoter-bound STAT3 by histone-modifying enzymes. Proc
Natl Acad Sci U S A. 2010;107:21499–504. Consent for publication 20. Kim E, Kim M, Woo DH, Shin Y, Shin J, Chang N, et al. Phosphorylation of
EZH2 activates STAT3 signaling via STAT3 methylation and promotes
tumorigenicity of glioblastoma stem-like cells. Cancer Cell. 2013;23:839–52. Authors’ contributions Y.T. and X.F. conceived the idea; S.Z., Q.T. and B.L. performed the literature
search and draft the manuscript; W.H. revised and edited the manuscript; X.F. and Y.T. supervised and revised the manuscript. The author(s) read and
approved the final manuscript. Page 15 of 19 Page 15 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 Ethics approval and consent to participate
Not applicable. 19. Ma L, Huang C, Wang XJ, Xin DE, Wang LS, Zou QC, et al. Lysyl Oxidase 3 Is
a Dual-Specificity Enzyme Involved in STAT3 Deacetylation and
Deacetylimination Modulation. Mol Cell. 2017;65:296–309. References 26. Huan L, Liang LH, He XH. Role of microRNAs in inflammation-associated
liver cancer. Cancer Biol Med. 2016;13:407–25. 1. Christofi T, Baritaki S, Falzone L, Libra M, Zaravinos A. Current Perspectives in
Cancer Immunotherapy. Cancers (Basel). 2019;11. 1. Christofi T, Baritaki S, Falzone L, Libra M, Zaravinos A. Current Perspectives in
Cancer Immunotherapy. Cancers (Basel). 2019;11. 27. Guo R, Wu Z, Wang J, Li Q, Shen S, Wang W, et al. Development of a Non-
Coding-RNA-based EMT/CSC Inhibitory Nanomedicine for In Vivo Treatment
and Monitoring of HCC. Adv Sci (Weinh). 2019;6:1801885. 2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. 2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N
Engl J Med. 2010;363:711–23. 2. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N
Engl J Med. 2010;363:711–23. 28. Yang Y, Ding L, Hu Q, Xia J, Sun J, Wang X, et al. MicroRNA-218 functions as
a tumor suppressor in lung cancer by targeting IL-6/STAT3 and negatively
correlates with poor prognosis. Mol Cancer. 2017;16:141. 3. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety
and activity of anti-PD-L1 antibody in patients with advanced cancer. N
Engl J Med. 2012;366:2455–65. 3. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety
and activity of anti-PD-L1 antibody in patients with advanced cancer. N
Engl J Med. 2012;366:2455–65. 29. Zhang X, Sai B, Wang F, Wang L, Wang Y, Zheng L, et al. Hypoxic BMSC-
derived exosomal miRNAs promote metastasis of lung cancer cells via
STAT3-induced EMT. Mol Cancer. 2019;18:40. 4. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF,
et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443–54. 30. Jin S, Yang X, Li J, Yang W, Ma H, Zhang Z. p53-targeted lincRNA-p21 acts
as a tumor suppressor by inhibiting JAK2/STAT3 signaling pathways in head
and neck squamous cell carcinoma. Mol Cancer. 2019;18:38. 5. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional
activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–21. 5. Darnell JE Jr, Kerr IM, Stark GR. Availability of data and materials
Not applicable. 18. Nie Y, Erion DM, Yuan Z, Dietrich M, Shulman GI, Horvath TL, et al. STAT3
inhibition of gluconeogenesis is downregulated by SirT1. Nat Cell Biol. 2009;
11:492–500. Ethics approval and consent to participate
Not applicable. Ethics approval and consent to participate
Not applicable. Author details
1 1Division of Endocrinology and Metabolism, National Clinical Research Center
for Geriatrics, State Key Laboratory of Biotherapy and Cancer Center, West
China Hospital, Sichuan University and Collaborative Innovation Center of
Biotherapy, Chengdu 610041, Sichuan, China. 2College of Life Sciences,
Sichuan University, Chengdu 610041, Sichuan, China. 3Department of
Integrated Traditional Chinese and Western Medicine, Sichuan Provincial
Pancreatitis Centre and West China-Liverpool Biomedical Research Centre,
West China Hospital, Sichuan University, Chengdu 610041, China. 22. Li LX, Fan LX, Zhou JX, Grantham JJ, Calvet JP, Sage J, et al. Lysine
methyltransferase SMYD2 promotes cyst growth in autosomal dominant
polycystic kidney disease. J Clin Invest. 2017;127:2751–64. 23. Zhou Z, Wang M, Li J, Xiao M, Chin YE, Cheng J, et al. SUMOylation and
SENP3 regulate STAT3 activation in head and neck cancer. Oncogene. 2016;
35:5826–38. Pancreatitis Centre and West China-Liverpool Biomedical Research Centre,
West China Hospital, Sichuan University, Chengdu 610041, China. Pancreatitis Centre and West China-Liverpool Biomedical Research Centre,
West China Hospital, Sichuan University, Chengdu 610041, China. 24. Lin W, Luo J, Sun Y, Lin C, Li G, Niu Y, et al. ASC-J9((R)) suppresses prostate
cancer cell invasion via altering the sumoylation-phosphorylation of STAT3. Cancer Lett. 2018;425:21–30. Received: 20 May 2020 Accepted: 4 September 2020 Funding
Th
k 14. Villarino AV, Kanno Y, O'Shea JJ. Mechanisms and consequences of Jak-STAT
signaling in the immune system. Nat Immunol. 2017;18:374–84. Funding
This work was supported by the Ministry of Science and Technology of
China (2018ZX09201018-005), the National Natural Science Foundation of
China (81970561, 81502631, 81570527, 91540113), the 1.3.5 Project for
Disciplines of Excellence, West China Hospital, Sichuan University
(ZYJC18049), and National Clinical Research Center for Geriatrics, West China
Hospital, Sichuan University (Z20191005, Z20201003). 15. Sgrignani J, Garofalo M, Matkovic M, Merulla J, Catapano CV, Cavalli A. Structural Biology of STAT3 and Its Implications for Anticancer Therapies
Development. Int J Mol Sci. 2018;19. 16. Nadiminty N, Lou W, Lee SO, Lin X, Trump DL, Gao AC. Stat3 activation of
NF-{kappa} B p100 processing involves CBP/p300-mediated acetylation. Proc
Natl Acad Sci U S A. 2006;103:7264–9. 16. Nadiminty N, Lou W, Lee SO, Lin X, Trump DL, Gao AC. Stat3 activation of
NF-{kappa} B p100 processing involves CBP/p300-mediated acetylation. Proc
Natl Acad Sci U S A. 2006;103:7264–9. 17. Gambi G, Di Simone E, Basso V, Ricci L, Wang R, Verma A, et al. The
Transcriptional Regulator Sin3A Contributes to the Oncogenic Potential of
STAT3. Cancer Res. 2019;79:3076–87. References 13. Kortylewski M, Kujawski M, Wang T, Wei S, Zhang S, Pilon-Thomas S, et al. Inhibiting Stat3 signaling in the hematopoietic system elicits
multicomponent antitumor immunity. Nat Med. 2005;11:1314–21. 38. Liu Y, Song J, Liu Y, Zhou Z, Wang X. Transcription activation of circ-STAT3
induced by Gli2 promotes the progression of hepatoblastoma via acting as Page 16 of 19 Page 16 of 19 Page 16 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 a sponge for miR-29a/b/c-3p to upregulate STAT3/Gli2. J Exp Clin Cancer
Res. 2020;39:101. 61. Shaim H, Estrov Z, Harris D, Hernandez Sanabria M, Liu Z, Ruvolo P, et al. The CXCR4-STAT3-IL-10 Pathway Controls the Immunoregulatory Function
of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide. Front
Immunol. 2017;8:1773. a sponge for miR-29a/b/c-3p to upregulate STAT3/Gli2. J Exp Clin Cancer
Res. 2020;39:101. 39. Iliopoulos D, Jaeger SA, Hirsch HA, Bulyk ML, Struhl K. STAT3 activation of
miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic
switch linking inflammation to cancer. Mol Cell. 2010;39:493–506. 62. Rebe C, Ghiringhelli F. STAT3, a Master Regulator of Anti-Tumor Immune
Response. Cancers (Basel). 2019;11. 40. Loffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermuller J,
Kretzschmar AK, et al. Interleukin-6 dependent survival of multiple myeloma
cells involves the Stat3-mediated induction of microRNA-21 through a
highly conserved enhancer. Blood. 2007;110:1330–3. 63. Huynh J, Chand A, Gough D, Ernst M. Therapeutically exploiting STAT3
activity in cancer - using tissue repair as a road map. Nat Rev Cancer. 2019;
19:82–96. 64. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells:
role of STAT3 in the tumour microenvironment. Nat Rev Immunol. 2007;7:
41–51. 41. Gao H, Liu R, Sun X. STAT3-induced upregulation of lncRNA SNHG17
predicts a poor prognosis of melanoma and promotes cell proliferation and
metastasis through regulating PI3K-AKT pathway. Eur Rev Med Pharmacol
Sci. 2019;23:8000–10. 65. Yoo SA, Kim M, Kang MC, Kong JS, Kim KM, Lee S, et al. Placental growth
factor regulates the generation of TH17 cells to link angiogenesis with
autoimmunity. Nat Immunol. 2019;20:1348–59. 42. Du C, Wang HX, Chen P, Chen CH. STAT3-induced upregulation of lncRNA
DUXAP8 functions as ceRNA for miR-577 to promote the migration and
invasion in colorectal cancer through the regulation of RAB14. Eur Rev Med
Pharmacol Sci. 2019;23:6105–18. 66. Lin F, Meng X, Guo Y, Cao W, Liu W, Xia Q, et al. References Epigenetic initiation of the
TH17 differentiation program is promoted by Cxxc finger protein 1. Sci Adv. 2019;5:eaax1608. 43. Chen JF, Wu P, Xia R, Yang J, Huo XY, Gu DY, et al. STAT3-induced lncRNA
HAGLROS overexpression contributes to the malignant progression of
gastric cancer cells via mTOR signal-mediated inhibition of autophagy. Mol
Cancer. 2018;17:6. 67. Mohammadpour H, MacDonald CR, Qiao G, Chen M, Dong B, Hylander BL,
et al. beta2 Adrenergic receptor-mediated signaling regulates the
immunosuppressive potential of myeloid-derived suppressor cells. J Clin
Invest. 2019. 44. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a
leading role for STAT3. Nat Rev Cancer. 2009;9:798–809. 68. Takenaka MC, Gabriely G, Rothhammer V, Mascanfroni ID, Wheeler MA,
Chao CC, et al. Control of tumor-associated macrophages and T cells in
glioblastoma via AHR and CD39. Nat Neurosci. 2019;22:729–40. 45. Pearce OMT, Delaine-Smith RM, Maniati E, Nichols S, Wang J, Bohm S, et al. Deconstruction of a Metastatic Tumor Microenvironment Reveals a
Common Matrix Response in Human Cancers. Cancer Discov. 2018;8:304–19. 69. Oweida AJ, Darragh L, Phan A, Binder D, Bhatia S, Mueller A, et al. STAT3
Modulation of Regulatory T Cells in Response to Radiation Therapy in Head
and Neck Cancer. J Natl Cancer Inst. 2019. 46. Azizi E, Carr AJ, Plitas G, Cornish AE, Konopacki C, Prabhakaran S, et al. Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor
Microenvironment. Cell. 2018;174:1293–308 e36. 70. Chen X, Song E. Turning foes to friends: targeting cancer-associated
fibroblasts. Nat Rev Drug Discov. 2019;18:99–115. 47. Martin JD, Cabral H, Stylianopoulos T, Jain RK. Improving cancer
immunotherapy using nanomedicines: progress, opportunities and
challenges. Nat Rev Clin Oncol. 2020. 71. Albrengues J, Bertero T, Grasset E, Bonan S, Maiel M, Bourget I, et al. Epigenetic switch drives the conversion of fibroblasts into proinvasive
cancer-associated fibroblasts. Nat Commun. 2015;6:10204. 48. Phuengkham H, Ren L, Shin IW, Lim YT. Nanoengineered Immune Niches
for Reprogramming the Immunosuppressive Tumor Microenvironment and
Enhancing Cancer Immunotherapy. Adv Mater. 2019;31:e1803322. 72. Yang X, Lin Y, Shi Y, Li B, Liu W, Yin W, et al. FAP Promotes
Immunosuppression by Cancer-Associated Fibroblasts in the Tumor
Microenvironment via STAT3-CCL2 Signaling. Cancer Res. 2016;76:4124–35 49. Herrmann A, Kortylewski M, Kujawski M, Zhang C, Reckamp K, Armstrong B,
et al. Targeting Stat3 in the myeloid compartment drastically improves the
in vivo antitumor functions of adoptively transferred T cells. Cancer Res. 2010;70:7455–64. 73. References Li X, Xu Q, Wu Y, Li J, Tang D, Han L, et al. A CCL2/ROS autoregulation loop
is critical for cancer-associated fibroblasts-enhanced tumor growth of oral
squamous cell carcinoma. Carcinogenesis. 2014;35:1362–70. 50. Iwata-Kajihara T, Sumimoto H, Kawamura N, Ueda R, Takahashi T, Mizuguchi
H, et al. Enhanced cancer immunotherapy using STAT3-depleted dendritic
cells with high Th1-inducing ability and resistance to cancer cell-derived
inhibitory factors. J Immunol. 2011;187:27–36. 74. Heichler C, Scheibe K, Schmied A, Geppert CI, Schmid B, Wirtz S, et al. STAT3
activation through IL-6/IL-11 in cancer-associated fibroblasts promotes
colorectal tumour development and correlates with poor prognosis. Gut. 2019. 51. Siegel AM, Heimall J, Freeman AF, Hsu AP, Brittain E, Brenchley JM, et al. A
critical role for STAT3 transcription factor signaling in the development and
maintenance of human T cell memory. Immunity. 2011;35:806–18. 75. Giovanelli P, Sandoval TA, Cubillos-Ruiz JR. Dendritic Cell Metabolism and
Function in Tumors. Trends Immunol. 2019;40:699–718. 76. Farren MR, Carlson LM, Netherby CS, Lindner I, Li PK, Gabrilovich DI, et al. Tumor-induced STAT3 signaling in myeloid cells impairs dendritic cell
generation by decreasing PKCbetaII abundance. Sci Signal. 2014;7:ra16. 52. Gotthardt D, Putz EM, Straka E, Kudweis P, Biaggio M, Poli V, et al. Loss of
STAT3 in murine NK cells enhances NK cell-dependent tumor surveillance. Blood. 2014;124:2370–9. 77. Calon A, Espinet E, Palomo-Ponce S, Tauriello DV, Iglesias M, Cespedes MV,
et al. Dependency of colorectal cancer on a TGF-beta-driven program in
stromal cells for metastasis initiation. Cancer Cell. 2012;22:571–84. 53. Wang T, Niu G, Kortylewski M, Burdelya L, Shain K, Zhang S, et al. Regulation
of the innate and adaptive immune responses by Stat-3 signaling in tumor
cells. Nat Med. 2004;10:48–54. 78. Ohlund D, Handly-Santana A, Biffi G, Elyada E, Almeida AS, Ponz-Sarvise M,
et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in
pancreatic cancer. J Exp Med. 2017;214:579–96. 54. Icardi L, Lievens S, Mori R, Piessevaux J, De Cauwer L, De Bosscher K, et al. Opposed regulation of type I IFN-induced STAT3 and ISGF3 transcriptional
activities by histone deacetylases (HDACS) 1 and 2. FASEB J. 2012;26:240–9. 79. Huang H, Zhang Y, Gallegos V, Sorrelle N, Zaid MM, Toombs J, et al. Targeting TGFbetaR2-mutant tumors exposes vulnerabilities to stromal
TGFbeta blockade in pancreatic cancer. EMBO Mol Med. 2019:e10515. y
y
55. Tsai MH, Lee CK. STAT3 Cooperates With Phospholipid Scramblase 2 to
Suppress Type I Interferon Response. Front Immunol. 2018;9:1886. 56. References Napabucasin
versus placebo in refractory advanced colorectal cancer: a randomised
phase 3 trial. Lancet Gastroenterol Hepatol. 2018;3:263–70. 97. Kim LH, Khadka S, Shin JA, Jung JY, Ryu MH, Yu HJ, et al. Nitidine
chloride acts as an apoptosis inducer in human oral cancer cells and a
nude mouse xenograft model via inhibition of STAT3. Oncotarget. 2017;
8:91306–15. 118. Reilley MJ, McCoon P, Cook C, Lyne P, Kurzrock R, Kim Y, et al. STAT3
antisense oligonucleotide AZD9150 in a subset of patients with heavily
pretreated lymphoma: results of a phase 1b trial. J Immunother Cancer. 2018;6:119. 98. Bai L, Zhou H, Xu R, Zhao Y, Chinnaswamy K, McEachern D, et al. A Potent
and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor
Regression In Vivo. Cancer Cell. 2019;36:498–511 e17. 119. Sen M, Thomas SM, Kim S, Yeh JI, Ferris RL, Johnson JT, et al. First-in-human
trial of a STAT3 decoy oligonucleotide in head and neck tumors:
implications for cancer therapy. Cancer Discov. 2012;2:694–705. 99. Schust J, Sperl B, Hollis A, Mayer TU, Berg T. Stattic: a small-molecule
inhibitor of STAT3 activation and dimerization. Chem Biol. 2006;13:1235–42. 120. Gupta V, Mesa RA, Deininger MW, Rivera CE, Sirhan S, Brachmann CB, et al. A phase 1/2, open-label study evaluating twice-daily administration of
momelotinib in myelofibrosis. Haematologica. 2017;102:94–102. 100. Akiyama Y, Nonomura C, Ashizawa T, Iizuka A, Kondou R, Miyata H, et al. The anti-tumor activity of the STAT3 inhibitor STX-0119 occurs via
promotion of tumor-infiltrating lymphocyte accumulation in temozolomide-
resistant glioblastoma cell line. Immunol Lett. 2017;190:20–5. 121. Berdeja J, Palandri F, Baer MR, Quick D, Kiladjian JJ, Martinelli G, et al. Phase
2 study of gandotinib (LY2784544) in patients with myeloproliferative
neoplasms. Leuk Res. 2018;71:82–8. 101. Soleimani AH, Garg SM, Paiva IM, Vakili MR, Alshareef A, Huang YH, et al. Micellar nano-carriers for the delivery of STAT3 dimerization inhibitors to
melanoma. Drug Deliv Transl Res. 2017;7:571–81. 122. Mascarenhas J, Hoffman R, Talpaz M, Gerds AT, Stein B, Gupta V, et al. Pacritinib vs Best Available Therapy, Including Ruxolitinib, in Patients
With Myelofibrosis: A Randomized Clinical Trial. JAMA Oncol. 2018;4:
652–9. 102. Siddiquee K, Zhang S, Guida WC, Blaskovich MA, Greedy B, Lawrence HR,
et al. Selective chemical probe inhibitor of Stat3, identified through
structure-based virtual screening, induces antitumor activity. Proc Natl Acad
Sci U S A. 2007;104:7391–6. 123. Kim DW, Saussele S, Williams LA, Mohamed H, Rong Y, Zyczynski T, et al. References Ji P, Xu X, Ma S, Fan J, Zhou Q, Mao X, et al. Novel 2-Carbonylbenzo [b]
thiophene 1,1-Dioxide Derivatives as Potent Inhibitors of STAT3 Signaling
Pathway. ACS Med Chem Lett. 2015;6:1010–4. 112. Holmstrom TH, Moilanen AM, Ikonen T, Bjorkman ML, Linnanen T, Wohlfahrt
G, et al. ODM-203, a Selective Inhibitor of FGFR and VEGFR, Shows Strong
Antitumor Activity, and Induces Antitumor Immunity. Mol Cancer Ther. 2019;18:28–38. 91. Lin L, Hutzen B, Zuo M, Ball S, Deangelis S, Foust E, et al. Novel STAT3
phosphorylation inhibitors exhibit potent growth-suppressive activity in
pancreatic and breast cancer cells. Cancer Res. 2010;70:2445–54. 113. Pei J, Zhang Y, Luo Q, Zheng W, Li W, Zeng X, et al. STAT3 inhibition
enhances CDN-induced STING signaling and antitumor immunity. Cancer
Lett. 2019;450:110–22. 92. Li Z, Zhu T, Xu Y, Wu C, Chen J, Ren Y, et al. A novel STAT3 inhibitor,
HJC0152, exerts potent antitumor activity in glioblastoma. Am J Cancer Res. 2019;9:699–713. 114. Lu C, Talukder A, Savage NM, Singh N, Liu K. JAK-STAT-mediated chronic
inflammation impairs cytotoxic T lymphocyte activation to decrease anti-
PD-1 immunotherapy efficacy in pancreatic cancer. Oncoimmunology. 2017;
6:e1291106. 93. Liu Z, Wang H, Guan L, Lai C, Yu W, Lai M. LL1, a novel and highly selective
STAT3 inhibitor, displays anti-colorectal cancer activities in vitro and in vivo. Br J Pharmacol. 2020;177:298–313. 94. Fuh B, Sobo M, Cen L, Josiah D, Hutzen B, Cisek K, et al. LLL-3 inhibits STAT3
activity, suppresses glioblastoma cell growth and prolongs survival in a
mouse glioblastoma model. Br J Cancer. 2009;100:106–12. 115. Yu GT, Mao L, Wu L, Deng WW, Bu LL, Liu JF, et al. Inhibition of SRC family
kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous
cell carcinoma. Cell Mol Life Sci. 2018;75:4223–34. 95. Zuo M, Li C, Lin J, Javle M. LLL12, a novel small inhibitor targeting STAT3 for
hepatocellular carcinoma therapy. Oncotarget. 2015;6:10940–9. 116. Yang H, Lee WS, Kong SJ, Kim CG, Kim JH, Chang SK, et al. STING activation
reprograms tumor vasculatures and synergizes with VEGFR2 blockade. J Clin
Invest. 2019;130:4350–64. 96. Wang H, Liu Z, Guan L, Li J, Chen S, Yu W, et al. LYW-6, a novel
cryptotanshinone derived STAT3 targeting inhibitor, suppresses colorectal
cancer growth and metastasis. Pharmacol Res. 2020;153:104661. 117. Jonker DJ, Nott L, Yoshino T, Gill S, Shapiro J, Ohtsu A, et al. References Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways
collaboratively link inflammation to cancer. Protein Cell. 2013;4:176–85. 57. Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer:
coming of age Nat Rev Immunol 2018;18:309–24 55. Tsai MH, Lee CK. STAT3 Cooperates With Phospholipid Scramblase 2 to
Suppress Type I Interferon Response. Front Immunol. 2018;9:1886. Suppress Type I Interferon Response. Front Immunol. 2018;9:1886. 56. Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways
collaboratively link inflammation to cancer. Protein Cell. 2013;4:176–85. 57. Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer: 80. Ham IH, Oh HJ, Jin H, Bae CA, Jeon SM, Choi KS, et al. Targeting interleukin-
6 as a strategy to overcome stroma-induced resistance to chemotherapy in
gastric cancer. Mol Cancer. 2019;18:68. 56. Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways
collaboratively link inflammation to cancer. Protein Cell. 2013;4:176–85. y
;
57. Taniguchi K, Karin M. NF-kappaB, inflammation, immunity and cancer:
coming of age. Nat Rev Immunol. 2018;18:309–24. 81. Lin Q, Ren L, Jian M, Xu P, Li J, Zheng P, et al. The mechanism of the
premetastatic niche facilitating colorectal cancer liver metastasis generated
from myeloid-derived suppressor cells induced by the S1PR1-STAT3
signaling pathway. Cell Death Dis. 2019;10:693. 58. Lee H, Herrmann A, Deng JH, Kujawski M, Niu G, Li Z, et al. Persistently
activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer
Cell. 2009;15:283–93. 82. Cheng JT, Deng YN, Yi HM, Wang GY, Fu BS, Chen WJ, et al. Hepatic
carcinoma-associated fibroblasts induce IDO-producing regulatory dendritic
cells through IL-6-mediated STAT3 activation. Oncogenesis. 2016;5:e198. 59. Garg B, Giri B, Modi S, Sethi V, Castro I, Umland O, et al. NFkappaB in
Pancreatic Stellate Cells Reduces Infiltration of Tumors by Cytotoxic T Cells
and Killing of Cancer Cells, via Up-regulation of CXCL12. Gastroenterology. 2018;155:880–91 e8. 83. Nagathihalli NS, Castellanos JA, Shi C, Beesetty Y, Reyzer ML, Caprioli R, et al. Signal Transducer and Activator of Transcription 3, Mediated Remodeling of
the Tumor Microenvironment Results in Enhanced Tumor Drug Delivery in a
Mouse Model of Pancreatic Cancer. Gastroenterology. 2015;149:1932–43 e9. 60. Saha A, Ahn S, Blando J, Su F, Kolonin MG, DiGiovanni J. Proinflammatory
CXCL12-CXCR4/CXCR7 Signaling Axis Drives Myc-Induced Prostate Cancer in
Obese Mice. Cancer Res. 2017;77:5158–68. Page 17 of 19 Page 17 of 19 Page 17 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. References Molecular Cancer (2020) 19:145 84. Sanz-Moreno V, Gaggioli C, Yeo M, Albrengues J, Wallberg F, Viros A, et al. ROCK and JAK1 signaling cooperate to control actomyosin contractility in
tumor cells and stroma. Cancer Cell. 2011;20:229–45. 106. Son DJ, Zheng J, Jung YY, Hwang CJ, Lee HP, Woo JR, et al. MMPP
Attenuates Non-Small Cell Lung Cancer Growth by Inhibiting the STAT3
DNA-Binding Activity via Direct Binding to the STAT3 DNA-Binding Domain. Theranostics. 2017;7:4632–42. 85. Gelain A, Mori M, Meneghetti F, Villa S. Signal Transducer and Activator of
Transcription Protein 3 (STAT3): An Update on its Direct Inhibitors as
Promising Anticancer Agents. Curr Med Chem. 2019;26:5165–206. 107. Huang W, Dong Z, Chen Y, Wang F, Wang CJ, Peng H, et al. Small-molecule
inhibitors targeting the DNA-binding domain of STAT3 suppress tumor
growth, metastasis and STAT3 target gene expression in vivo. Oncogene. 2016;35:783–92. 86. Beebe JD, Liu JY, Zhang JT. Two decades of research in discovery of
anticancer drugs targeting STAT3, how close are we? Pharmacol Ther. 2018;
191:74–91. 108. Souissi I, Ladam P, Cognet JA, Le Coquil S, Varin-Blank N, Baran-Marszak F,
et al. A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates
between STAT1 and STAT3 and induces death in a human colon carcinoma
cell line. Mol Cancer. 2012;11:12. 87. Nagel-Wolfrum K, Buerger C, Wittig I, Butz K, Hoppe-Seyler F, Groner B. The
interaction of specific peptide aptamers with the DNA binding domain and
the dimerization domain of the transcription factor Stat3 inhibits
transactivation and induces apoptosis in tumor cells. Mol Cancer Res. 2004;
2:170–82. 109. Koppikar P, Abdel-Wahab O, Hedvat C, Marubayashi S, Patel J, Goel A, et al. Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-
induced thrombocytosis and myelofibrosis. Blood. 2010;115:2919–27. 88. Miccoli A, Dhiani BA, Mehellou Y. Phosphotyrosine prodrugs: design,
synthesis and anti-STAT3 activity of ISS-610 aryloxy triester
phosphoramidate prodrugs. Medchemcomm. 2019;10:200–8. 110. Lo MC, Peterson LF, Yan M, Cong X, Hickman JH, Dekelver RC, et al. JAK
inhibitors suppress t(8;21) fusion protein-induced leukemia. Leukemia. 2013;
27:2272–9. 89. Turkson J, Ryan D, Kim JS, Zhang Y, Chen Z, Haura E, et al. Phosphotyrosyl
peptides block Stat3-mediated DNA binding activity, gene regulation, and
cell transformation. J Biol Chem. 2001;276:45443–55. 111. Lu X, Zhang T, Zhu SJ, Xun Q, Tong L, Hu X, et al. Discovery of JND3229 as
a New EGFR(C797S) Mutant Inhibitor with In Vivo Monodrug Efficacy. ACS
Med Chem Lett. 2018;9:1123–7. 90. References Xu H, Du X, Xu J, Zhang Y, Tian Y, Liu G, et al. Pancreatic beta cell
microRNA-26a alleviates type 2 diabetes by improving peripheral insulin
sensitivity and preserving beta cell function. PLoS Biol. 2020;18:e3000603. 129. Winter GE, Buckley DL, Paulk J, Roberts JM, Souza A, Dhe-Paganon S, et al. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo
target protein degradation. Science. 2015;348:1376–81. 152. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell
immunotherapy for human cancer. Science. 2018;359:1361–5. 130. Lai AC, Toure M, Hellerschmied D, Salami J, Jaime-Figueroa S, Ko E, et al. Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. Angew Chem Int Ed Eng. 2016;55:807–10. 153. Kennedy LB, Salama AKS. A review of cancer immunotherapy toxicity. CA
Cancer J Clin. 2020. 154. O'Reilly EM, Oh DY, Dhani N, Renouf DJ, Lee MA, Sun W, et al. Durvalumab
With or Without Tremelimumab for Patients With Metastatic Pancreatic
Ductal Adenocarcinoma: A Phase 2 Randomized Clinical Trial. JAMA Oncol. 2019. 131. Burslem GM, Smith BE, Lai AC, Jaime-Figueroa S, McQuaid DC, Bondeson
DP, et al. The Advantages of Targeted Protein Degradation Over Inhibition:
An RTK Case Study. Cell Chem Biol. 2018;25:67–77 e3. 132. Khan S, Zhang X, Lv D, Zhang Q, He Y, Zhang P, et al. A selective BCL-XL
PROTAC degrader achieves safe and potent antitumor activity. Nat Med. 2019;25:1938–47. 155. Zhou Y, Fei M, Zhang G, Liang WC, Lin W, Wu Y, et al. Blockade of the
Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances
P2X7R-Dependent STING Activation by Tumor-Derived cGAMP. Immunity. 2020;52:357–73 e9. 133. Njatcha C, Farooqui M, Kornberg A, Johnson DE, Grandis JR, Siegfried JM. STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small
Cell Lung Cancer. Mol Cancer Ther. 2018;17:1917–26. 156. Hellmann MD, Paz-Ares L, Bernabe Caro R, Zurawski B, Kim SW, Carcereny
Costa E, et al. Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung
Cancer. N Engl J Med. 2019;381:2020–31. 134. Hong D, Kurzrock R, Kim Y, Woessner R, Younes A, Nemunaitis J, et al. AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3
with early evidence of clinical activity in lymphoma and lung cancer. Sci
Transl Med. 2015;7:314ra185. 157. Atsaves V, Tsesmetzis N, Chioureas D, Kis L, Leventaki V, Drakos E, et al. PD-
L1 is commonly expressed and transcriptionally regulated by STAT3 and
MYC in ALK-negative anaplastic large-cell lymphoma. Leukemia. 2017;31:
1633–7. 135. References PD-
1 up-regulation on CD4(+) T cells promotes pulmonary fibrosis through
STAT3-mediated IL-17A and TGF-beta1 production. Sci Transl Med. 2018;10. 142. Li H, Liu A, Zhao Z, Xu Y, Lin J, Jou D, et al. Fragment-based drug design
and drug repositioning using multiple ligand simultaneous docking (MLSD):
identifying celecoxib and template compounds as novel inhibitors of signal
transducer and activator of transcription 3 (STAT3). J Med Chem. 2011;54:
5592–6. 165. Celada LJ, Rotsinger JE, Young A, Shaginurova G, Shelton D, Hawkins C,
et al. Programmed Death-1 Inhibition of Phosphatidylinositol 3-Kinase/AKT/
Mechanistic Target of Rapamycin Signaling Impairs Sarcoidosis CD4(+) T
Cell Proliferation. Am J Respir Cell Mol Biol. 2017;56:74–82. 166. Krasilnikov M, Ivanov VN, Dong J, Ronai Z. ERK and PI3K negatively regulate
STAT-transcriptional activities in human melanoma cells: implications
towards sensitization to apoptosis. Oncogene. 2003;22:4092–101. 143. Huang R, Jing X, Huang X, Pan Y, Fang Y, Liang G, et al. Bifunctional
Naphthoquinone Aromatic Amide-Oxime Derivatives Exert Combined
Immunotherapeutic and Antitumor Effects through Simultaneous Targeting
of Indoleamine-2,3-dioxygenase and Signal Transducer and Activator of
Transcription 3. J Med Chem. 2020. 167. Mace TA, Shakya R, Pitarresi JR, Swanson B, McQuinn CW, Loftus S, et al. IL-6
and PD-L1 antibody blockade combination therapy reduces tumour
progression in murine models of pancreatic cancer. Gut. 2018;67:320–32. 144. Buchert M, Burns CJ, Ernst M. Targeting JAK kinase in solid tumors:
emerging opportunities and challenges. Oncogene. 2016;35:939–51. 168. Liu H, Shen J, Lu K. IL-6 and PD-L1 blockade combination inhibits
hepatocellular carcinoma cancer development in mouse model. Biochem
Biophys Res Commun. 2017;486:239–44. 145. Kang S, Tanaka T, Narazaki M, Kishimoto T. Targeting Interleukin-6 Signaling
in Clinic. Immunity. 2019;50:1007–23. 146. Liu L, Nam S, Tian Y, Yang F, Wu J, Wang Y, et al. 6-Bromoindirubin-3'-oxime
inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma
cells. Cancer Res. 2011;71:3972–9. 169. Luo F, Luo M, Rong QX, Zhang H, Chen Z, Wang F, et al. Niclosamide, an
antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint
blockade in non-small cell lung cancer. J Immunother Cancer. 2019;7:245. 170. Bagley SJ, O'Rourke DM. Clinical investigation of CAR T cells for solid
tumors: Lessons learned and future directions. Pharmacol Ther. 2019;107419. 147. Tian Y, Nam S, Liu L, Yakushijin F, Yakushijin K, Buettner R, et al. Spirooxindole
derivative SOID-8 induces apoptosis associated with inhibition of JAK2/STAT3
signaling in melanoma cells. PLoS One. 2012;7:e49306. 171. References Odate S, Veschi V, Yan S, Lam N, Woessner R, Thiele CJ. Inhibition of STAT3
with the Generation 2.5 Antisense Oligonucleotide, AZD9150, Decreases
Neuroblastoma Tumorigenicity and Increases Chemosensitivity. Clin Cancer
Res. 2017;23:1771–84. 158. Song TL, Nairismagi ML, Laurensia Y, Lim JQ, Tan J, Li ZM, et al. Oncogenic
activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-
cell lymphoma. Blood. 2018;132:1146–58. 136. Shastri A, Choudhary G, Teixeira M, Gordon-Mitchell S, Ramachandra N,
Bernard L, et al. Antisense STAT3 inhibitor decreases viability of
myelodysplastic and leukemic stem cells. J Clin Invest. 2018;128:5479–88. 159. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, Rodriguez GA,
et al. Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2
Expression. Cell Rep. 2017;19:1189–201. 137. Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and
challenges. Nat Rev Drug Discov. 2017;16:181–202. 160. Yoyen-Ermis D, Tunali G, Tavukcuoglu E, Horzum U, Ozkazanc D, Sutlu T,
et al. Myeloid maturation potentiates STAT3-mediated atypical IFN-gamma
signaling and upregulation of PD-1 ligands in AML and MDS. Sci Rep. 2019;
9:11697. 138. Esposito CL, Nuzzo S, Catuogno S, Romano S, de Nigris F, de Franciscis V. STAT3 Gene Silencing by Aptamer-siRNA Chimera as Selective Therapeutic
for Glioblastoma. Mol Ther Nucleic Acids. 2018;10:398–411. 161. Herrmann A, Lahtz C, Nagao T, Song JY, Chan WC, Lee H, et al. CTLA4
Promotes Tyk2-STAT3-Dependent B-cell Oncogenicity. Cancer Res. 2017;77:
5118–28. 139. Yoon S, Wu X, Armstrong B, Habib N, Rossi JJ. An RNA Aptamer Targeting the
Receptor Tyrosine Kinase PDGFRalpha Induces Anti-tumor Effects through
STAT3 and p53 in Glioblastoma. Mol Ther Nucleic Acids. 2019;14:131–41. 162. Hsu P, Santner-Nanan B, Hu M, Skarratt K, Lee CH, Stormon M, et al. IL-10
Potentiates Differentiation of Human Induced Regulatory T Cells via STAT3
and Foxo1. J Immunol. 2015;195:3665–74. 140. Heppler LN, Frank DA. Targeting Oncogenic Transcription Factors:
Therapeutic Implications of Endogenous STAT Inhibitors. Trends Cancer. 2017;3:816–27. 163. Keremu A, Aimaiti A, Liang Z, Zou X. Role of the HDAC6/STAT3 pathway in
regulating PD-L1 expression in osteosarcoma cell lines. Cancer Chemother
Pharmacol. 2019;83:255–64. 141. Khan MW, Saadalla A, Ewida AH, Al-Katranji K, Al-Saoudi G, Giaccone ZT,
et al. The STAT3 inhibitor pyrimethamine displays anti-cancer and immune
stimulatory effects in murine models of breast cancer. Cancer Immunol
Immunother. 2018;67:13–23. 164. Celada LJ, Kropski JA, Herazo-Maya JD, Luo W, Creecy A, Abad AT, et al. References Outcomes of switching to dasatinib after imatinib-related low-grade
adverse events in patients with chronic myeloid leukemia in chronic phase:
the DASPERSE study. Ann Hematol. 2018;97:1357–67. 103. Turkson J, Zhang S, Palmer J, Kay H, Stanko J, Mora LB, et al. Inhibition of
constitutive signal transducer and activator of transcription 3 activation by
novel platinum complexes with potent antitumor activity. Mol Cancer Ther. 2004;3:1533–42. 124. Antonarakis ES, Heath EI, Posadas EM, Yu EY, Harrison MR, Bruce JY, et al. A
phase 2 study of KX2-391, an oral inhibitor of Src kinase and tubulin
polymerization, in men with bone-metastatic castration-resistant prostate
cancer. Cancer Chemother Pharmacol. 2013;71:883–92. 104. Buettner R, Corzano R, Rashid R, Lin J, Senthil M, Hedvat M, et al. Alkylation
of cysteine 468 in Stat3 defines a novel site for therapeutic development. ACS Chem Biol. 2011;6:432–43. 125. Finn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, et al. Atezolizumab
plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med. 2020;382:1894–905. 105. Escobar Z, Bjartell A, Canesin G, Evans-Axelsson S, Sterner O, Hellsten R, et al. Preclinical Characterization of 3beta-(N-Acetyl l-cysteine methyl ester)-2abeta,3-
dihydrogaliellalactone (GPA512), a Prodrug of a Direct STAT3 Inhibitor for the
Treatment of Prostate Cancer. J Med Chem. 2016;59:4551–62. 126. D'Angelo SP, Shoushtari AN, Keohan ML, Dickson MA, Gounder MM, Chi P,
et al. Combined KIT and CTLA-4 Blockade in Patients with Refractory GIST Page 18 of 19 Page 18 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 149. Zhang Q, Liu RX, Chan KW, Hu J, Zhang J, Wei L, et al. Exosomal transfer of
p-STAT3 promotes acquired 5-FU resistance in colorectal cancer cells. J Exp
Clin Cancer Res. 2019;38:320. and Other Advanced Sarcomas: A Phase Ib Study of Dasatinib plus
Ipilimumab. Clin Cancer Res. 2017;23:2972–80. and Other Advanced Sarcomas: A Phase Ib Study of Dasatinib plus
Ipilimumab. Clin Cancer Res. 2017;23:2972–80. 127. Turkson J, Kim JS, Zhang S, Yuan J, Huang M, Glenn M, et al. Novel
peptidomimetic inhibitors of signal transducer and activator of transcription
3 dimerization and biological activity. Mol Cancer Ther. 2004;3:261–9. 150. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of
exosomes. Science. 2020;367. 128. Lai AC, Crews CM. Induced protein degradation: an emerging drug
discovery paradigm. Nat Rev Drug Discov. 2017;16:101–14. 151. References Du H, Hirabayashi K, Ahn S, Kren NP, Montgomery SA, Wang X, et al. Antitumor Responses in the Absence of Toxicity in Solid Tumors by
Targeting B7-H3 via Chimeric Antigen Receptor T Cells. Cancer Cell. 2019;35:
221–37 e8. 148. Wen W, Wu J, Liu L, Tian Y, Buettner R, Hsieh MY, et al. Synergistic anti-
tumor effect of combined inhibition of EGFR and JAK/STAT3 pathways in
human ovarian cancer. Mol Cancer. 2015;14:100. Page 19 of 19 Page 19 of 19 Zou et al. Molecular Cancer (2020) 19:145 Zou et al. Molecular Cancer (2020) 19:145 172. Majzner RG, Theruvath JL, Nellan A, Heitzeneder S, Cui Y, Mount CW, et al. CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent
Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clin
Cancer Res. 2019;25:2560–74. 173. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD19 chimeric antigen receptor
(CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24:
563–71. 174. Kagoya Y, Tanaka S, Guo T, Anczurowski M, Wang CH, Saso K, et al. A novel
chimeric antigen receptor containing a JAK-STAT signaling domain
mediates superior antitumor effects. Nat Med. 2018;24:352–9. 175. Wang Y, Jiang H, Luo H, Sun Y, Shi B, Sun R, et al. An IL-4/21 Inverted
Cytokine Receptor Improving CAR-T Cell Potency in Immunosuppressive
Solid-Tumor Microenvironment. Front Immunol. 2019;10:1691. 176. Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev
Clin Oncol. 2019;16:372–85. 177. Guha P, Gardell J, Darpolor J, Cunetta M, Lima M, Miller G, et al. STAT3
inhibition induces Bax-dependent apoptosis in liver tumor myeloid-derived
suppressor cells. Oncogene. 2019;38:533–48. 178. Corrales L, McWhirter SM, Dubensky TW Jr, Gajewski TF. The host STING
pathway at the interface of cancer and immunity. J Clin Invest. 2016;126:
2404–11. 179. Ramanjulu JM, Pesiridis GS, Yang J, Concha N, Singhaus R, Zhang SY, et al. Design of amidobenzimidazole STING receptor agonists with systemic
activity. Nature. 2018;564:439–43. 180. Shae D, Becker KW, Christov P, Yun DS, Lytton-Jean AKR, Sevimli S, et al. Endosomolytic polymersomes increase the activity of cyclic dinucleotide
STING agonists to enhance cancer immunotherapy. Nat Nanotechnol. 2019;
14:269–78. 181. Zhu Q, Man SM, Gurung P, Liu Z, Vogel P, Lamkanfi M, et al. Cutting edge:
STING mediates protection against colorectal tumorigenesis by governing
the magnitude of intestinal inflammation. J Immunol. 2014;193:4779–82. 182. References Zhang CX, Ye SB, Ni JJ, Cai TT, Liu YN, Huang DJ, et al. STING signaling
remodels the tumor microenvironment by antagonizing myeloid-derived
suppressor cell expansion. Cell Death Differ. 2019;26:2314–28. 183. Hsia HC, Hutti JE, Baldwin AS. Cytosolic DNA Promotes Signal Transducer
and Activator of Transcription 3 (STAT3) Phosphorylation by TANK-binding
Kinase 1 (TBK1) to Restrain STAT3 Activity. J Biol Chem. 2017;292:5405–17. 184. Moreira D, Adamus T, Zhao X, Su YL, Zhang Z, White SV, et al. STAT3
Inhibition Combined with CpG Immunostimulation Activates Antitumor
Immunity to Eradicate Genetically Distinct Castration-Resistant Prostate
Cancers. Clin Cancer Res. 2018;24:5948–62. 185. Nefedova Y, Nagaraj S, Rosenbauer A, Muro-Cacho C, Sebti SM, Gabrilovich
DI. Regulation of dendritic cell differentiation and antitumor immune
response in cancer by pharmacologic-selective inhibition of the janus-
activated kinase 2/signal transducers and activators of transcription 3
pathway. Cancer Res. 2005;65:9525–35. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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http://www.ajnr.org/content/ajnr/37/11/2123.full.pdf
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English
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Orbital Fat Volumetry and Water Fraction Measurements Using T2-Weighted FSE-IDEAL Imaging in Patients with Thyroid-Associated Orbitopathy
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American journal of neuroradiology
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cc-by
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Thyroid-Associated Orbitopathy
FSE-IDEAL Imaging in Patients with
Measurements Using T2-Weighted
Orbital Fat Volumetry and Water Fraction
Takahashi, Y. Akiyama and K. Awai
Y. Kaichi, K. Tanitame, H. Itakura, H. Ohno, M. Yoneda, Y.
http://www.ajnr.org/content/37/11/2123
https://doi.org/10.3174/ajnr.A4859
doi:
2016, 37 (11) 2123-2128
AJNR Am J Neuroradiol of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/37/11/2123
https://doi.org/10.3174/ajnr.A4859
doi:
2016, 37 (11) 2123-2128
AJNR Am J Neuroradiol ORIGINAL RESEARCH
HEAD & NECK ORIGINAL RESEARCH
HEAD & NECK Received January 28, 2016; accepted after revision May 8.
From the Department of Diagnostic Radiology (Y.K., K.A.), Graduate School and
Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Ja-
pan; Department of Radiology (K.T.), Chugoku Rosai Hospital, Kure, Japan; Depart-
ment of Ophthalmology and Visual Science (H.I.) and Department of Molecular
and Internal Medicine (H.O., M.Y.), Institute of Biomedical and Health Sciences,
Hiroshima University, Hiroshima, Japan; and Department of Clinical Radiology (Y.T.,
Y.A.), Hiroshima University Hospital, Hiroshima, Japan.
Please address correspondence to Yoko Kaichi, MD, Diagnostic Radiology, Gradu-
ate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3,
Minami-ku, Hiroshima 734-8551, Japan; e-mail: kaichi@hiroshima-u.ac.jp
http://dx.doi.org/10.3174/ajnr.A4859 Orbital Fat Volumetry and Water Fraction Measurements
Using T2-Weighted FSE-IDEAL Imaging in Patients with
Thyroid-Associated Orbitopathy X Y. Kaichi, X K. Tanitame, X H. Itakura, X H. Ohno, X M. Yoneda, X Y. Takahashi, X Y. Akiyama, and AJNR Am J Neuroradiol 37:2123–28
Nov 2016
www.ajnr.org http://dx.doi.org/10.3174/ajnr.A4859 Received January 28, 2016; accepted after revision May 8. ABSTRACT BACKGROUND AND PURPOSE: The quantitative evaluation of orbital fat proliferation and edema and the assessment of extraocular
muscles are useful for diagnosing and monitoring thyroid-associated orbitopathy. To evaluate therapy-induced quantitative changes in
the orbital fat of patients with thyroid-associated orbitopathy, we performed volumetric and water fraction measurements by using
T2-weighted FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation (FSE-IDEAL) imaging. MATERIALS AND METHODS: Orbital FSE-IDEAL images of 30 volunteers were acquired twice within 1 week. Nine patients with thyroid-
associated orbitopathy underwent FSE-IDEAL imaging before and after methylprednisolone pulse therapy, and the treatment results were
assessed by using their pre- and post-methylprednisolone pulse therapy clinical activity scores. We performed volumetric and water
fraction measurements of orbital fat by using FSE-IDEAL imaging and evaluated interscan differences in the volunteers. In patients with
thyroid-associated orbitopathy, we compared pre- and posttherapy orbital fat measurements and assessed the correlation between the
pretherapy values and clinical activity score improvement. RESULTS: The reproducibility of results obtained by the quantitative evaluation of orbital fat in volunteers was acceptable. After
methylprednisolone pulse therapy, the water fraction in the orbital fat of patients with thyroid-associated orbitopathy was significantly
decreased (P .001). There was a significant positive correlation between the pretherapy water fraction and clinical activity score
improvement (right, r 0.82; left, r 0.79) and a significant negative correlation between the pretherapy volume and clinical activity score
improvement (bilateral, r 0.84). CONCLUSIONS: Volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging are feasible and useful for monitoring
the effects of therapy and for predicting the response of patients with thyroid-associated orbitopathy to methylprednisolone pulse therapy. CONCLUSIONS: Volumetric and water fraction measurements of orbital fat by using FSE-IDEAL imaging are feasible and useful for monitoring
the effects of therapy and for predicting the response of patients with thyroid-associated orbitopathy to methylprednisolone pulse therapy. Please address correspondence to Yoko Kaichi, MD, Diagnostic Radiology, Gradu-
ate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3,
Minami-ku, Hiroshima 734-8551, Japan; e-mail: kaichi@hiroshima-u.ac.jp ABSTRACT Two of them underwent local injection
of triamcinolone acetonide into the or-
bit, and 5 of them followed a conserva-
tive
wait-and-see
approach
because
their TAO was slight and the risk of
MPPT would exceed the benefit. Conse-
quently, the 9 patients (2 men, 7 women;
median age, 57 12.5 years; 18 eyes)
who received MPPT (500 mg of methyl-
prednisolone
administered
intrave-
nously once a day for 3 consecutive
days) were included in this study and
underwent FSE-IDEAL imaging again
1.6 0.7 months thereafter. Re-evalua-
tion of the TAO activity was based on the
latest CAS obtained around the time of
g study (the interval, 1–15 days; median, FIG 1. Axial water (A) and fat (B) images of a healthy volunteer. The ROIs are encircled in yellow. We also recruited 16 patients older
than 20 years of age who had a clinical
diagnosis of TAO between December
2013 and November 2015. The recorded
activity of their TAO was based on their
clinical activity score (CAS), including
pain, eyelid erythema or edema, con-
junctival hyperemia and chemosis, and a
swollen caruncle.20 All 16 patients un-
derwent orbital FSE-IDEAL imaging. Two of them underwent local injection
of triamcinolone acetonide into the or-
bit, and 5 of them followed a conserva-
tive
wait-and-see
approach
because
their TAO was slight and the risk of
MPPT would exceed the benefit. Conse-
quently, the 9 patients (2 men, 7 women;
median age, 57 12.5 years; 18 eyes)
who received MPPT (500 mg of methyl-
prednisolone
administered
intrave-
nously once a day for 3 consecutive
days) were included in this study and
underwent FSE-IDEAL imaging again
1.6 0.7 months thereafter. Re-evalua-
tion of the TAO activity was based on the
latest CAS obtained around the time of
he second MR imaging study (the interval, 1–15 days; median,
5 days). e encircled in yellow. at is light green. We
manually removed ex-
ge SI of 207 and an SD
of 88. Therefore the
EAL images, we then
asured the orbital fat FIG 1. Axial water (A) and fat (B) images of a healthy volunteer. The ROIs are encircled in yellow. FIG 2. Axial fat image (A) of a healthy volunteer. The selected orbital fat is light green. We
separated fat tissue by using an adequate signal-intensity threshold and manually removed ex-
traorbital fat. ABSTRACT ABBREVIATIONS: CAS clinical activity score; FSE-IDEAL FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation; ABBREVIATIONS: CAS clinical activity score; FSE-IDEAL FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation;
MPPT methylprednisolone pulse therapy; SI signal intensity; TAO thyroid-associated orbitopathy ABBREVIATIONS: CAS clinical activity score; FSE-IDEAL FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation;
MPPT methylprednisolone pulse therapy; SI signal intensity; TAO thyroid-associated orbitopathy ABBREVIATIONS: CAS clinical activity score; FSE-IDEAL FSE iterative decomposition of water and fat with echo asymmetry and least-squares estimation;
MPPT methylprednisolone pulse therapy; SI signal intensity; TAO thyroid-associated orbitopathy T
hyroid-associated orbitopathy (TAO) is the most common
extrathyroid manifestation of Graves disease. The enlarge-
ment of orbital fat and extraocular muscles in the relatively fixed
volume space imposed by the bony orbit may produce proptosis,
ocular motility loss, and decreased visual acuity.1 Histologic stud-
ies showed lymphocytic infiltration and edema due to the accu- mulation of hydrophilic, interstitial glycosaminoglycans in the
orbital fat and extraocular muscles,2,3 attributable to antigenic
cross-reactivity between thyroid proteins and orbital fibroblasts.4 T The course of TAO can be divided into the active, regressing,
and a burnt-out phase.5 Early active disease is treated with com-
bined immunosuppression.6,7 The identification of patients who
stand to benefit from this therapy is important but clinically dif-
ficult, despite the availability of activity scoring systems. Conse-
quently, objective methods are needed. CT has been used to quantify the volume of extraocular mus-
cles and orbital fat in TAO.8-11 However, ocular radiation expo-
sure limits its clinical use. Because MR imaging without ionizing
radiation yields orbital images with excellent soft-tissue contrast
in any plane, it has been used for evaluating TAO.12-15 The quantitative evaluation of the orbital fat volume on T1-
weighted images was reported useful for assessing the severity of
TAO and for monitoring the treatment response.12,16 Recently, 2123 We also recruited 16 patients older
than 20 years of age who had a clinical
diagnosis of TAO between December
2013 and November 2015. The recorded
activity of their TAO was based on their
clinical activity score (CAS), including
pain, eyelid erythema or edema, con-
junctival hyperemia and chemosis, and a
swollen caruncle.20 All 16 patients un-
derwent orbital FSE-IDEAL imaging. ABSTRACT In this volunteer, the internal rectus muscle showed an average SI of 207 and an SD
of 112, whereas the orbital fat showed an average SI of 1636 and an SD of 88. Therefore the
threshold value was [(207 112) (1636 88) / 2 933]. Using FSE-IDEAL images, we then
produced 3D reconstruction images of the bilateral orbital fat (B) and measured the orbital fat
volume on a workstation. FIG 2. Axial fat image (A) of a healthy volunteer. The selected orbital fat is light green. We
separated fat tissue by using an adequate signal-intensity threshold and manually removed ex-
traorbital fat. In this volunteer, the internal rectus muscle showed an average SI of 207 and an SD
of 112, whereas the orbital fat showed an average SI of 1636 and an SD of 88. Therefore the
threshold value was [(207 112) (1636 88) / 2 933]. Using FSE-IDEAL images, we then
produced 3D reconstruction images of the bilateral orbital fat (B) and measured the orbital fat
volume on a workstation. Higashiyama et al17 demonstrated that after methylprednisolone
pulse therapy (MPPT), the orbital fat volume was unchanged,
while the total volume of extraocular muscles was decreased on
T2-weighted images. However, in our search of the literature, we
found no reports on quantitative changes in orbital fat edema
after MPPT or orbital irradiation. Consequently, the correlation
between the volume and edema of orbital fat and the treatment
response of patients with TAO remains to be elucidated. Orbital Fat Water Fraction and Volume Measurements Orbital Fat Water Fraction and Volume Measurements
We placed an ROI in the orbital fat on FSE-IDEAL images of water
and fat and measured the average signal intensity (SI) in the ROIs. Next, we defined and calculated the water fraction of the orbital
fat as [SI Water / (SI Water SI Fat)] to assess the orbital fat
edema on the basis of the fat fraction (SI Fat / [SI Water SI
Fat]),18 defined to quantify fatty infiltration (Fig 1). The orbital
fat volume of both eyes was measured on a workstation (Vir-
tual Place Raijin; AZE Ltd, Tokyo, Japan). We first separated
fat tissue from other structures by using the threshold value,
recorded as the mean value between the average SI plus the SD
of the ROI in the internal rectus muscle and the average SI
minus the SD of the ROI in the orbital fat, considering some
dispersion of signal intensities in the ROIs. Then we manually
removed the fatty marrow of the orbital bone and outer fat; the
orbital fat volume was automatically measured on the work-
station (Fig 2). MR Imaging
All i All images were acquired on a 3T scanner (Signa Excite HD 3.0;
GE Healthcare, Milwaukee, Wisconsin; gradient strength, 40
mT/m; slew rate, 150 T/m/s) by using an 8-channel phased array
brain coil. We optimized the T2-weighted FSE-IDEAL sequence
(TR/TE, 6000/100 ms; flip angle, 90°; image matrix, 288 160;
FOV, 160 160 mm; section thickness/gap, 2/0 mm; asymmetric
echo shifts, /6, /2, 7/6; number of acquisitions, 3; number
of sections, 32; scan time, 2 minutes 42 seconds) and obtained
orbital water and fat images for all subjects. FSE iterative decomposition of water and fat with echo asym-
metry and least-squares estimation (FSE-IDEAL), a novel 3-point
Dixon method, is useful for separating the fat signal from the
water signal18,19 but has not been used to quantify the orbital
structures in TAO. To evaluate the feasibility of FSE-IDEAL im-
aging for the precise quantitative evaluation of orbital fat, we sub-
jected healthy volunteers to 2 FSE-IDEAL imaging studies per-
formed during 1 week. We measured the volume and the water
fraction of their orbital fat and ascertained the reproducibility of
the measurement results. We also acquired FSE-IDEAL images
in patients with TAO to assess MPPT-induced changes in the
volume and water fraction of their orbital fat and evaluated the
correlation between the quantitative values of orbital fat and
the improvement in their symptoms. 2124
Kaichi
Nov 2016
www.ajnr.org MATERIALS AND METHODS
Subjects
Thi
i
d This prospective study was approved by the Ethics Committee of
Hiroshima University. Informed consent was obtained from all
participants before entry into the study. We recruited 30 healthy volunteers (15 men, 15 women; me-
dian age, 29 7.6 years). They underwent orbital FSE-IDEAL
imaging twice within 1 week to evaluate the reproducibility of the
water fraction and the volume measurements of the orbital fat. 2124
Kaichi
Nov 2016
www.ajnr.org 2124 -0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.15
0.17
0.19
0.21
0.23
0.25
0.27
CI (95%)
1st scan – 2nd scan
Mean of 1st and 2nd scan
Bias
-4
-3
-2
-1
0
1
2
3
4
9
11
13
15
17
19
21
CI (95%)
1st scan – 2nd scan (ml)
Mean of 1st and 2nd scan (ml)
Bias
A
B
FIG 3. Bland-Altman analysis confirming the interscan reproducibility of the water fraction (A) and the volume of orbital fat (B) on FSE-IDEAL
images of the volunteers. -0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.15
0.17
0.19
0.21
0.23
0.25
0.27
CI (95%)
1st scan – 2nd scan
Mean of 1st and 2nd scan
Bias
A -4
-3
-2
-1
0
1
2
3
4
9
11
13
15
17
19
21
CI (95%)
1st scan – 2nd scan (ml)
Mean of 1st and 2nd scan (ml)
Bias
B 1st scan – 2nd scan (ml) B A FIG 3. Bland-Altman analysis confirming the interscan reproducibility of the water fraction (A) and the volume of orbital fat (B) on FSE-IDEAL
images of the volunteers FIG 3. Bland-Altman analysis confirming the interscan reproducibility of the water fraction (A) and the volume of orbital fat (B) on FSE-IDEAL
images of the volunteers. Patient profiles
Characteristics
Median age (yr) (range)
57 (43–82)
Sex: male/female
2/7
Median duration of the treatment for GD (mo) (range)
11 (1–168)
Median duration from the onset of TAO (mo) (range)
4 (1–8)
Smoking: yes/no
3/6
Median pretherapy CAS (range)
2 (1–4)
Involvement of extraocular muscle: yes/no
7/2
Note:—GD indicates Graves disease. Patient profiles
Characteristics
Median age (yr) (range)
57 (43–82)
Sex: male/female
2/7
Median duration of the treatment for GD (mo) (range)
11 (1–168)
Median duration from the onset of TAO (mo) (range)
4 (1–8)
Smoking: yes/no
3/6
Median pretherapy CAS (range)
2 (1–4)
Involvement of extraocular muscle: yes/no
7/2
Note:—GD indicates Graves disease. Volunteers The difference in the water fraction and the volume of orbital fat
on the first and second scans was 1.1% 11.9% and 0.5%
3.6%, respectively. Bland-Altman analysis of the measurement
results showed that their reproducibility was adequate (water
fraction: r 0.71, bias 0.002; 95% CI bias, 0.012–0.007;
95% CI, 0.052–0.047; volume: r 0.99, bias 0.066; 95% CI
bias, 0.235–0.102; 95% CI, 0.951–0.819) (Fig 3). DISCUSSION To the best of our knowledge, this is the first quantitative eval-
uation of orbital fat by using FSE-IDEAL imaging. We docu-
ment an MPPT-induced reduction in the water fraction of or-
bital fat in patients with TAO and report the positive
correlation between the pretreatment water fraction and the Statistical Analyses All statistical analyses were performed with commercially avail-
able software (XLSTAT, version 2015.6.01.244, Addinsoft;
https://www.xlstat.com/en/company/about-us) and Excel 2010
(Microsoft, Redmond, Washington). We used Bland-Altman analysis and the Pearson correlation
coefficient to evaluate interscan differences in the volunteers and
used the mean value of the water fraction and the volume of the
bilateral orbit in our analyses. In patients with TAO, we performed paired-sample t tests to
compare the water fraction and the volume of orbital fat before
and after MPPT. The right and left orbits were examined separately
because patients with unilateral TAO manifested unilateral excess fat
exophthalmos.10 To ascertain that the values obtained before and
aftertreatmentweresignificantlydifferent,weappliedthe2-samplet
test to compare the therapy-induced changes with the values ob-
tained in the volunteers. We defined the therapeutic effect of MPPT
as the following formula: [CAS Improvement Ratio (Pretherapy
CAS Posttherapy CAS) / Pretherapy CAS)] and determined the
correlation between the measured values before MPPT and the CAS
improvement ratio with the Pearson correlation coefficient test. Dif-
ferences of P .05 were considered statistically significant. 12.4% 3.8%, P .001; left, 13.6% 6.5%, P .001; Fig 4A). The treatment-induced reduction in the water fraction was sig-
nificantly larger than the interscan difference observed in
the volunteers (right, P .002; left, P .001). There was no
significant difference in the orbital fat volume pre- and
posttreatment (right, 2.0% 7.8%, P .37; left, 1.9% 3.8%,
P .17, Fig 4B). The pre- and posttreatment CAS fell by 3 points in 2 patients,
by 2 points in 1 patient, and by 1 point in 3 patients. In the other
3, there was no change. The positive correlation between the pre-
treatment water fraction and the CAS improvement rate (right,
r 0.82, P .007; left, r 0.79, P .012) showed that the higher
the pretreatment water fraction, the greater was the posttreatment
CAS improvement. On the other hand, there was a negative cor-
relation between the fat volume and CAS improvement (right, r
0.84, P .005; left, r 0.84, P .005), indicating that the
larger the pretreatment orbital fat volume, the lower the post-
treatment CAS improvement rate. Patients The reproducibility of water fraction and fat volume
measurements on all iteratively acquired FSE-IDEAL images
was sufficient. In Japan, MPPT is generally applied in patients with TAO with
low CAS scores because the Japanese patients with TAO often
have orbital inflammation despite low CAS scores.30 We found
that the higher the pretreatment water fraction, the greater the
posttreatment decrease in the CAS, despite the low pretreatment
CAS scores of our study patients. An increase in the water fraction
is indicative of an increase in the tissue water content and may
reflect acute inflammatory changes. Earlier studies on extraocular
muscles found a correlation between the SI increase on pretreat-
ment STIR images and a good therapeutic response31-33 and be-
tween prolonged pretreatment T2 values and a good response to
systemic corticosteroids or orbital radiation therapy.33 These
findings indicate that treatment at an early and immunologi-
cally active stage of orbitopathy is important.11,34,35 Our quan-
titative MR imaging study by using the FSE-IDEAL sequence
showed that MPPT was useful in patients with TAO whose
orbital fat contained an elevated water fraction. On the other
hand, the larger the pretreatment orbital fat volume, the lower
the posttreatment CAS improvement rate. Because a longer
duration of TAO is associated with a larger orbital fat mass and
fibrosis,36 we think that a response to steroids is less likely
when the disease is in its late, inactive stage with more
fibrosis.33,34 Others12,21 used a 1.5T MR imaging scanner and a receiver
surface coil to measure orbital fat volumes. Although surface coils
can yield orbital images with high spatial resolution, the signal
strength decreases as the distance from the coil increases. Our
use of a 3T MR imaging scanner and an 8-channel brain coil
resulted in orbital images with a high enough signal-to-noise
ratio for the estimation of the orbital fat volume and the water
fraction. Orbital images with high spatial and temporal resolution and
fewer motion artifacts due to eye movement can be acquired on
multidetector row CT scanners.11 However, the low soft-tissue
contrast on multidetector row CT images makes it difficult to
separate out orbital fat. In addition, ocular radiation exposure is a
serious concern. Therefore, MR imaging with high soft-tissue
contrast is safer for orbital imaging. We found that MPPT reduced the water fraction in the orbital
fat of patients with TAO, possibly because it decreased the severity
of edema. Patients The clinical characteristics of the 9 patients with TAO are sum-
marized in the Table. After MPPT, the water fraction in the
orbital fat on both sides was significantly decreased (right, AJNR Am J Neuroradiol 37:2123–28
Nov 2016
www.ajnr.org
2125 0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
right, p < 0.001
le, p < 0.001
pre
post
pre
post
right, p = 0.37
10
20
30
40
50
60
10
20
30
40
50
60
Le, p = 0.17
ml
ml
pre
post
pre
post
A
B
FIG 4. Comparison of the pre- and posttreatment water fraction (A) and the volume of orbital fat (B) in patients with TAO. Methylprednisolone
pulse therapy significantly decreased the water fraction. The volume was unchanged. right, p = 0.37
10
20
30
40
50
60
ml
pre
post
B le, p < 0.001
pre
post 0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
right, p < 0.001
pre
post
A 10
20
30
40
50
60
Le, p = 0.17
ml
pre
post B A FIG 4. Comparison of the pre- and posttreatment water fraction (A) and the volume of orbital fat (B) in patients with TAO. Methylprednisolone
pulse therapy significantly decreased the water fraction. The volume was unchanged. CAS improvement rate and the negative correlation between
the pretreatment orbital fat volume and the CAS improvement
rate. found no difference in the degree of exophthalmos, which is
related to an increase in orbital fat, in patients with TAO
who had undergone MPPT. Expansion of the adipose tissue
volume is elicited by glycosaminoglycan-related edema and the
emergence of a population of newly differentiated fat cells
in these tissues.29 Although MPPT can reduce orbital fat
edema, the increase in the number of fat cells may persist after
therapy. We used FSE-IDEAL imaging to acquire axial images of the
bilateral orbits and measured the water fraction and the volume of
orbital fat in healthy volunteers and patients with TAO. Although
many images contained motion artifacts due to voluntary and
involuntary eye movements, it was possible to differentiate the SI
of orbital fat and other intraorbital structures by applying an
adequate threshold in the orbital fat volumetry. To measure
the water fraction in orbital fat, we placed ROIs on the intraor-
bital fat; measurement of the SI was not hampered by motion
artifacts. REFERENCES 1. KendlerDL,LippaJ,RootmanJ.Theinitialclinicalcharacteristicsof
Graves’ orbitopathy vary with age and sex. Arch Ophthalmol 1993;
111:197–201 CrossRef Medline 1. KendlerDL,LippaJ,RootmanJ.Theinitialclinicalcharacteristicsof
Graves’ orbitopathy vary with age and sex. Arch Ophthalmol 1993;
111:197–201 CrossRef Medline 21. Tian S, Nishida Y, Isberg B, et al. MRI measurements of normal
extraocular muscles and other orbital structures. Graefes Arch Clin
Exp Ophthalmol 2000;238:393–404 CrossRef Medline 2. Winand RJ, Cornet G, Etienne-Decerf J, et al. Original acquisition in
the pathogenesis and the treatment of endocrine ophthalmopathy. Metab Pediatr Syst Ophthalmol 1988;11:126–32 Medline 2. Winand RJ, Cornet G, Etienne-Decerf J, et al. Original acquisition in
the pathogenesis and the treatment of endocrine ophthalmopathy. Metab Pediatr Syst Ophthalmol 1988;11:126–32 Medline 22. Hoh HB, Laitt RD, Wakely C, et al. The STIR sequence MRI in the
assessment of extraocular muscles in thyroid eye disease. Eye 1994;
8:506–10 CrossRef Medline 3. Kahaly G, Hansen C, Beyer J, et al. Plasma glycosaminoglycans
in endocrine ophthalmopathy. J Endocrinol Invest 1994;17:45–50
CrossRef Medline 23. Laitt RD, Hoh B, Wakeley C, et al. The value of the short tau inver-
sion recovery sequence in magnetic resonance imaging of thyroid
eye disease. Br J Radiol 1994;67:244–47 CrossRef Medline 4. Ludgate M, Baker G. Unlocking the immunological mechanisms of
orbitalinflammationinthyroideyedisease.ClinExpImmunol2002;
127:193–98 CrossRef Medline 24. Bailey CC, Kabala J, Laitt R, et al. Magnetic resonance imaging in
thyroid eye disease. Eye 1996;10:617–19 CrossRef Medline 5. Rundle FF, Wilson CW. Development and course of exophthal-
mos and ophthalmoplegia in Graves’ disease with special refer-
ence to the effect of thyroidectomy. Clin Sci 1945;5:177–94
Medline 25. Higashiyama T, Nishida Y, Morino K, et al. Use of MRI signal inten-
sity of extraocular muscles to evaluate methylprednisolone pulse
therapy in thyroid-associated ophthalmopathy. Jpn J Ophthalmol
2015;59:124–30 CrossRef Medline 6. Claridge KG, Ghabrial R, Davis G, et al. Combined radiotherapy and
medical immunosuppression in the management of thyroid eye
disease. Eye 1997;11:717–22 CrossRef Medline 6. Claridge KG, Ghabrial R, Davis G, et al. Combined radiotherapy and
medical immunosuppression in the management of thyroid eye
disease. Eye 1997;11:717–22 CrossRef Medline 26. Bartalena L, Baldeschi L, Dickinson AJ, et al. Consensus statement of
the European Group on Graves’ Orbitopathy (EUGOGO) on man-
agement of Graves’ orbitopathy. Thyroid 2008;18:333–46 CrossRef
Medline y
7. Mayer EJ, Herdman G, Burnett C, et al. Serial STIR magnetic reso-
nance imaging correlates with clinical score of activity in thyroid
disease. Eye 2001;15:313–18 CrossRef Medline 27. Patients Earlier studies7,22-25 reported that the SI of extraocular
muscles on short inversion recovery sequences correlated well
with the disease activity assessed with clinical methods. The cor-
relation was maintained as disease activity changed. Although the
reversibility of the relaxation time in muscles with prolonged T2
values was explained as a therapy-induced decrease in the water
content,8 treatment-induced changes in orbital fat have not been
documented. Our study has several limitations. The number of patients with
TAO who underwent MPPT was small, and the lack of age-
matched controls limits the comparison between patients with
TAO and healthy individuals. In addition, we used the T2-
weighted FSE-IDEAL sequence provided by GE Healthcare. It is a
novel, 3-point Dixon method that applies iterative algorithms and
the region-growing technique to estimate local field inhomoge-
neities.37-39 Application of the 3-point Dixon method developed
by different vendors may result in significantly different measure-
ment results with respect to the water fraction of orbital fat. In
addition, the demarcation between orbital and upper or lower Like Higashiyama et al,17 we detected no significant difference
in the pre- and posttreatment orbital fat volume. Others26-28
2126
Kaichi
Nov 2016
www.ajnr.org Like Higashiyama et al,17 we detected no significant difference
in the pre- and posttreatment orbital fat volume. Others26-28 eyelid fat is ambiguous, and this feature may introduce some in-
terobserver variability in the orbital fat volume. Last, the correct
cutoff value for the water fraction and the volume of the orbital fat
between patients with TAO and their controls remains to be
determined. ophthalmopathy. Clin Endocrinol (Oxf) 2001;54:205–09 CrossRef
Medline 15. Mayer EJ, Fox DL, Herdman G, et al. Signal intensity, clinical activ-
ity and cross-sectional areas on MRI scans in thyroid eye disease. Eur J Radiol 2005;56:20–24 CrossRef Medline 16. Comerci M, Elefante A, Strianese D, et al. Semiautomatic regional
segmentation to measure orbital fat volumes in thyroid-associated
ophthalmopathy: a validation study. Neuroradiol J 2013;26:373–79
CrossRef Medline CONCLUSIONS The estimation of the water fraction and the volume of orbital
fat on FSE-IDEAL images is feasible, and the reproducibility of
these measurements is adequate. FSE-IDEAL imaging is useful
for monitoring the effects of therapy and for predicting the
response of patients with TAO to methylprednisolone pulse
therapy. 17. Higashiyama T, Nishida Y, Ohji M. Changes of orbital tissue vol-
umes and proptosis in patients with thyroid extraocular muscle
swelling after methylprednisolone pulse therapy. Jpn J Ophthalmol
2015;59:430–35 CrossRef Medline 18. Hu HH, Kim HW, Nayak KS, et al. Comparison of fat-water MRI
and single-voxel MRS in the assessment of hepatic and pancreatic
fat fractions in humans. Obesity (Silver Spring) 2010;18:841–47
CrossRef Medline Disclosures: Kazuo Awai—UNRELATED: Consultancy: GE Healthcare; Grants/
Grants Pending: Toshiba Medical Systems,* Hitachi Medical Corporation,* Bayer
Yakuhin,* Eizai,* Daiichi Sankyo,* Comments: research grants. *Money paid to the
institution. 19. Takasu M, Tani C, Sakoda Y, et al. Iterative decomposition of water
and fat with echo asymmetry and least-squares estimation (IDEAL)
imaging of multiple myeloma: initial clinical efficiency results. Eur
Radiol 2012;22:1114–21 CrossRef Medline 20. Bartalena L, Baldeschi L, Dickinson A, et al; European Group on
Graves’ Orbitopathy (EUGOGO). Consensus statement of the
European Group on Graves’ Orbitopathy (EUGOGO) on man-
agement of GO. Eur J Endocrinol 2008;158:273–85 CrossRef
Medline 2128
Kaichi
Nov 2016
www.ajnr.org REFERENCES Kauppinen-Ma¨kelin R, Karma A, Leinonen E, et al. High dose intra-
venous methylprednisolone pulse therapy versus oral prednisone
for thyroid-associated ophthalmopathy. Acta Ophthalmol Scand
2002;80:316–21 CrossRef Medline 8. Kahaly GJ. Imaging in thyroid-associated orbitopathy. Eur J Endo-
crinol 2001;145:107–18 CrossRef Medline 9. Feldon SE, Lee CP, Muramatsu SK, et al. Quantitative computed
tomography of Graves’ ophthalmopathy: extraocular muscle and
orbital fat in development of optic neuropathy. Arch Ophthalmol
1985;103:213–15 CrossRef Medline 28. van Geest RJ, Sasim IV, Koppeschaar HP, et al. Methylprednisolone
pulse therapy for patients with moderately severe Graves’
orbitopathy: a prospective, randomized, placebo-controlled study. Eur J Endocrinol 2008;158:229–37 CrossRef Medline 10. Peyster RG, Ginsberg F, Silber JH, et al. Exophthalmos caused by
excessivefat:CTvolumetricanalysisanddifferentialdiagnosis.AJR
Am J Roentgenol 1986;146:459–64 CrossRef Medline 29. Bahn RS, Heufelder AE. Pathogenesis of Graves’ ophthalmopathy. N Engl J Med 1993;329:1468–75 CrossRef Medline 11. FangZJ,ZhangJY,HeWM.CTfeaturesofexophthalmosinChinese
subjects with thyroid-associated ophthalmopathy. Int J Ophthalmol
2013;6:146–49 CrossRef Medline 30. Watanabe N, Noh JY, Kozaki A, et al. Radioiodine-associated exac-
erbation of Graves’ orbitopathy in the Japanese population: ran-
domized prospective study. J Clin Endocrinol Metab 2015;100:
2700–08 CrossRef Medline 12. Nishida Y, Tian S, Isberg B, et al. Significance of orbital fatty
tissue for exophthalmos in thyroid-associated ophthalmopathy. Graefes Arch Clin Exp Ophthalmol 2002;240:515–20 CrossRef
Medline 31. Hiromatsu Y, Kojima K, Ishisaka N, et al. Role of magnetic resonance
imaging in thyroid-associated ophthalmopathy: its predictive
value for therapeutic outcome of immunosuppressive therapy. Thyroid 1992;2:299–305 CrossRef Medline 13. Kvetny J, Puhakka KB, Rohl L. Magnetic resonance imaging deter-
mination of extraocular eye muscle volume in patients with thy-
roid-associated ophthalmopathy and proptosis. Acta Ophthalmol
Scand 2006;84:419–23 CrossRef Medline 32. Just M, Kahaly GJ, Higer HP, et al. Graves ophthalmopathy: role of
MR imaging in radiation therapy. Radiology 1991;179:187–90
CrossRef Medline 32. Just M, Kahaly GJ, Higer HP, et al. Graves ophthalmopathy: role of
MR imaging in radiation therapy. Radiology 1991;179:187–90
CrossRef Medline 14. Prummel MF, Gerding MN, Zonneveld FW, et al. The usefulness
of quantitative orbital magnetic resonance imaging in Graves’ 33. Ohnishi T, Noguchi S, Murakami N, et al. Extraocular muscles in 33. Ohnishi T, Noguchi S, Murakami N, et al. Extraocular muscles in AJNR Am J Neuroradiol 37:2123–28
Nov 2016
www.ajnr.org
2127 AJNR Am J Neuroradiol 37:2123–28
Nov 2016
www.ajnr.org
2127 is a rather late phenomenon. Eur J Endocrinol 2015;173:149–53
CrossRef Medline Gravesophthalmopathy:usefulnessofT2relaxationtimemeasure-
ments. Radiology 1994;190:857–62 CrossRef Medline is a rather late phenomenon. REFERENCES Eur J Endocrinol 2015;173:149–53
CrossRef Medline ments. Radiology 1994;190:857–62 CrossRef Medline 37. Costa DN, Pedrosa I, McKenzie C, et al. Body MRI using IDEAL. AJR
Am J Roentgenol 2008;190:1076–84 CrossRef Medline 34. Kendall-Taylor P, Crombie AL, Stephenson AM, et al. Intravenous
methylprednisolone in the treatment of Graves’ ophthalmopathy. BMJ 1988;297:1574–78 CrossRef Medline g
38. Gerdes CM, Kijowski R, Reeder SB. IDEAL imaging of the musculo-
skeletal system: robust water fat separation for uniform fat sup-
pression, marrow evaluation, and cartilage imaging. AJR Am J
Roentgenol 2007;189:284–91 Medline 35. Bartalena L, Marcocci C, Bogazzi F, et al. Use of corticosteroids to
prevent progression of Graves’ ophthalmopathy after radioiodine
therapy for hyperthyroidism. N Engl J Med 1989;321:1349–52
CrossRef Medline g
39. Humbert IA, Reeder SB, Porcaro EJ, et al. Simultaneous estimation
of tongue volume and fat fraction using IDEAL-FSE. J Magn Reson
Imaging 2008;28:504–08 CrossRef Medline 36. Potgieser PW, Wiersinga WM, Regensburg NI, et al. Some studies on
the natural history of Graves’ orbitopathy: increase in orbital fat 128
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Nov 2016
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https://openalex.org/W2088735728
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https://europepmc.org/articles/pmc2409882?pdf=render
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The utility of a multimedia education program for prostate cancer patients: a formative evaluation
|
British journal of cancer
| 2,004
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cc-by
| 6,612
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Received 12 February 2003; revised 17 February 2004; accepted 14 June
2004; published online 3 August 2004
*Correspondence: Dr P van Schaik; E-mail: P.Van-Schaik@tees.ac.uk The utility of a multimedia education program for prostate cancer
patients: a formative evaluation D Flynn1, P van Schaik*,1, A van Wersch1, T Ahmed2 and D Chadwick2
1School of Social Sciences and Law, University of Teesside, Middlesbrough TS1 3BA, UK; 2Department of Urology, James Cook University Hospital,
Middlesbrough TS4 3BW, UK D Flynn , P van Schaik
, A van Wersch , T Ahmed and D Chadwick
1School of Social Sciences and Law, University of Teesside, Middlesbrough TS1 3BA, UK; 2Department of Urology, James Cook University Hospital,
Middlesbrough TS4 3BW, UK A multimedia program (MMP) was developed to educate patients with prostate cancer about their disease. A within-subjects design
was used to investigate the changes in levels of cancer-related knowledge, psychosocial functioning, treatment decision-making role
and information needs immediately after browsing the MMP. The participants were 67 men recently diagnosed with prostate cancer. Psychosocial functioning was assessed with 20 items describing common emotional states and coping strategies employed by cancer
patients. Treatment decision-making role was assessed with the Control Preference Scale. A principle component analysis of the 20
psychosocial items yielded three components: distress, positive approach and nonacceptance. After browsing the MMP significant
increases in knowledge and reductions in distress were reported. Marital status was significantly associated with knowledge gain. Married men and those attending the study session with their spouse displayed a significant shift towards a more active role in
treatment decisions. The majority of information needs were fulfilled by the MMP; however, information related to the likelihood of a
cure, treatment side effects, coping strategies and aetiology were not completely satisfied by the MMP. Implications of the findings and
suggestions for future work on the design and evaluation of the MMP are discussed. gg
g
British Journal of Cancer (2004) 91, 855–860. doi:10.1038/sj.bjc.6602071
www.bjcancer.com
P bli h d
li
3 A
t 2004 gg
g
British Journal of Cancer (2004) 91, 855–860. doi:10.1038/sj.bjc.6602071
www.bjcancer.com
Published online 3 August 2004
& 2004 C
R
h UK eywords: prostate cancer; knowledge; psychosocial functioning; decision-making; multimedia; information needs In the UK, one in 14 men are at a lifetime risk of developing
prostate cancer, which is the second leading cause of death in men
after lung cancer, accounting for 9280 deaths in 2000 (Cancer
Research UK, 2002a, b). Treatments for prostate cancer include
surgery, hormone therapy, radiotherapy, chemotherapy and active
monitoring (or watchful waiting). British Journal of Cancer (2004) 91, 855 – 860
& 2004 Cancer Research UK
All rights reserved 0007 – 0920/04
$30.00
www.bjcancer.com British Journal of Cancer (2004) 91, 855 – 860
& 2004 Cancer Research UK
All rights reserved 0007 – 0920/04
$30.00
www.bjcancer.com British Journal of Cancer (2004) 91, 855 – 860
& 2004 Cancer Research UK
All rights reserved 0007 – 0920/04
$30.00 www.bjcancer.com Patient education Patient education (information provision) has been proven to be
an effective strategy for alleviating psychosocial problems in both
men and women with cancer (Fallowfield et al, 1997; Davison et al,
2003). Information provides a sense of control, reduces distress,
facilitates adaptive coping, and increases participation in shared
decision-making (SDM) with physicians (Gregoire et al, 1997; van
Wersch et al, 1997a, b; Davison et al, 2003). Effective information
provision should enrich doctor–patient interactions by transform-
ing consultations into negotiations between expert patients and
expert physicians; however, in reality it is clinicians, not patients,
who are in possession of the knowledge required to make an
informed decision (Crawford et al, 1997). Despite the general The utility of a multimedia education program for prostate cancer
patients: a formative evaluation Treatment side effects are
numerous and can occur for short periods, whereas others such as
incontinence and impotence have long-term effects that impact
negatively upon quality of life. However, the probability of
experiencing side effects associated with particular treatments is
unclear from the literature, with large differences in frequency,
duration and severity between studies. Furthermore, the relative
survival benefit of different treatments has yet to be elucidated;
consequently, there is still no unequivocal evidence to support one
treatment over another (Holmberg et al, 2002). problems such as social role changes, financial worries, anger,
depression and anxiety regarding treatment and potential death
(Gregoire et al, 1997; Gray et al, 1999). Research has also reported
that men with cancer rarely seek help for psychosocial problems
(Jorm, 1994); have little awareness of coping strategies (Whitrod,
1996); are often denied information on positive coping by
clinicians (Fitch et al, 1999); and are prone to relying upon
avoidance-coping strategies associated with poor psychological
outcomes and decreased survival rates (Shrock et al, 1999). Furthermore, men appear to receive little emotional support other
than from their spouse (Helgason et al, 2001) who also experience
psychosocial problems in response to their partner’s diagnosis
(Gray et al, 1999). Participants The participants were 67 men recently diagnosed (1 week or less)
with prostate cancer. The men were selected based on consultant
urologists’ assessment of their suitability for inclusion in the study. The age range was 48–89 with a mean age of 65.7 years
(SD ¼ 7.95). The
percentage
of
participants
with
secondary
(school, aged p16), further (college, aged X16) and higher
education (university, ages X18) was 50, 36 and 14% respectively. The majority were married (90%), retired (76%), resided in their
own homes with at least one other person (84%) and attended the
study session with their spouse (70%). Multimedia patient education A multimedia program (MMP) is a computer-based application
that combines text, sound, graphics, video and interactivity, which
serve to reinforce and complement one another to facilitate
learning. Multimedia programs presently offer the most compre-
hensive method of information provision that address several of
the shortcomings associated with other media such as printed
information. Multimedia programs can be easily and quickly
updated to incorporate new treatment approaches and evidence
from clinical trails that may refute previous information. Inter-
activity can provide autonomy as it enables patients to dictate the
pace, type and the order information is viewed in the MMP, which
enables more knowledgeable patients to access salient information
more quickly without attending to previously accessed informa-
tion. Multimedia programs provide all the benefits of patient
education without increasing staff costs or time, and are capable of
being accessed at home via the Internet or CD-ROM. Disadvan-
tages of MMPs are initial development and start-up costs and
technology acceptance by clinicians and patients. Design A within-subjects design was used to evaluate the utility of the
MMP. The independent variables were study condition (pretrial –
immediately before using the MMP and post-trial – immediately
after using the MMP), patient age, education, living circumstances
and employment status. The outcome measures were the level of
cancer-related knowledge, psychosocial functioning, treatment
decision-making role and information needs. The main disadvantages of printed information (and other
media such as audio and videotapes) are that reading level is often
inappropriate;
these
media
possess
limited
information
for
patients who wish to pursue a deeper understanding; they are
unable to adapt quickly to new information; salient topics are often
missing; uncertainties are ignored; and they fail to provide a
balanced account of the effectiveness of available treatments
(Smith and Timoney, 1997; Coulter et al, 1999). Psychosocial functioning Verbal consultations may fulfil these information needs
but this information is subject to poor recall and understanding by
patients (Michie et al, 1997). Consequently, patients are increas-
ingly given printed information to reinforce, or in many cases to
replace, verbal information provided by clinicians (Frank-Strom-
borg and Cohen, 1991). Currently, a combination of information
provision (verbal and printed) with support from healthcare
professionals is considered ‘good clinical practice’. This enables
the healthcare professional to respond according to an individual
patient’s information needs. Multimedia program An MMP was developed using previous research on the informa-
tion needs of prostate cancer patients (e.g. Davison et al, 1995) and
a working committee consisting of two consultant urologists, a
health
psychologist,
a
psychologist
specialising
in human–
computer interaction and a multimedia developer. The MMP
combined text with sound, narration, images, animation and
streaming video. The MMP was comprised of six cancer-related
modules: (a) prostate anatomy, (b) disease stages, aetiology and
symptoms, (c) diagnostic techniques, (d) treatment options
(surgery, hormone therapy and radiotherapy) and side effects,
which included a research update, (e) coping strategies and (f)
further information (self-help groups, prostate cancer organisa-
tions, further reading and a cancer glossary). The MMP was
operated on a stand-alone PC and participants navigated through
the MMP using a mouse. The interface used a selection of on-
screen buttons (forward, back, exit) that controlled interaction and
navigation through the MMP. Participants were instructed how to
use the MMP by a research assistant who was present throughout
the study session. gy
p
y
p
Clinical trials of MMPs as health education tools have reported
positive results for increasing patients’ knowledge, information-
seeking and participation in SDM (Krishna et al, 1997). Multi-
media programs for patients with benign prostatic hyperplasia
have reported positive results for reducing self-assessed prostate
symptoms (van Schaik et al, 1999) and facilitating SDM (Barry
et al, 1995; Shepperd et al, 1995; Wagner et al, 1995; Murray et al,
2001). Pilot studies of MMPs for prostate cancer have reported
patient satisfaction with navigability, layout and content (Jenkin-
son et al, 1998; Brink et al, 2000). Patient outcomes such as
participation in SDM were neglected, although Brink et al (2000)
reported increased knowledge of cancer staging and brachytherapy
(the only treatment that was included in the MMP) including
increased patient self-efficacy for discussing brachytherapy with
physicians. These studies demonstrated that MMPs can be effective
media for increasing knowledge of the entire spectrum of
treatment options for prostate cancer. Psychosocial functioning Psychosocial problems experienced by men with cancer have
received sparse attention compared to women in the research
literature. This difference in attention is hard to justify as research
has shown that men with prostate cancer experience psychosocial Multimedia education program for prostate cancer
D Flynn et al Multimedia education program for prostate cancer D Flynn et al 856 agreement that men should be involved in treatment decisions, the
type and amount of information needed for SDM has failed to
reach a consensus (Feldman-Stewart et al, 1998). Davison et al
(1995) reported the following hierarchical structure of information
needs of men recently diagnosed with prostate cancer: likelihood
of cure, stage of disease, available treatments, side effects on usual
social activity, self-care, treatment side effects, hereditary risks,
effects upon family and friends, and treatment effects upon sexual
activity. Verbal consultations may fulfil these information needs
but this information is subject to poor recall and understanding by
patients (Michie et al, 1997). Consequently, patients are increas-
ingly given printed information to reinforce, or in many cases to
replace, verbal information provided by clinicians (Frank-Strom-
borg and Cohen, 1991). Currently, a combination of information
provision (verbal and printed) with support from healthcare
professionals is considered ‘good clinical practice’. This enables
the healthcare professional to respond according to an individual
patient’s information needs. patients recently diagnosed with prostate cancer. A formative
evaluation
is an
evaluation
that
takes place
before
actual
implementation of a final product, and which influences the
development of the product (Preece et al, 1994). The results of the
formative evaluation will be used to conduct a future ‘summative
evaluation’ (undertaken after implementation of the final product,
with the aim of testing the functioning of a product) of the final
version of the MMP. Involving patients in the formative evaluation
is in line with guidelines for conducting research affecting patients
in the UK (DoH, 2001). agreement that men should be involved in treatment decisions, the
type and amount of information needed for SDM has failed to
reach a consensus (Feldman-Stewart et al, 1998). Davison et al
(1995) reported the following hierarchical structure of information
needs of men recently diagnosed with prostate cancer: likelihood
of cure, stage of disease, available treatments, side effects on usual
social activity, self-care, treatment side effects, hereditary risks,
effects upon family and friends, and treatment effects upon sexual
activity. MATERIALS AND METHODS Clinical British Journal of Cancer (2004) 91(5), 855 – 860 & 2004 Cancer Research UK Study questionnaire Rotation method: direct Oblimin
with Kaiser normalisation. The stem question preceding the 20 psychosocial
functioning items was ‘please tick one box for each of the following statements to
indicate how you feel now about your prostate cancer’ (very much, a little or not at
all). 857 Table 2
Percentage and cumulative percentage of variance explained
per component, and component loading matrix from the principle
components factor analysis of the psychosocial functioning items Clinical which has been used previously to assess treatment decision-
making preferences of prostate cancer patients (e.g. Davison et al,
1995). Participants indicated their preferred role in treatment
decision-making (active, passive or collaborative) by selecting the
category indicative of their status. Overall
12.62 (3.90)
14.38
(3.00)
aMean. bs.d. Overall Information needs were assessed with free-response items
that asked participants to state their most important information
need at pre- and post-trial. Participants were also asked to
state the most important knowledge they had acquired at
post-trial. aMean. bs.d. Study questionnaire A
questionnaire
in
hard-copy
format
was
used
to
assess
psychosocial functioning, cancer-related knowledge, treatment
decision-making roles and information needs. The first part of
the questionnaire described the aims and objectives of the research
and requested demographical information from the participants. A
questionnaire
in
hard-copy
format
was
used
to
assess
psychosocial functioning, cancer-related knowledge, treatment
decision-making roles and information needs. The first part of
the questionnaire described the aims and objectives of the research
and requested demographical information from the participants. Study questionnaire Cancer-related knowledge was assessed using 20 statements that
were representative of the information presented in the six cancer-
related modules of the MMP: cancer in general and prostate Therefore, the objective of the current study was to conduct a
‘formative evaluation’ by investigating the effect of the MMP on
knowledge acquisition, psychosocial functioning, preference for
participating in treatment decisions and information needs of Cancer-related knowledge was assessed using 20 statements that
were representative of the information presented in the six cancer-
related modules of the MMP: cancer in general and prostate British Journal of Cancer (2004) 91(5), 855 – 860 & 2004 Cancer Research UK Multimedia education program for prostate cancer
D Flynn et al Table 1
The 20 statements used to assess cancer-related knowledge
Knowledge
Pretrial
Post-trial
Cancer in general and prostate anatomy
2.49a (1.01)b
2.82 (0.75)
Male hormones are produced by the brain
Cancer is a type of infection of tissue
The prostate is part of the penis
The prostate surrounds the first part of the tube
which carries urine from the bladder to the penis
Disease advancement
2.75 (1.29)
3.13 (1.08)
Cancer is a lump of cells that may invade and
destroy surrounding tissues
Prostate cancer can spread to other parts of the
body
Prostate cancer never spreads outside the prostate
Growth of prostate cancer is driven by the male
hormones
Aims and side effects of surgery
3.07 (0.98)
3.22 (0.94)
The aim of prostate surgery is to remove part or all
of the tumour in the prostate
The aim of prostate surgery is to remove the
testicles
Possible side effects of prostate surgery include
problems in control of the bladder
After prostate surgery most prostate patients are
incontinent
Aims and side effects of radiotherapy
2.39 (1.05)
2.67 (0.68)
A possible side effect of radiotherapy is breast
enlargement
Possible side ffects of radiotherapy include tiredness
and nausea
The aim of radiotherapy is to destroy cancer cells
while doing as little harm as possible to normal cells
The aim of radiotherapy is to remove the prostate
Aims and side effects of hormone therapy
1.92 (1.27)
2.55 (1.00)
The aim of hormone therapy is to increase the
amount of male hormones
P
ibl
id
ff
t
f h
th
i
l d
Table 2
Percentage and cumulative percentage of variance explained
per component, and component loading matrix from the principle
components factor analysis of the psychosocial functioning items
Component
Item
Distress
Positive
approach
Nonacceptance
Shocked
0.77
I cannot believe this has
happened to me
0.53
I expected it
Angry
Anxious
0.92
Frightened
0.79
Uncertain
Blame myself
0.84
Miserable
Having a good cry
Running away
0.94
Pray to God
Talk to other patient
Talking to someone I trust
0.52
I feel like going to another
doctor to make sure it is true
0.74
Finding out more on prostate
cancer
Do not think about it
Carry on with your life
0.75
Fight this disease
0.74
Enjoy myself as much as I can
0.86
% age of variance explained
24.7
12.6
9.7
Cumulative %
24.7
37.3
47.0
Extraction method: principle components analysis. & 2004 Cancer Research UK British Journal of Cancer (2004) 91(5), 855 – 860 Procedure anatomy; disease advancement; and aims and side effects of
surgery, radiotherapy and hormone therapy (see Table 1). Participants responded to each statement as ‘true’, ‘false’ or ‘don’t
know’. Ethical approval for the study was granted by the Trust on the
basis that it was part of existing practice to provide practical
advice, guidance and support to men with newly diagnosed with
prostate cancer. After the ‘bad news’ consultation the urologist or
prostate cancer nurse informed the participant about the study and
provided them with a study information sheet (that detailed the
study aims and rationale) and a consent form. Participants were
given the choice of participating immediately, or within 1 week
after the initial bad news consultation. They were also given the
choice of attending the study session with a significant other or
alone. A private room situated within the urology department was
used to conduct the study. Prior to browsing the MMP, the
participants were asked to complete the study questionnaire, Previous research on emotional states and coping strategies
employed by cancer patients was used to design a 20-item checklist
to assess psychosocial functioning. Six items used adjectives as
descriptors for internal emotional states (e.g. angry); the remain-
ing 14 items used brief statements related to coping strategies for
dealing with cancer (see Table 2). The participants responded to
each item on a three-point Likert scale (not at all, a little and very
much). The Control Preference Scale developed by Degner and Sloan
(1992) was used to assess the treatment decision-making role, British Journal of Cancer (2004) 91(5), 855 – 860 Multimedia education program for prostate cancer
D Fl
l D Flynn et al D Flynn et al 858 Table 3
Information needs at pre- and post-trial and most important
knowledge acquired which was followed by instructions on how to use the MMP. No
time limit was imposed on patients for browsing the MMP. After
browsing
the
MMP,
participants
were
requested
to
complete the study questionnaire for a second time. They were
then fully debriefed and thanked for their time, cooperation and
patience. Knowledge acquisition Overall numbers of correct responses to the 20 knowledge items
increased between the pre- and post-trial conditions (see Table 1). A related t-test revealed that overall levels of correct responses
significantly increased between the pre- and post-trial conditions
(t [59] ¼ 4.49, Po0.001). A multiple regression analysis showed
that being married was a significant predictor of overall knowledge
gain
between
the
pre-
and
post-trial
conditions
(b ¼ 0.31,
R2 ¼ 0.10, Po0.05). Clinical Correct responses for each of the five knowledge domains
increased between the pre- and post-trial conditions (see Table 1). A two-factor repeated measures ANOVA revealed a significant
main effect of knowledge domain (F [3.5, 206.6] ¼ 15.67, Po0.001,
MSknowledge
domain ¼ 12.30, Greenhouse–Geisser correction ap-
plied) and a significant interaction effect between knowledge
domain and study condition (F [3.19, 187.89] ¼ 12.22, Po0.001,
MSinteraction ¼ 12.91). A series of simple effect tests revealed that
the following knowledge domains increased significantly between
the pre- and post-trial conditions: cancer in general and prostate
anatomy
(t
[59] ¼ 2.34,
Po0.05),
disease
advancement
(t
[59] ¼ 2.92, Po0.01), aims and side effects of radiotherapy (t
[59] ¼ 2.25, Po0.05) and hormone therapy (t [59] ¼ 4.51,
Po0.001). Knowledge gain for aims and side effects of surgery
failed to reach significance. Note: Percentages may not equal 100 due to rounding. exclusive
categories
(see
Table
3). A
frequency
analysis
revealed six categories of primary information needs at pretrial
with the following hierarchical structure: likelihood of a cure
(28%), treatment side effects (15%), coping strategies (13%),
diagnostic tests (12%), treatment duration (7%) and aetiology
(4%). In total, 19% stated that they had no information needs at
pretrial. exclusive
categories
(see
Table
3). A
frequency
analysis
revealed six categories of primary information needs at pretrial
with the following hierarchical structure: likelihood of a cure
(28%), treatment side effects (15%), coping strategies (13%),
diagnostic tests (12%), treatment duration (7%) and aetiology
(4%). In total, 19% stated that they had no information needs at
pretrial. At post-trial, five categories of information needs displayed
at least a 40% decrease, with only aetiology displaying a
negligible
increase. Approximately
66%
of
the
participants
indicated that they required no further information needs
at
post-trial. Knowledge acquisition Seven
categories
were
reported
as
the
most
important knowledge acquired with the following hierarchical
structure: hereditary risks (30%), aetiology (24%), likelihood of a
cure (4%), disease advancement (4%), coping strategies (2%),
diagnostic tests (2%) and treatment side effects (2%). Approxi-
mately 33% could not decide upon the most important knowledge
they had acquired. DISCUSSION Consistent with previous research evaluating patient education
tools, patients in the current study reported significantly less
distress (Gregoire et al, 1997; Davison et al, 2003), more cancer-
related knowledge (Glajchen and Moul, 1996), and a desire for a
more active role in treatment decisions (if they attended the
session with their spouse/partner or were married) immediately
after browsing the MMP. Procedure Most
important
information
need
Most
important
information
still required
Most
important
knowledge
acquired
Pretrial
Post-trial
Post-trial
F
% F
F
% F
F
% F
Likelihood of a cure
19
28
10
15
3
4
Treatment side effects
10
15
4
6
1
2
Coping strategies
9
13
4
6
1
2
Diagnostic tests
8
12
0
0
1
2
Treatment duration
5
7
1
2
0
0
Aetiology
3
4
4
6
16
24
Hereditary risks
0
0
0
0
20
30
Disease advancement
0
0
0
0
3
4
Cannot decide
13
19
44
66
22
33
Totals
67
100
67
100
67
100
Note: Percentages may not equal 100 due to rounding. Treatment decision-making In the pre- and
post-trial
study conditions,
68 and 71%
respectively of participants preferred an active or collaborative
role in treatment decisions. Wilcoxon tests revealed no significant
differences in treatment decision-making roles between the pre-
and post-trial study conditions; however, a significant shift in
preferences for a more active role in treatment decisions was
reported for (a) participants who attended the study session with
their spouse or partner (z [42] ¼ 2.49, Po0.05) and (b)
participants who were married (z [47] ¼ 1.98, Po0.05). g
The current study also demonstrated that men expressed similar
patterns of psychosocial problems as women with cancer as they
reported anxiety, fear and shock (distress) and utilised both
positive (positive approach) and negative (nonacceptance) coping
strategies (Fallowfield et al, 1997; van Wersch et al, 1997a, b). The
association between distress and nonacceptance was also consis-
tent with previous research that found men with cancer are prone
to relying upon avoidance coping strategies in response to the
stress of a life-threatening disease such as cancer (Vingerhoets and
Van Heck, 1990). The reduction in distress is an important finding
given that less distressed patients are better able to make sense of
their experience with cancer and seek desired information (Leydon
et al, 2000). Psychosocial functioning Responses to the 20 psychosocial functioning items at pretrial were
subjected to a principle components analysis. A three-component
solution was extracted that explained 47% of the variance (see
Table 2). Each component possessed adequate factor loadings
(0.52–0.94) and internal reliability with Cronbach’s alpha values of
X0.68. The three components were subsequently named distress
(feelings of shock and fear), positive approach (optimism and a
fighting spirit) and nonacceptance (denial) that explained 25, 13
and 10% of the variance respectively. Distress was significantly
positively associated with both positive approach (r [61] ¼ 0.32,
Po0.05) and nonacceptance (r [60] ¼ 0.31, Po0.05) in both study
conditions. A related t-test revealed that distress decreased
significantly
between
the
pre-
and
post-trial
conditions
(t [58] ¼ 2.35, Po0.05). & 2004 Cancer Research UK Future research The use of the MMP for prostate cancer patients at this stage of
evaluation cannot be recommended until prospective randomised
control trials to compare the utility of the MMP with good clinical
practice have been completed. In a summative evaluation,
important factors such as ‘usability’ may have influenced the
outcome measures in the current study. Usability refers to the ease
of use and acceptability of a product for particular types of user to
perform specific tasks in a given context, which is influenced by
cost, convenience, availability, prerequisite training and organisa-
tional issues (Bevan and McLeod, 1994). Therefore, a combination
of performance measures and assessments of user satisfaction is
required to determine the usability of the MMP in both clinical and
residential environments as a function of style and properties of
the interface (e.g. methods used to communicate between the user
and computer), dialogue structure, functionality (e.g. browsing
content), efficiency (e.g. navigation structure), reliability (e.g. fault
tolerance), user characteristics (e.g. age) including the combina-
tion of attributes that provide the greatest level of satisfaction for
the majority of users. Particular attention should be given to
obtaining information from patients who dislike or feel uncom-
fortable using computers. Ergonomic factors such as postural
demands may also be related to perceived ease of use, especially in
elderly men, and deserve consideration in future applications of
this MMP. Browsing the MMP developed a need to acquire information
other than those anticipated at pretrial and to reprioritise
information needs. However, the hierarchical structure of infor-
mation needs was inconsistent with previous research (Davison
et al, 1995, 2002). The discrepancies with previous research may be
due to using free-response questions, only requesting primary
information needs, and 19% of men in the pretrial condition
indicating they did not have any information needs, which
increased to 66% at post-trial. Information needs The
information
needs
reported
by
the
study
participants
were examined for common themes and coded into mutually British Journal of Cancer (2004) 91(5), 855 – 860 Multimedia education program for prostate cancer
D Flynn et al Multimedia education program for prostate cancer 859 disease stage could be followed by a description of how to develop
positive coping strategies to deal with treatment side effects. The results of the current study were inconsistent with previous
research using the Control Preference Scale (CPS) that reported the
majority of men within 0–13 weeks of receiving their diagnosis
preferred a passive decision-making role (Davison et al, 1995). However, more recent studies utilising the CPS are congruent with
the current study reporting that 68% (Davison and Degner, 1997),
75% (Wong et al, 2000) and as many as 93% (Davison et al, 2002)
of men recently diagnosed prefer either an active or collaborative
role in treatment decisions. This trend in the current study could
be attributed to the relatively low mean age of the study
participants (Davison et al, 2002), and/or spousal support that
served as a catalyst to learn and take part in shared decision-
making (Ptacek et al, 1999). p
p
g
g
An assessment of reading level required to comprehend
the information presented in the MMP needs to be conducted
to
ensure
understanding
by
all
patients
irrespective
of
educational background. A self-test at the end of each topic
that provides feedback on performance (and delivering reassur-
ance and support in the case of poor performance) could also
enhance learning via the use of positive reinforcement (operant
conditioning). The inclusion of a search function would also
facilitate learning as patients could pinpoint salient information
needs more quickly and avoid the frustration of being unable to
locate desired information. Following a summative evaluation,
algorithms built into the MMP could suggest an appropriate
treatment modality based on a patient’s unique status (clinical
profile and preferences regarding both positive and negative
treatment outcomes). The MMP adequately fulfilled information needs for treatment
side effects, coping strategies, diagnostic tests and treatment
duration. The failure of the MMP to completely satisfy information
needs related to the likelihood of a cure and aetiology is probably
attributable to the state of medical knowledge and the lack of
prospective clinical trials of sufficient quality comparing one
treatment with another rather than shortcomings of the MMP. Limitations Despite these encouraging results, there are several methodological
issues that may have had a confounding influence upon the
outcome measures. The sampling method (selection by urologist)
employed to select participants may have produced an unrepre-
sentative sample as reasons for noninclusion were not recorded. Other methodological issues that potentially reduce generalisa-
bility of the results include the failure to record the participants’
disease stage and functional status. Furthermore, given the
generally late onset of prostate cancer, the mean age (66 years)
of the study participants was relatively young. The psychosocial functioning scale utilised in the current study
needs to be further validated in future research, as only internal
reliability and internal validity was assessed. A research assistant
also supported the participants throughout the study session,
which may have impacted upon the participants’ level of distress. Furthermore, the question of whether the level of support provided
is necessary in future applications of the MMP needs to be
investigated. High usability of the MMP is essential if the MMP is to be used
in patients’ homes or accessed via the WWW, to ensure that the
MMP can successfully compete with the perennial increase in the
number of cancer-related Web sites on the Internet. In particular,
it must be established if the MMP confers benefits over time in
terms of outcomes assessed in the current study, including
satisfaction with care, quality of life and ultimately survival. Furthermore, future work should be conducted to determine if the
MMP can fulfil post-treatment information needs and those of
partner-caregivers who are reported to have information needs
equivalent to those of patients (Davison et al, 2002). Suggested improvements to the MMP The inclusion of a decision-aid to communicate quantitative
outcome information to patients could address the shortcomings
of the MMP in terms of fulfilling information needs. However, the
decision-aid used to present outcome data must be processed with
a high degree of accuracy by patients, otherwise it can influence
the perception of probability (Chatterton, 1999). According to
Feldman-Stewart et al (2000) a 10 10 matrix of shaded ovals is
the most efficacious format for presenting information on
probabilities to patients, although this must be explained with
support from clinicians. & 2004 Cancer Research UK British Journal of Cancer (2004) 91(5), 855 – 860 REFERENCES Br Med J 318: 318–322
f
d
h
h Crawford ED, Bennett CL, Stone NN, Knight SJ, DeAntoni E, Sharp L,
Garnick MB, Poterfield HA (1997) Comparison of perspectives on
prostate cancer: analyses of survey data. Urology 50: 366–372 Michie S, McDonald V, Marteau TM (1997) Genetic counselling: informa-
tion given, recall and satisfaction. Patient Educ Couns 32: 101–106 y
y
gy
Davison BJ, Degner LF (1997) Empowerment of men newly diagnosed with
prostate cancer. Cancer Nurs 20: 187–197 Murray E, Davis H, Tai SS, Coulter A, Gray A, Haines A (2001) Randomised
control trial of an interactive multimedia decision aid on benign
prostatic hypertrophy in primary care. Br Med J 323: 1–6 Davison BJ, Degner LF, Morgan TR (1995) Information and decision-
making preferences of men with prostate cancer. Oncol Nurs Forum 22:
1401–1408 Preece J, Sharp H, Benyan D, Holland S, Carey T (1994) Human–Computer
Interaction. Reading, MA: Addison-Wesley Davison BJ, Gleave ME, Goldenberg SL, Degner LF, Hoffart D, Berkowitz J
(2002) Assessing information and decision preferences of men with
prostate cancer and their partners. Cancer Nurs 25: 42–49 Ptacek JT, Pierce GR, Ptack JJ, Nogel C (1999) Stress and coping processes
in men with prostate cancer: the divergent views of husbands and wives. J Soc Clin Psychol 18: 299–324 y
Shepperd S, Coulter A, Farmer A (1995) Using interactive videos in general
practice to inform patients about treatment choices: a pilot study. Fam
Pract 12: 443–447 Davison BJ, Goldenberg L, Gleave ME, Degner LF (2003) Provision of
individualized information to men and their partners to facilitate
treatment decision making in prostate cancer. Oncol Nurs Forum 30:
107–114 Shrock D, Palmer RF, Taylor B (1999) Effects of a psychosocial intervention
on survival among patients with stage I breast and prostate cancer: a
matched case–control study. Altern Ther Health Med 5: 49–55 Degner LF, Sloan JA (1992) Decision-making during serious illness: what
role do patients really want to play? J Clin Epidemiol 45: 941–950 y
Smith D, Timoney A (1997) Patient information systems. Br J Urol 80:
27–30 DoH (2001) The Expert Patient: A New Approach to Chronic Disease
Management for the 21st Century. London: HMSO Fallowfield LJ, Hall A, McGuire GP, Baum S (1997) Psychological outcomes
of different treatment policies in women with early breast cancer outside
the clinical trial. REFERENCES 50-80-year-old prostate cancer patients. Scand J Urol Nephrol 35:
97–101 50-80-year-old prostate cancer patients. Scand J Urol Nephrol 35:
97–101 Barry MJ, Fowler JF, Mulley AG, Henderson JV, Wennberg JE (1995)
Patient reactions to a program designed to facilitate patient participation
in treatment decisions for benign prostatic hyperplasia. Med Care 33:
771–782 Clinical Holmberg L, Bill-Axelson A, Helgesen F, Salo JO, Folmerz P, Haggman M,
Andersson SO, Spangberg A, Busch C, Nordling S, Palmgren J, Adami
HO, Johansson JE, Norlen BJ (2002) A randomized trial comparing
radical prostatectomy with watchful waiting in early prostate cancer. N
Engl J Med 347: 781–789 Bevan N, McLeod M (1994) Usability measurement in context. Behav
Inform Technol 13: 132–145 f
Brink SG, Birney AJ, McFarren AE (2000) Charting your course: formative
evaluation of a prostate cancer treatment decision-aid. Int Electron J
Health Educ 3: 44–54 Jenkinson
J,
Wilson-Pauwels
L,
Jewett
MAS,
Woolridge
N
(1998)
Development of a hypermedia program designed to assist patients with
localised prostate cancer in making treatment decisions. J Biocommun
25: 2–11 Cancer Research UK (2002a) CancerStats: Incidence – UK, Retrieved
December 22, 2002 from http://www.cancerresearchuk.org/aboutcancer/
statistics/29375/29902 Jorm AF (1994) Characteristics of Australians who reported consulting a
psychologist for a health problem: an analysis of data from the 1989–90
National Health Survey. Aust Psychol 29: 212–215 Cancer Research UK (2002b) CancerStats: Mortality – UK, Retrieved
December 22, 2002 from http://www.cancerresearchuk.org/aboutcancer/
statistics/29375/29902 y
y
Krishna S, Balas EA, Spencer DC, Griffin JZ, Boren SA (1997) Clinical trials
of interactive computerised patient education: implications for family
practice. J Fam Pract 45: 25–33 Chatterton HT (1999) Efficacy, risk and the determination of value: shared
decision-making in the age of information. J Fam Pract 48: 505–507
Coulter A, Entwistle V, Gilbert D (1999) Sharing decisions with patients: is
the information good enough? Br Med J 318: 318–322 Chatterton HT (1999) Efficacy, risk and the determination of value: shared
decision-making in the age of information. J Fam Pract 48: 505–507 p
Leydon GM, Boulton M, Moynihan C, Jones A, Mossman J, Boudioni M,
McPherson K (2000) Cancer patients’ information needs and information
seeking behaviour: in depth interview study. Br Med J 320: 909–913 g
g
Coulter A, Entwistle V, Gilbert D (1999) Sharing decisions with patients: is
the information good enough? REFERENCES Br Med J 301: 575–580 Van Schaik P, Ahmed T, Suvakovic N, Hindmarsh J (1999) The design of an
educational multimedia interactive prostate program for BPH. Cleveland
Med J 1: 16–22 Feldman-Stewart D, Brundage MD, Hayter C, Groome P, Nickel JC, Downes
H, Mackillop WJ (1998) What prostate cancer patients should know:
variation in professionals’ opinions. Radiother Oncol 49: 111–123 Van Wersch A, Bonnema J, Prinsen B, Pruyn J, Wiggers T, van Geel AN
(1997a) Continuity of information for breast cancer patients: the
development, use and evaluation of a multidisciplinary care-protocol. Patient Educ Couns 30: 175–186 Feldman-Stewart D, Brundage MD, McConnnell BA, MacKillop WJ (2000)
Practical issues in shared decision-making. Health Expect 3: 46–54 Van Wersch A, de Boer MF, van der Does E, de Jong P, Knegt P, Meeuwis
CA, Stringer P, Pruyn JF (1997b) Continuity of information in cancer
care: evaluation of a logbook. Patient Educ Couns 31: 223–236 Fitch MI, Johnson B, Gray R, Franssen E (1999) Survivors’ perspectives on
the impact of prostate cancer: implications for oncology nurses. Can
Oncol Nurs J 9: 23–28 g
Vingerhoets AJ, van Heck GL (1990) Gender, coping and psychosomatic
symptoms. Psychol Med 20: 125–135 Frank-Stromborg M, Cohen R (1991) Evaluating written patient education
materials. Semin Oncol Nurs 7: 128–134 Wagner EH, Barrett P, Barry MJ, Barlow W, Fowler FJ (1995) The effect of a
shared decision-making program on rates of surgery for benign prostate
hyperplasia. Med Care 33: 765–770 Glajchen M, Moul JW (1996) Teleconferencing as a method of educating
men about managing advanced prostate cancer and pain. J Psychosoc
Oncol 14: 73–87 y
Weed LL (1997) New connections between medical knowledge and patient
care. Br Med J 315: 231–235 Gray RE, Fitch MI, Phillips C, Labrecque M, Klotz L (1999) Presurgery
experiences of prostate cancer patients and their spouses. Cancer Pract 7:
130–135 Whitrod R (1996) Improved quality of life for men with advanced prostate
cancer: the need for an increased contribution by psychologists. Aust
Psychol 31: 127–132 Gregoire I, Kalogeropoulos D, Corcos J (1997) The effectiveness of a
professionally led support group for men with prostate cancer. Urol Nurs
17: 58–66 Wong F, Stewart DE, Dancey J, Meana M, McAndrews MP, Bunston T,
Cheung AM (2000) Men with prostate cancer: influence of psychological
factors on informational needs and decision-making. ACKNOWLEDGEMENTS staff time to deliver the information. Multimedia programs are
unlikely to replace the ‘human touch’ associated with traditional
doctor–patient interactions, although, in the present climate of
health care reform, which is geared toward the cost-effective
delivery of quality services, MMPs will become increasingly
commonplace tools for patient education if they are demonstrated
to be more effective than good clinical practice. We thank all the clinical staff at the department of urology at
James Cook University Hospital for their support in conducting
this study. We would also like to express our deepest gratitude to
all the participants who agreed to take part for their patience, time
and co-operation. CONCLUSION The incidence of prostate cancer is expected to increase in
developed countries due to ageing populations, increased use of
PSA screening and declines in other major causes of mortality. This will result in concomitant cost increases to health care
providers and it is unlikely that the slow and expensive process of
training biomedical practitioners occupying the central role in
health care will meet the increased demand for their ‘expert
knowledge’ (Weed, 1997). However, empowered with sufficient
knowledge patients can make informed decisions about their
treatment in collaboration with clinicians without an investment of Coping knowledge may be enhanced by amalgamating informa-
tion on positive coping strategies with other salient topics such as
likelihood of cure. To enable men to vicariously learn positive
coping strategies, streaming video of a real-patient (or trained
actor) describing the probability of a cure associated with each British Journal of Cancer (2004) 91(5), 855 – 860 Multimedia education program for prostate cancer
D Flynn et al D Flynn et al 860 REFERENCES J Psychosom Res 49:
13–19 Helgason
AR,
Dickman
PW,
Adolfsson
J,
Steineck
G
(2001)
Emotional isolation: prevalence and the effect on well-being among British Journal of Cancer (2004) 91(5), 855 – 860 & 2004 Cancer Research UK & 2004 Cancer Research UK
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Spontaneous calcific cerebral embolization revealing a calcified rheumatic mitral stenosis: a case report
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background Cerebral cardiac embolism accounts for an increasing proportion of ischemic strokes and transient
ischemic attacks. Calcified cerebral emboli are rare and mostly iatrogenic secondary to heart or aorta catheteriza-
tion. However, spontaneous cerebral calcified embolism in the case of calcified aortic valve is very rare and there are
less than 10 case reports in the literature. And a more interesting fact is that such an event, in the context of calcified
mitral valve disease, has never been reported, at least to our knowledge. We are reporting a case of spontaneous
calcified cerebral embolism revealing a calcified rheumatic mitral valve stenosis. Case presentation We report a case of a 59 year-old Moroccan patient, with a history of rheumatic fever at the age
of 14 and no history of recent cardiac intervention or aortic/carotid manipulation, who was admitted to the emer-
gency department after a transient ischemic attack. Physical examination at admission found normal blood pressure
of 124/79 mmHg and heart rate of 90 bpm. A 12-lead electrocardiogram showed an atrial fibrillation, no other anoma-
lies. Unenhanced cerebral computed tomography imaging was performed, revealing calcified material inside both
middle cerebral arteries. Transthoracic echocardiography was performed, showing severe mitral leaflets calcification
with a severe mitral stenosis, probably due to rheumatic heart disease. Cervical arteries Duplex was normal. A vitamin
K antagonist (acenocoumarol) was prescribed, targeting an international normalized ratio of 2–3 and mitral valve
replacement surgery was performed using mechanical prosthesis. Short- and long-term health, with a 1-year follow-
up, were good and the patient did not experience any stroke. Conclusion Spontaneous calcified cerebral emboli secondary to mitral valve leaflet calcifications is an extremely
rare condition. Replacement of the valve is the only option to prevent recurrent emboli and outcomes are still to be
determined. Keywords Cerebral embolism, Calcified mitral stenosis, Transient ischemic attack Spontaneous calcific cerebral embolization
revealing a calcified rheumatic mitral stenosis:
a case report M. Haboub1*, S. Abouradi1, H. Mechal1, G. Minko1, A. Moukhliss1, S. Arous1, M. E. G. Benouna1, A. Drighil1,
L. Azzouzi1 and R. Habbal1 Open Access Open Access Haboub et al. Journal of Medical Case Reports (2023) 17:254
https://doi.org/10.1186/s13256-023-03982-2 Haboub et al. Journal of Medical Case Reports (2023) 17:254
https://doi.org/10.1186/s13256-023-03982-2 (2023) 17:254 Journal of
Medical Case Reports Background Cerebral cardiac embolism accounts for an increasing
proportion of ischemic strokes and transient ischemic
attacks [1]. Calcified cerebral emboli are rarely reported,
but potentially cause of strokes and transient ischemic
attacks and may be the first manifestation of vascular
or cardiac disease. Identification of the source of embo-
lization is crucial to prevent future emboli, neurological
damage, and death. Non-contrast computed tomography *Correspondence:
M. Haboub
haboubmeryem@gmail.com
1 Cardiology Department, Hospital University Ibn Rochd, Casablanca,
Morocco *Correspondence:
M. Haboub
haboubmeryem@gmail.com
1 Cardiology Department, Hospital University Ibn Rochd, Casablanca,
Morocco © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Haboub et al. Journal of Medical Case Reports (2023) 17:254 Page 2 of 4 Fig. 1 Chest computed tomography confirming mitral leaflet
calcification (CT) scan of the head is the most common imaging pro-
cedure used as the initial assessment of suspected stroke
or transient ischemic attack [2].i Cerebral calcified embolus can occur after percuta-
neous and surgical intervention in the context of calci-
fied aortic or mitral valve disease [3]. These emboli are
presumed to occur because of valve trauma. However,
spontaneous cerebral calcified embolism in the case of
calcified aortic valve is very rare and there are less than
ten case reports in the literature [4]. And a more interest-
ing fact is that such an event in the context of calcified
mitral valve disease has never been reported, at least to
our knowledge.i We are reporting a case of spontaneous calcified cer-
ebral embolism revealing a calcified rheumatic mitral
valve stenosis. Fig. 1 Chest computed tomography confirming mitral leaflet
calcification Case presentation Cervical arteries Duplex showed normal carotid and
vertebral arteries. Cervical arteries Duplex showed normal carotid and
vertebral arteries. We are reporting a case of a 59-year-old Moroccan man
presenting to the emergency department after a transient
ischemic attack (right hemiparesia and left central facial
paralysis resolving briefly and spontaneously). There was
a history of rheumatic fever at age 14 and stage II New
York Heart Association (NYHA) dyspnea on moderate
exertion for 2 years, with no history of recent cardiac
intervention or aortic/carotid manipulation and no other
symptoms. Physical examination on admission found an
irregular heart rhythm of 90 bpm, blood pressure (BP)
of 124/79 mmHg, mid-diastolic rumble at apex, no signs
of heart congestion, and no signs of neurological impair-
ment. A 12-lead-electrocardiogram showed an atrial
fibrillation without other anomalies. Two-dimensional
(2D) transthoracic echocardiograms revealed important
mitral valve leaflets calcifications, probably related to
rheumatic heart disease; planimetry of the valve was not
possible. Continuous wave Doppler interrogation of the
mitral valve found a severe mitral stenosis with a mean
gradient of 15 mmHg and a continuity equation surface
of 1 cm2. The aortic valve was thickened, but not calci-
fied, a moderate aortic regurgitation was noticed. Left
ventricular (LV) function was normal and the LV ejection
fraction (LVEF) was at 55%. There was a right ventricu-
lar (RV) longitudinal systolic dysfunction: TAPSE 11 mm
and S’VD 6 cm/second. Tricuspid valve was thin with
a mild tricuspid regurgitation. Continuous wave Dop-
pler interrogation of the tricuspid valve allowed to esti-
mate systolic pulmonary artery pressure at 69 mmHg. Mitral valve calcification was best shown on a cardiac CT
(Fig. 1). A vitamin K antagonist (acenocoumarol) was pre-
scribed, targeting an international normalized ratio (INR)
of 2–3 and replacement of mitral valve using a mechani-
cal prosthesis was performed with a good short-term
and long-term outcome. The patient did not experience
ischemic stroke during a 1-year follow-up. Discussion In our patient, transient ischemic attack (TIA) was cer-
tainly due to atrial fibrillation (AF), and cerebral imaging
lead to the discovery of calcified emboli. i
Approximately 6%–31% of TIA are caused by a car-
diogenic cerebral embolism (cardioembolic TIA) [5, 6]. Determining TIA etiology is important before adminis-
tering therapy. Permanent or paroxysmal, valvular and
non-valvular, AF is associated with a three- to five-fold
increased risk of TIA and stroke [7, 8]. Cardiogenic cer-
ebral embolization is common among patients with
any cause of AF, but particularly in AF resulting from
rheumatic and arteriosclerotic heart disease [9]. It is
recommended to prescribe these patients oral antico-
agulant therapy in case of valvular AF and, according to
CHA2DS2VASc score, in case of non-valvular AF.i Calcified cerebral emboli are an infrequent, but
increasingly recognized cause of TIA and ischemic
stroke, although recognition among general radiolo-
gists and clinicians can be limited. Unenhanced CT
and computed tomography angiography (CTA) are the
imaging techniques of choice for the diagnosis [10, 11]. First described on CT by Yock in 1981, calcified cere-
bral emboli were previously thought to be unusual, and
to most commonly arise following instrumentation of
calcified cardiac valves or direct aortic/carotid artery Unenhanced cerebral CT was performed, revealing cal-
cified emboli in both middle cerebral arteries (M3 and
M4 segments) (Fig. 2). Susceptibility weighted magnetic
resonance cerebral sequences result is reported in Fig. 3. Page 3 of 4 Haboub et al. Journal of Medical Case Reports (2023) 17:254 Fig. 2 Unenhanced cerebral computed tomography with axial reconstructions showing calcified emboli in both middle cerebral arteries. These
arteries were permeable Fig. 2 Unenhanced cerebral computed tomography with axial reconstructions showing calcified emboli in both middle cerebral arteries. These
arteries were permeable Fig. 3 Susceptibility weighted cerebral magnetic resonance sequences showing absence of signal inside both middle cerebral arteries Fig. 3 Susceptibility weighted cerebral magnetic resonance sequences showing absence of signal inside both middle cer manipulation [12, 13]. However, there is growing evi-
dence that spontaneous calcified cerebral embolism is
more common, with a recent study and review of pub-
lished cases reporting a 2.7% prevalence among a group
of patients presenting with suspected stroke over a
1-year period. In this report, the middle cerebral artery
was the site of 83% calcified emboli. Funding Funding
The authors have no funding to declare. The authors have no funding to declare. 19. Raghib MF, Mutzenback JS, Rosler C, et al. Acute treatment of stroke due
to spontaneous calcified cerebral emboli causing large vessel occlusion. J
Clin Neurosci. 2018;47:56–61. Availability of data and materials The published information is available from the corresponding author on
reasonable request. 20. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in
systemic calciphylaxis. Neurology. 1998;51:295–7. 20. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in
systemic calciphylaxis. Neurology. 1998;51:295–7. 20. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in
systemic calciphylaxis. Neurology. 1998;51:295–7. Consent for publication Written informed consent was obtained from the patient for publication of
this case report and any accompanying images. A copy of the written consent
is available for review by the Editor-in-Chief of this journal. There are some reported cases of cerebral calcific
emboli following open heart mitral valvotomy and per-
cutaneous mitral valvuloplasty. Mitral calcification
accounts for fewer than 1% of cerebral cardiac embolism,
and in all described cases, it was secondary to mitral
valve intervention [16]. However, there is not any case
report published describing spontaneous calcified cer-
ebral emboli in the context of calcified rheumatic mitral
stenosis.i Author contributions 14. Salka S, Almassi H, Leitshuh M. Spontaneous coronary artery embolus
associated with calcific artery stenosis. Chest. 1994;105:1289–90. 14. Salka S, Almassi H, Leitshuh M. Spontaneous coronary artery embolus
associated with calcific artery stenosis. Chest. 1994;105:1289–90. MH conceived the study, participated in its design, acquired the data, per-
formed a literature review, and drafted the manuscript. SA participated in the
design of the study and helped with the literature review. HM helped with the
literature review. GM helped with the literature review. AM helped with the
literature review. SA participated in the design of the study. MEGB helped with
the literature review, and helped draft and edit the manuscript. AD helped
draft the manuscript. LA helped draft and edit the manuscript. RH helped draft
and edit the manuscript. All authors read and approved the final manuscript. i
15. Wilson JH, Cranley JJ. Recurrent calcium emboli in a patient with aortic
stenosis. Chest. 1989;96:1433–4. 15. Wilson JH, Cranley JJ. Recurrent calcium emboli in a patient with aortic
stenosis. Chest. 1989;96:1433–4. 16. Hickey TBM. Iatrogenic embolization following cardiac intervention:
postmortem analysis of 110 cases. Cardiovasc Pathol. 2019;40:12–8. 16. Hickey TBM. Iatrogenic embolization following cardiac intervention:
postmortem analysis of 110 cases. Cardiovasc Pathol. 2019;40:12–8. 17. O’Cearbhaill RM, Moriarty HK, Crosbie I, et al. Calcified cerebral emboli: a
case series and review of the literature. J Syst Int Neurosci. 2016;2:180–3. 17. O’Cearbhaill RM, Moriarty HK, Crosbie I, et al. Calcified cerebral emboli: a
case series and review of the literature. J Syst Int Neurosci. 2016;2:180–3. 18. Halloran JI, Bekavac I. Unsuccessful tissue plasminogen activator treat-
ment of acute stroke caused by a calcific embolus. J Neuroimaging. 2004;14:385–7. 18. Halloran JI, Bekavac I. Unsuccessful tissue plasminogen activator treat-
ment of acute stroke caused by a calcific embolus. J Neuroimaging. 2004;14:385–7. References In case of stroke secondary to calcified emboli, the role
of thrombolysis remains uncertain, as there are conflict-
ing reports regarding its efficacy in this setting [17–19]. There is debate and very limited experience regarding the
place of mechanical thrombectomy [18, 20]. Subsequent
imaging evaluation of this subgroup of patients who have
suffered from ischemic strokes requires caution because
the calcified nature of the embolus may be obscured
on CT angiography or magnetic resonance imaging
(MRI). Clinical evaluation should include consideration
of potential proximal source of calcified material and
recent cardiac intervention or aortic/carotid manipula-
tion. Although there is no data showing benefit of valve
replacement, most authors advocate valve replacement to
remove the source of emboli. 1. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL,
Marsh EE. Classification of subtype of acute ischemic stroke. Definitions
for use in a multicenter clinical trial TOAST Trial of Org 10172 in Acute
Stroke Treatment. Stroke. 1993;24:35–41. 1. Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL,
Marsh EE. Classification of subtype of acute ischemic stroke. Definitions
for use in a multicenter clinical trial TOAST Trial of Org 10172 in Acute
Stroke Treatment. Stroke. 1993;24:35–41. 2. Walker BS. Calcified cerebral emboli, a “do not miss” imaging diagno-
sis: 22 new cases and review of the literature. AJNR Am J Neuroradiol. 2014;35:1515–9. https://doi.org/10.3174/ajnr.A3892. 3. Kapila A, Hart R. Calcific cerebral emboli and aortic stenosis: detection by
computed tomography. Stroke. 1986;17:619–21. 3. Kapila A, Hart R. Calcific cerebral emboli and aortic stenosis: detection by
computed tomography. Stroke. 1986;17:619–21. 4. Khetarpal V. Calcific aortic valve and spontaneous embolic stroke: a
review of literature. J Neurol Sci. 2009;287:32–5. 5. Bogousslavsky J, Hachinski VC, Boughner DR. Cardiac and arterial lesions
in carotid transient ischemic attacks. Arch Neurol. 1986;43:223–8. 3. Kapila A, Hart R. Calcific cerebral emboli and aortic stenosis: detection by
computed tomography. Stroke. 1986;17:619–21. 4. Khetarpal V. Calcific aortic valve and spontaneous embolic stroke: a
review of literature. J Neurol Sci. 2009;287:32–5. computed tomography. Stroke. 1986;17:619–21. 4. Khetarpal V. Calcific aortic valve and spontaneous embolic stroke: a
review of literature. J Neurol Sci. 2009;287:32–5. 5. Bogousslavsky J, Hachinski VC, Boughner DR. Cardiac and arterial lesions
in carotid transient ischemic attacks. Arch Neurol. 1986;43:223–8. 6. Sempere AP, Duarte J, Cabezas C, et al. Etiopathogenesis of transient
ischemic attacks and minor ischemic strokes: a community-based study
in Segovia, Spain. Stroke. References 1998;29:40–5. 7. Wolf PA, Abbott RD, Kannel WB. Atrial fibrillation as an independent risk
factor for stroke: the Framingham Study. Stroke. 1991;22:983–8. g
y
8. Wolf PA, Dawber TR, Thomas HE Jr, Kannel WB. Epidemiologic assessment
of chronic atrial fibrillation and risk of stroke: the Framingham study. Neurology. 1978;28:973–7. Acknowledgements
Not applicable. 13. Khaw N, Gailloud P. CT of calcific cerebral emboli after carotidmanipula-
tion. Am J Roentgenol. 2000;174:1467. 13. Khaw N, Gailloud P. CT of calcific cerebral emboli after carotidmanipula-
tion. Am J Roentgenol. 2000;174:1467. Discussion Cardiac valvu-
lar disease was more common than carotid atheroma-
tous disease, with calcified aortic stenosis three times more common than mitral valve disease as the embolic
source [3].i Iatrogenic calcified embolus following cardiac sur-
gery or catheterization is common [4, 14, 15]. These
emboli are presumed to occur because of valve trauma. According to a most recent postmortem analysis of iatro-
genic embolization cases, the source of calcified cerebral
emboli was attributed to dislodgement and displace-
ment of calcified material from calcified aortic valves and Haboub et al. Journal of Medical Case Reports (2023) 17:254 Page 4 of 4 ulcerated aortic atherosclerotic plaques during therapeu-
tic and investigative procedures [16].hi Competing interests The authors declare that they have no competing interests. Received: 11 March 2020 Accepted: 10 May 2023 Received: 11 March 2020 Accepted: 10 May 2023 Conclusion y
9. Stirling J. Cerebral embolism as a cause of stroke and transient ischemic
attack. Echocardiography. 1996;13(5):513–8. Spontaneous calcified cerebral emboli, secondary to
mitral valve leaflet calcification is an extremely rare con-
dition. Replacement of the valve is the only option to
prevent recurrent emboli and outcomes are still to be
determined. 10. Rancurel G, Marelle L, Vincent D, Catala M, Arzimanaglou A, Vacheron A. Spontaneous calcific cerebral embolus from a calcific aortic stenosis in a
middle cerebral artery infarct. Stroke. 1989;20:691–3. 11. Oliveira-Filho J, Massaro AR, Yamamoto F, Bustamante L, Scaff M. Stroke
as the first manifestation of calcific aortic stenosis. Cerebrovasc Dis. 2000;10:413–6. 12. Yock DH. CT demonstration of cerebral emboli. J Comput Assist Tomogr. 1981;5:190–6. 12. Yock DH. CT demonstration of cerebral emboli. J Comput Assist Tomogr. 1981;5:190–6.i Publisher’s Note
S Springer Nature remains neutral with regard to jurisdictional claims in pub-
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lished maps and institutional affiliations. Ethics approval and consent to participate Ethics approval and consent to participate
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Relationships of Gut Microbiota Composition, Short-Chain Fatty Acids and Polyamines with the Pathological Response to Neoadjuvant Radiochemotherapy in Colorectal Cancer Patients
|
International journal of molecular sciences
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Correspondence: jaime.gomez@ibima.eu (J.G.-M.); maribel.queipo@ibima.eu (M.I.Q.-O.) Citation: Sánchez-Alcoholado, L.;
Laborda-Illanes, A.; Otero, A.;
Ordóñez, R.; González-González, A.;
Plaza-Andrades, I.; Ramos-Molina, B.;
Gómez-Millán, J.; Queipo-Ortuño,
M.I. Relationships of Gut Microbiota
Composition, Short-Chain Fatty
Acids and Polyamines with the
Pathological Response to
Neoadjuvant Radiochemotherapy in
Colorectal Cancer Patients. Int. J. Mol. Sci. 2021, 22, 9549. https://doi.org/
10.3390/ijms22179549 Abstract: Emerging evidence has suggested that dysbiosis of the gut microbiota may influence
the drug efficacy of colorectal cancer (CRC) patients during cancer treatment by modulating drug
metabolism and the host immune response. Moreover, gut microbiota can produce metabolites that
may influence tumor proliferation and therapy responsiveness. In this study we have investigated
the potential contribution of the gut microbiota and microbial-derived metabolites such as short
chain fatty acids and polyamines to neoadjuvant radiochemotherapy (RCT) outcome in CRC patients. First, we established a profile for healthy gut microbiota by comparing the microbial diversity and
composition between CRC patients and healthy controls. Second, our metagenomic analysis revealed
that the gut microbiota composition of CRC patients was relatively stable over treatment time with
neoadjuvant RCT. Nevertheless, treated patients who achieved clinical benefits from RTC (responders,
R) had significantly higher microbial diversity and richness compared to non-responder patients
(NR). Importantly, the fecal microbiota of the R was enriched in butyrate-producing bacteria and had
significantly higher levels of acetic, butyric, isobutyric, and hexanoic acids than NR. In addition, NR
patients exhibited higher serum levels of spermine and acetyl polyamines (oncometabolites related
to CRC) as well as zonulin (gut permeability marker), and their gut microbiota was abundant in
pro-inflammatory species. Finally, we identified a baseline consortium of five bacterial species that
could potentially predict CRC treatment outcome. Overall, our results suggest that the gut microbiota
may have an important role in the response to cancer therapies in CRC patients. Academic Editors: Rustam I. Aminov
and Catalina Carrasco-Pozo Received: 18 August 2021
Accepted: 30 August 2021
Published: 2 September 2021 Received: 18 August 2021
Accepted: 30 August 2021
Published: 2 September 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: colorectal cancer; gut microbiota; SCFAs; gut permeability; radiochemotherapy; treat-
ment outcome Article
Relationships of Gut Microbiota Composition, Short-Chain
Fatty Acids and Polyamines with the Pathological Response to
Neoadjuvant Radiochemotherapy in Colorectal Cancer Patients Lidia Sánchez-Alcoholado 1, Aurora Laborda-Illanes 1, Ana Otero 2, Rafael Ordóñez 2,
Alicia González-González 1
, Isaac Plaza-Andrades 1, Bruno Ramos-Molina 3
, Jaime Gómez-Millán 2,*
and María Isabel Queipo Ortuño 1,* Lidia Sánchez-Alcoholado 1, Aurora Laborda-Illanes 1, Ana Otero 2, Rafael Ordóñez 2,
Alicia González-González 1
, Isaac Plaza-Andrades 1, Bruno Ramos-Molina 3
, Jaime Gómez-Millán 2,*
and María Isabel Queipo-Ortuño 1,* 1
Unidad de Gestión Clínica Intercentros de Oncología Médica, Hospitales Universitarios Regional y Virgen de
la Victoria, Instituto de Investigación Biomédica de Málaga (IBIMA)-CIMES-UMA, 29010 Málaga, Spain;
l.s.alcoholado@gmail.com (L.S.-A.); auroralabordaillanes@gmail.com (A.L.-I.);
agonzalez.bq@gmail.com (A.G.-G.); isaacplazaandrade@gmail.com (I.P.-A.) 1
Unidad de Gestión Clínica Intercentros de Oncología Médica, Hospitales Universitarios Regional y Virgen de
la Victoria, Instituto de Investigación Biomédica de Málaga (IBIMA)-CIMES-UMA, 29010 Málaga, Spain;
l.s.alcoholado@gmail.com (L.S.-A.); auroralabordaillanes@gmail.com (A.L.-I.);
agon ale bq@gmail com (A G G ) isaacpla aandrade@gmail com (I P A ) g
p
g
g
3
Grupo de Obesidad y Metabolismo, Instituto Murciano de Investigación Biosanitaria (IM
30120 Murcia, Spain; brunoramosmolina@gmail.com International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences 1. Introduction Colorectal cancer (CRC) is the second most common malignant cancer in Western
countries. The global burden of CRC is expected to substantially increase in the next
two decades as a consequence of adopting Western lifestyles [1]. In recent years, several
works have demonstrated that the gut microbiome could be a critical environmental factor
that contributes to the tumorigenesis and progression of CRC, potentially by inducing
pro-inflammatory responses, by producing microbial oncometabolites, and by interfering
with the energy balance in cancer cells. Moreover, CRC is frequently associated with a
dysbiosis in the microbial composition of the tumor and adjacent mucosa [2–4]. Several Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 9549. https://doi.org/10.3390/ijms22179549 Int. J. Mol. Sci. 2021, 22, 9549 2 of 25 studies have suggested that the composition of the gut microbiota could affect the body’s
response to a variety of cancer therapies, including chemotherapy, radiotherapy, and
immunotherapy [5–7]. py
Preoperative radiochemotherapy (RCT) followed by surgery has become the standard
treatment for patients with CRC [8,9]. Recent studies have suggested that the gut microbiota
may influence drug response (efficacy and toxicity) in CRC patients through several
mechanisms such as immunomodulation, reduced diversity, translocation, metabolism, and
ecological variation [10]. Specific gut bacteria have been shown to affect cancer treatment by
modulating drug metabolism and the host immune response [11,12]. Thus, several phyla
are known to mediate drug metabolism via different reactions such as isoxazole scission,
denitration, proteolytic degradation, acetylation/deacetylation, deconjugation, physical
adherence to the drugs as well as by amine formation and/or hydrolysis [13]. Scott et al. described that the gut microbiota was able to influence the efficacy of one of the first-line
treatments for CRC, such asfluoropyrimidines, through drug interconversion involving
bacterial vitamin B6 and B9 and ribonucleotide metabolism [14]. In addition, the effect
5-fluorouracil treatment in CRC cells could be mediated by gut microbial metabolites [15]. Remarkably, Fusobacterium nucleatum is able to promote CRC resistance to chemotherapy
by targeting both TLR4 and MYD88 innate immune signaling [16]. Furthermore, radiation
may also lead to alterations in gut microbiota composition in animal models [17]. 1. Introduction However,
the clinical impact of radiotherapy on gut microbiota in cancer patients remains mostly
unexplored although it has been proposed that the gut microbiota might play a role in the
immunogenic effect of radiotherapy [18]. On the other hand, the gut microbiome produces bacteria-derived metabolites that
could affect cancer proliferation and chemotherapy responsiveness. Thus, previous studies
describe that SCFAs (such as butyric acid, isobutyric acid and acetic acid) inhibit the growth
of cultured human colorectal cancer cells and that butyric acid is the strongest inhibitor [19]. Ross et al. reported an association between the levels of the short-chain fatty acids (SCFAs)
propionate and butyrate in patients with early stage breast cancer with a pathological
complete response (pCR) to neoadjuvant chemotherapy [20]. Coutzac et al. suggested that
SCFA limits anti-CTLA-4 activity in patients with metastatic melanoma [21]. y
p
In addition, lower SCFA (especially butyrate) concentrations might induce a dysfunc-
tion in the gut epithelial barrier, thereby activating proinflammatory cytokines such as
interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which damage epithelial cells
and their junctions [22]. Other bacteria-derived metabolites, such as the polyamines (PAs) (spermine, spermi-
dine and putrescine), have been involved in almost all the steps of colorectal tumorigenesis. PAs are molecules that are indispensable in normal cell growth and gene expression and
are needed in cell proliferation, but their concentrations increase during the transition from
a healthy cell to a tumor cell [23]. Recently, it was shown that the level of acetylated PAs is
more specific for cancer. For example, N1, N12-diacetylspermine (DiAcSPM) was increased
in CRC and in dysplastic colorectal lesions [24]. Therefore, taking all of the evidence together, we hypothesized a bidirectional interac-
tion between the neoadjuvant RCT and the gut microbiome in CRC patients: RCT might
induce alterations in the gut microbiome, and these alterations might, in turn, influence
the effectiveness of RCT by directly interacting with the treatment and/or by stimulating
the host’s immune response. In this study, we aimed to identify the possible relationship between the gut micro-
biome, the fecal SCFAs levels, the serum levels of the polyamines and acetyl derivatives of
polyamines, and the intestinal permeability to neoadjuvant RCT outcome in CRC patients. 2.1. Clinical Characteristics of the Patients and Healthy Controls CRC patients and healthy controls had comparable eating habits to exclude the influ-
ence of dietary differences. CRC patients and healthy controls followed a Mediterranean Int. J. Mol. Sci. 2021, 22, 9549 3 of 25 3 of 25 diet consisting in a high consumption of olive oil, fruits, legumes, vegetables, nuts, whole
grains, and fish and a low intake of red meat and dairy products. Adherence to the Mediter-
ranean diet was assessed by using a validated 14-item food frequency questionnaire in all
study patients. All CRC patients completed the neoadjuvant RCT and underwent surgical
resection. There was no significant difference between CRC patients and healthy controls in
terms of age, sex, BMI, and biochemical data (Table 1). A total of 28 of the 40 CRC patients
(70%) had a good response to the neoadjuvant RCT (responders, R) (TGR 1–2), and 12 (30%)
had a poor or non-response (non-responders, NR) (TGR 3–5) to therapy. Both R and NR
patients were similar in terms of sex, age, BMI, and stage of the cancer, as shown in Table 1. Table 1. Clinical characteristics of study groups. Table 1. Clinical characteristics of study groups. Table 1. Clinical characteristics of study groups. 2.1. Clinical Characteristics of the Patients and Healthy Controls Healthy
Controls
(N = 20)
CRC-Patients
(N = 40)
* p
R Patients
(N = 28)
NR Patients
(N = 12)
* p
Age (years)
61.42 ± 7.40
63.35 ± 6.97
0.326
62.93 ± 8.27
63.12 ± 6.34
0.928
Gender, n (M/F)
10/10
23/17
0.783
16 /12
7/5
0.780
BMI (kg/m2)
25.45 ± 3.23
26.42 ± 4.71
0.412
26.22 ± 4.22
25.92 ± 3.92
0.835
Constipation, n (%)
6 (20%)
10 (25%)
0.914
7 (25%)
3 (25%)
0.690
Alcohol consumption,
n (%)
4 (13.3%)
6 (15%)
0.831
4 (14.28%)
2 (16.16%)
0.740
Current smoking, n (%)
9 (30%)
15 (37.5%)
0.774
11 (39.28%)
4 (33.33%)
0.990
Biochemical data
Glucose (mg/dl)
94.85 ± 19.86
104.79 ± 27.94
0.161
102.83 ± 26.38
104.15 ± 23.56
0.882
Total cholesterol (mg/dl)
175.2 ± 33.6
183.95 ± 25.71
0.268
184.17 ± 21.64
181.67 ± 26.12
0.755
Triglycerides (mg/dl)
112.67 ± 34.51
114.85 ± 33.62
0.815
109.25 ± 32.12
118.32 ± 27.12
0.398
HDL-cholesterol (mg/dl)
60.7 ± 15.1
54.83 ± 18.23
0.219
55.32 ± 16.21
53.89 ± 18.34
0.807
LDL-cholesterol (mg/dl)
107.78 ± 27.12
112.07 ± 33.45
0.621
109.68 ± 30.29
112.36 ± 33.21
0.805
Histological variables
Disease stage
II
22 (55%)
-
15 (53.57%)
7 (58.33%)
0.945
III
18 (45%)
-
13 (46.42%)
5 (41.66%)
0.950
Tumor depth
penetration (T)
T2–T3
26 (65%)
-
18(64.28%)
8 (66.66%)
0.828
T4
14 (35%)
-
10 (35.71%)
4 (33.33%)
0.832
Grade of differentiation
G1
18 (45%)
-
12 (42.85%)
6 (50%)
0.944
G2
10 (25%)
-
7 (25%)
3 (25%)
0.690
G3
7 (17.5%)
-
5 (18.85%)
2 (16.16%)
0.806
No differentiation
5 (12.5%)
-
3 (10.71%)
2 (16.66%)
0.777
CRC: colorectal cancer; R: responders; NR: non-responders; BMI: body mass index; HDL: high density lipoprotein; LDL: low density
lipoprotein. Values are expressed as mean ± SD. * p < 0.05 was considered statistically significant. CRC: colorectal cancer; R: responders; NR: non-responders; BMI: body mass index; HDL: high density lipoprotein; LDL: low density
lipoprotein. Values are expressed as mean ± SD. * p < 0.05 was considered statistically significant. 2.2. Differences in Taxonomic Composition and Diversity of Gut Microbiota between CRC Patients
and Healthy Controls The analysis of stool samples revealed 17,496,823 reads of the 16S rRNA gene (hyper-
variable V2–V9 regions), with an average of 105,632 (±10,825) reads for each sample in
a range between 359 and 39,873. After trimming and filtering, 52,844 high-quality reads
were selected. 2.1. Clinical Characteristics of the Patients and Healthy Controls A total of 15,326 OTUs were obtained in the OTUs clustering process, and
after the alignment of the OTU representative sequences, 2582 OTUs were identified to
have a relative abundance >1% in at least four samples (97% similarity cut-off). For the
taxonomic assignment of these OTUs, QIIME2 pipeline and Greengenes v13.8 were used,
and the OTUs were binned into 7 phyla, 39 families, 45 genera, and 53 species. p y
g
p
We first compared the landscape of the gut microbiome in the stool samples of all
CRC patients at baseline and in healthy controls in order to define a normal gut microbiota
profile. As expected, we found significantly higher diversity and richness (defined by the
Shannon and Chao1 indexes, respectively) in the fecal samples of healthy controls with Int. J. Mol. Sci. 2021, 22, 9549 4 of 25 respect to those of CRC patients (Shannon p = 0.026 and Chao1 p = 0.001) (Figure S1A,B). The beta diversity (Bray–Curtis dissimilarity) comparison of the baseline CRC patients
and the healthy controls indicated that the two cohorts had significantly different genus
compositions of intestinal bacteria (p = 0.0001, ANOSIM) (Figure S1C). Furthermore, the analysis of the gut microbiota profiles between the CRC patients and
the healthy controls at baseline revealed significant differences in the abundance at different
taxonomic levels. At phylum level, the relative abundance of Fusobacteria (q < 0.001),
Firmicutes (q < 0.001), Lentisphaerae (q = 0.007), and Proteobacteria (q = 0.003) were
significantly increased in patients with CRC, while the relative abundance of Bacteroidetes
(q < 0.001) and Actinobacteria (q = 0.034) were significantly decreased in CRC patients
when compared to the controls (Figure 1A). At the genus level, the results indicated significant differences in the microbial com-
position of the dominant genera between the CRC patients at baseline and the healthy
controls. Compared to the healthy controls, patients with CRC displayed an enrichment
in the genera Prevotella (q < 0.001), Oscillospira (q < 0.001), Fusobacterium (q = 0.001), Enter-
obacter (q = 0.020), Victivallis (q = 0.012), Escherichia (q = 0.046), and Desulfovibrio (q < 0.001). Conversely, the abundance of Bacteroides (q = 0.003), Roseburia (q < 0.001), Ruminococcus
(q = 0.006), Faecalibacterium (q = 0.01), Bifidobacterium (q = 0.023), and Blautia (q = 0.014)
were enriched in the healthy controls compared to in the CRC patients (Figure 1B). 2.1. Clinical Characteristics of the Patients and Healthy Controls y
p
p
g
At species level, while healthy subjects showed a significantly higher abundance of
Bifidobacterium bifidum (q = 0.034) and Faecalibacterium prausnitzii (q = 0.040) with respect to
the CRC patients, Fusobacterium nucleatum (q = 0.020), Bacteroides fragilis (q = 0.024), and
Escherichia coli (q = 0.016) were significantly increased in the fecal samples of CRC patients
in comparison to the controls. 2.3. Changes in Gut Microbiota Diversity and Composition in Response to Neoadjuvant RCT
Treatment in CRC Patients 2.3. Changes in Gut Microbiota Diversity and Composition in Response to Neoadjuvant RCT
Treatment in CRC Patients We compared the gut microbiota communities at baseline (T0) versus at post-treatment
time points (T1, T2, and T3) to study the effect of neoadjuvant RCT on the gut microbial
diversity and composition in CRC patients. The alpha diversity comparison showed no
significant differences in the levels of richness (Chao 1) and diversity (Shannon) between the
baseline and the different time points (Shannon p = 0.75 and Chao1 p = 0.61) (Figure 2A,B). Moreover, the PCoA plot based on the beta diversity (Bray–Curtis dissimilarity) revealed
that the differences in the gut microbial community at T1, T2, and T3 compared to at
baseline (T0) were not significant (p = 0.716, ANOSIM) (Figure 2C). (
)
g
(p
) (
g
)
The main bacterial phyla (Firmicutes and Bacteroidetes) remained stable over time,
while other, less abundant phyla, such as Fusobacteria and Proteobacteria, were signif-
icantly decreased at T3 compared to at T0 (q = 0.042 and q = 0.039, respectively) in the
CRC patients. Although the bacterial family and genera proportions differed between the
different time points, they were not significantly altered by the RCT treatment (Wilcoxon
test p > 0.05), apart from the genera Fusobacterium (q = 0.015), Escherichia (q = 0.04) and
Klebsiella (q = 0.035), which were significantly decreased after treatment, and the genus Bifi-
dobacterium (q = 0.049), which was significantly increased at T3 compared to T0 (Figure 3). 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to
Neoadjuvant RCT in CRC Patients To evaluate the relationship between the microbial community and the treatment
outcome, we classified the patients based on their response to RCT into categories such as
responders (R) and non-responders (NR). As shown in Table 1, no significant differences
in terms of stage of cancer, sex, age, and BMI were observed between the study groups
(R vs. NR). 5 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 1. Relative abundance at phylum (A) and genera (B) levels of differentially abundant bacteria in the stool samples
CRC patients at baseline (CRC-T0) and healthy controls (HC). * p < 0.05, ** p < 0.001. Figure 1. Relative abundance at phylum (A) and genera (B) levels of differentially abundant bacteria in the stool samples of
CRC patients at baseline (CRC-T0) and healthy controls (HC). * p < 0.05, ** p < 0.001. 6 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 2. Gut microbiota diversity and richness at baseline and during RTC treatment and post-
treatment points in CRC patients. (A) Shannon index (p = 0.75); (B) Chao1 index (p = 0.61); (C) princi-
pal component analysis representation based on Bray–Curtis distance matrix of patient distribution
based on bacterial genera composition at baseline and during RTC treatment and at post-treatment
points (p = 0.716). The first two coordinates are plotted with the percentage of variability, which is
explained and indicated on the axis. Figure 2. Gut microbiota diversity and richness at baseline and during RTC treatment and post-
treatment points in CRC patients. (A) Shannon index (p = 0.75); (B) Chao1 index (p = 0.61); (C) princi-
pal component analysis representation based on Bray–Curtis distance matrix of patient distribution
based on bacterial genera composition at baseline and during RTC treatment and at post-treatment
points (p = 0.716). The first two coordinates are plotted with the percentage of variability, which is
explained and indicated on the axis. 7 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 3. Heatmap diagram of the gut microbiota composition at different taxa levels for baseline (CRC-T0), treatment
points with neoadjuvant RCT (CRC-T1, CRC-T2 and CRC-T3), and the healthy control subjects (HC). The 29 phylum and
genera that were shared by all of the tested samples (core microbiome) are displayed. Figure 3. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to
Neoadjuvant RCT in CRC Patients Heatmap diagram of the gut microbiota composition at different taxa levels for baseline (CRC-T0), treatment
points with neoadjuvant RCT (CRC-T1, CRC-T2 and CRC-T3), and the healthy control subjects (HC). The 29 phylum and
genera that were shared by all of the tested samples (core microbiome) are displayed. Figure 3. Heatmap diagram of the gut microbiota composition at different taxa levels for baseline (CRC-T0), treatment
points with neoadjuvant RCT (CRC-T1, CRC-T2 and CRC-T3), and the healthy control subjects (HC). The 29 phylum and
genera that were shared by all of the tested samples (core microbiome) are displayed. An analysis of the alpha diversity at T3 revealed that the R group had higher diversity
(Shannon index, q < 0.001; Simpson index, q = 0.039) and richness that the NR group (Chao1
index, q = 0.015) at genus level (Figure 4A,B). Furthermore, the ordination plot based on
Bray–Curtis dissimilarities and the Jaccard index showed different intestinal microbial
compositions at the genus level between both the R and the NR groups at T3 (Bray–Curtis
index, q = 0.038; Jaccard index, q = 0.035; non-parametric ANOSIM test) (Figure 4C). Next, we searched for differentially abundant taxa in the gut microbiome of R versus
NR at T3. The analysis revealed that at the phylum level, there was a significant enrichment
in the Actinobacteria (q = 0.0025) and Firmicutes (q = 0.0017) populations and a significant
decrease in the Fusobacterias (q = 0.025) and Proteobacterias (q = 0.037) populations in the
R group in comparison to the NR group (Figure 5A,B). At the family level, a significantly
higher abundance of Ruminococcaceae (q = 0.004) and Bifidobacteriaceae (q = 0.03) accom-
panied with a significantly lower abundance of Prevotellaceae (q = 0.045), Enterobactericeae
(q = 0.027), and Fusobacteriaceae (q = 0.014) were shown in the R group compared to in the
NR group (Figure 5A,C). 8 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 4. Comparison of alpha and beta diversity in CRC patients according to their response to therapy. (A) Shannon
index; (B) Chao1index; (C) principal component plot based on the Bray–Curtis distance matrix and the Jaccard indices from
the responder (R) and non-responder (NR) patients at genus-level. The first two coordinates are plotted with the percentage
of variability, which is explained and indicated on the axis. Figure 4. Comparison of alpha and beta diversity in CRC patients according to their response to therapy. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to
Neoadjuvant RCT in CRC Patients (A) Shannon
index; (B) Chao1index; (C) principal component plot based on the Bray–Curtis distance matrix and the Jaccard indices from
the responder (R) and non responder (NR) patients at genus level The first two coordinates are plotted with the percentage Figure 4. Comparison of alpha and beta diversity in CRC patients according to their response to therapy. (A) Shannon
index; (B) Chao1index; (C) principal component plot based on the Bray–Curtis distance matrix and the Jaccard indices from
the responder (R) and non-responder (NR) patients at genus-level. The first two coordinates are plotted with the percentage
of variability, which is explained and indicated on the axis. In addition, at the genera level, we identified a significant increase in Ruminococcus
(q = 0.035), Bilophila (q = 0.008), Collinsiella (q = 0.015), Bifidobacterium (q = 0.024), Roseburia
(q = 0.032), and Faecalibacterium (q = 0.041) in R patients with respect to the NR, while a
significant increase in Prevotella (q = 0.05), Fusobacterium (q = 0.045), Escherichia (q = 0.037),
Bacteroides (q = 0.027), and Klebsiella (q = 0.035) were observed in the NR patients compared
to the R group (Figure 6A,B). Finally, at the species level, we found a significant overabun-
dance of Prevotella copri (q < 0.001), Escherichia coli (q = 0.029), Fusobacterium nucleatum
(q = 0.015), and Bacteroides fragilis (q = 0.029) in the NR group, while the R group displayed
a significantly higher abundance of Bifidobacterium bifidum (q = 0.043), Ruminococcus albus
(q = 0.019), Collinsella aerofaciens (q = 0.018), and Faecalibacterium prausnitzii (q = 0.027). 2.5. Baseline Microbiota Composition Could Predict Response to RCT Treatment in CRC Patients After describing the significant differences in the intestinal microbial composition
between the R and NR after RCT treatment, we next assessed the predictive power of the
gut microbiome related to neoadjuvant RCT response. We used random forest (RF) to
build a predictive model based on the overall gut microbiota profile using the species-level
abundance data as input. After RF analysis with 500 bootstrap samples, we found that Int. J. Mol. Sci. 2021, 22, 9549 9 of 25 9 of 25 the overall gut microbiota composition data had a significant accuracy of 80% and an
area under the curve (AUC) of 0.71. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to
Neoadjuvant RCT in CRC Patients The main species accounting for this stratification
were Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium
nucleatum, and Bacteroides fragilis, and when the proportions of these bacterial species were
only used for testing the accuracy of the RF classifier, this increased to 96% (AUC = 0.92). Thus, the response to RTC or the lack of it were identified with an accuracy of 94%
(AUC = 0.95) and of 91% (AUC = 0.92), respectively (Figure 7A). The validation cohort
consisted of 84 CRC patients under neoadjuvant RCT (45 R patients and 39 NR patients)
(data collected from the Genome Sequence Archive in National Genomics Data Center,
accession number CRA002850). After RF analysis in this validation cohort, an accuracy
of 92.0% (AUC = 0.93) and 90.0% (AUC = 0.91) were obtained for the response to RTC
or the lack of it, respectively (Figure 7B). Among the five species variables, Fusobacterium
nucleatum, and Bacteroides fragilis were biomarkers of R patients, and Ruminococcus albus,
Bifidobacterium bifidum, and Faecalibacterium prausnitzii were biomarkers of NR patients. Figure 5. Cont. Figure 5. Cont. Figure 5. Cont. 10 of 25 Int. J. Mol. Sci. 2021, 22, 9549 e 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non-
nder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the
amples of N patients compared to the NR patients. * p < 0.05. Figure 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non-
responder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the
stool samples of N patients compared to the NR patients. * p < 0.05. Figure 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non-
responder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the
stool samples of N patients compared to the NR patients. * p < 0.05. Figure 5. Heatmap of the fecal microbiota composition at the phylum and family levels in the responder (R) and non-
responder (NR) patients (A). Relative abundance at phylum (B) and family (C) levels of differentially abundant OTUs in the
stool samples of N patients compared to the NR patients. * p < 0.05. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to
Neoadjuvant RCT in CRC Patients 11 of 25 Int. J. Mol. Sci. 2021, 22, 9549 gure 6. Heatmap of the fecal microbiota composition at genera level in the responder (R) and non-responder (NR) patient
A). Relative abundance at genera level of differentially abundant OTUs in the stool samples of the N patients compared t
e NR patients. * p < 0.05 (B). Figure 6. Heatmap of the fecal microbiota composition at genera level in the responder (R) and non-responder (NR) patients
(A). Relative abundance at genera level of differentially abundant OTUs in the stool samples of the N patients compared to
the NR patients. * p < 0.05 (B). 12 of 25 12 of 25 Int. J. Mol. Sci. 2021, 22, 9549 Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed using microbial
variables (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides
fragilis). (A) Training cohort. The area under the ROC curve (AUC) was 0.95, and the 95% confidence interval (CI) was
0.901–1 for the R patients (green), and the AUG was 0.92 and 95% the IC was 0.827–1 for the NR patients (red). (B) Validation
cohort. The AUG was 0.93 and the 95% IC was 0.877–0.987 for the R patients (green), and the AUG was 0.91 and 95% the IC
was 0.835–0.984 for the NR patients (red). er operating characteristic (ROC) curve based on the random forest classifier constructed using microbial Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed using microbial
variables (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides
fragilis). (A) Training cohort. The area under the ROC curve (AUC) was 0.95, and the 95% confidence interval (CI) was
0.901–1 for the R patients (green), and the AUG was 0.92 and 95% the IC was 0.827–1 for the NR patients (red). (B) Validation
cohort. The AUG was 0.93 and the 95% IC was 0.877–0.987 for the R patients (green), and the AUG was 0.91 and 95% the IC
was 0.835–0.984 for the NR patients (red). Figure 7. Receiver operating characteristic (ROC) curve based on the random forest classifier constructed using microbial
variables (Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nucleatum, and Bacteroides
fragilis). (A) Training cohort. 2.4. Post-Treatment Microbiota Diversity and Composition Is Associated to Clinical Response to
Neoadjuvant RCT in CRC Patients The area under the ROC curve (AUC) was 0.95, and the 95% confidence interval (CI) was
0.901–1 for the R patients (green), and the AUG was 0.92 and 95% the IC was 0.827–1 for the NR patients (red). (B) Validation
cohort. The AUG was 0.93 and the 95% IC was 0.877–0.987 for the R patients (green), and the AUG was 0.91 and 95% the IC
was 0.835–0.984 for the NR patients (red). 2.6. Differences in the Gut Microbiota Functions between Responder and Non-Responder KEGG pathway enrichment analysis of the metagenomic data showed that genes for
energy metabolism such as methane metabolism (q < 0.004), carbohydrate metabolism, such
as the pentose phosphate pathway (q = 0.0022), pyruvate metabolism (q-value < 0.001),
starch and sucrose metabolism (q = 0.008), galactose metabolism (q = 0.007), butanoate
metabolism (q = 0.005), and glycolysis-gluconeogenesis (q = 0.0028); for xenobiotic biodegra-
dation and metabolism pathways, including benzoate degradation (q = 0.038) and nitro-
toluene degradation (q = 0.005); and membrane transport, such as ABC transporters
(q = 0.012) and transporters (q = 0.012), were significantly depleted in NR compared to
R patients. Nevertheless, compared to the R patients, in the NR patients, there was a signif-
icant over-representation of genes for lipid metabolism, such as for araquidonic acid
metabolism (q = 0.006); amino acid metabolism pathways, such as for arginine and pro-
line metabolism (q = 0.029); for glycine, serine, and threonine metabolism (q = 0.001); in
genes for the metabolism of other amino acids such as glutathione metabolism (q = 0.003);
for the metabolism of cofactors and vitamins such as riboflavin metabolism (q = 0.003),
ubiquinone, and other terpenoid metabolism (q < 0.001); folate biosynthesis (q = 0.014),
glycan biosynthesis, and metabolism, such as lipopolysaccharide biosynthesis (q = 0.007);
lipopolysaccharide biosynthesis proteins (q = 0.001); cellular processes and signaling that
contain cell motility and secretion (q = 0.0018); oxidative phosphorylation (q < 0.001); and
for pathways in cancer (q < 0.001) (Figure 8). 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients Significant differences in the serum levels of several polyamines and acetyl derivatives
of polyamines were found in the R and NR patients at post-treatment point (T3). Then,
in the NR patients, we found a significant increase in the levels of spermine, N1-acetyl
spermine (N1-AcSP), N1, N12-diacetylspermine (N1, N12-DiAcSP), N1-acetylspermidine Int. J. Mol. Sci. 2021, 22, 9549 13 of 25 13 of 25 (N1-AcSPD), N1, N8- diacetylspermidine (N1, N8-DiAcSPD), and N1-acetylputrescine
(N1-AcPUT) compared to those in the R patients. On the other hand, within-group, there
were also significant changes in the levels of N1-AcSPD and spermine in both the R and
NR patients and in the serum levels of N8-AcSPD only in the NR group (Table 2). Figure 8. Heatmap of bacterial gene functional predictions using the PICRUSt algorithm from the
fecal samples from the responder (R) patients and the non-responder (NR) patients. Table 2. Serum polyamines levels at baseline (T0) and post-treatment (T3). R Patients
(N = 28)
NR Patients
(N = 12)
Between-Group
Difference 1
p 2
Agmatine (ng/mL)
Baseline
Post-treatment
Change
0.11 ± 0.13
0.25 ± 0.24
0.14 (−0.27, −0.13)
0.13 ± 0.15
0.17 ± 0.15
0.035 (−0.13, 0.061)
0.025 (−0.11, 0.63)
0.571
Arginine (µg/mL)
Baseline
Post-treatment
Change
23.18 ± 4.20
22.82 ± 4.16
−0.36 (−1.5, 2.27)
24.54 ± 4.76
23.10 ± 4.48
−1.43 (−1.13, 4.0)
−1.35 (−4.05, 1.35)
0.319 Table 2. Serum polyamines levels at baseline (T0) and post-treatment (T3). Int. J. Mol. Sci. 2021, 22, 9549 14 of 25 Table 2. Cont. Table 2. Cont. Table 2. Cont. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients R Patients
(N = 28)
NR Patients
(N = 12)
Between-Group
Difference 1
p 2
Ornithine (µg/mL)
Baseline
Post-treatment
Change
19.46 ± 5.74
20.21 ± 4.16
0.74 (−3.69, 2.19)
23.31 ± 8.06
22.80 ± 7.55
−0.51 (−3.72, 4.74)
−3.85 (−8.07, 0,37)
0.073
N1,N12-diacetylspermine (ng/mL)
Baseline
Post-treatment
Change
1.08 ± 0.43
0.90 ± 0.52
−0.18 (0.017, 0.34)
1.68 ± 1.34
1.22 ± 0.57
0.46 (−0.152, 1.07)
−0.59 (−1.20, 0.06)
0.015
N1,N8-diacetylspermidine (ng/mL)
Baseline
Post-treatment
Change
0.71 ± 0.26
0.74 ± 0.34
0.03 (−0.13, 0.059)
0.99 ± 1.03
0.88 ± 0.38
−0.11 (−0.34, 0.57)
−0.28 (−0.74, 0.17)
0.007
N1-acetylspermidine (ng/mL)
Baseline
Post-treatment
Change
22.47 ± 7.10
23.42 ± 8.26
0.94 (−3.88, 1.99) *
27.68 ± 13.47
28.89 ± 10.38
1.20 (−6.10, 3.68) *
−5.21 (−11.73, 1.3)
0.021
N8-acetylspermidine (ng/mL)
Baseline
Post-treatment
Change
14.52 ± 3.48
14.69 ± 3.39
0.16 (−0.90, 0.57)
14.88 ± 3.27
16.10 ± 2.33
1.22 (−2.42, −0.20) *
−0.35 (−2.38, 1.67)
0.727
N1-acetylputrescine (ng/mL)
Baseline
Post-treatment
Change
5.04 ± 1.60
4.77 ± 1.70
−0.27 (−1.78, 1.09)
5.92 ± 5.38
5.39 ± 3.79
−0.53 (−1.01, 3.32)
−0.88 (−3.29, 1.53)
0.030
Putrescine (ng/mL)
Baseline
Post-treatment
Change
8.84 ± 4.40
8.06 ± 3.89
−0.78 (−0.39, 1.96)
7.95 ± 3.52
7.47 ± 3.09
−0.47 (−1.07, 2.02)
0.89 (−1.49, 3.28)
0.457
Spermidine (ng/mL)
Baseline
Post-treatment
Change
17.14 ± 7.19
20.42 ± 12.40
3.28 (−7.42, 0.85)
22.26 ± 12.69
20.90 ± 10.81
−1.35 (−2.01, 4.73)
−4.11 (−11.36, 1.12)
0.106
N1-acetylspermine (ng/mL)
Baseline
Post-treatment
Change
0.89 ± 0.33
1.19 ± 0.63
0.29 (−0.55, −0.046)
1.48 ± 0.70
1.33 ± 0.62
−0.14 (−0.11, 0.40)
−0.58 (−0.92, −0.25)
0.014
Spermine (ng/mL)
Baseline
Post-treatment
Change
3.77 ± 1.30
4.80 ± 2.88
1.03 (−2.17, 0.107) *
12.10 ± 7.85
7.35 ± 3.66
−4.74 (1.71, 7.77) *
−7.32 (−11.74, −4,89)
0.001
Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-
MS/MS). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and
post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-
MS/MS). 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients NR Patients
(N = 12)
Between-Group
Difference 1
p 2
0.71 ± 0.15
0.77 ± 0.17
0.06 (−0.30, 0.18)
0.26 (−0.03, 0.56)
0.012
2.02 ± 1.35
1.70 ± 1.52
−0.32 (−0.9, 0.36)
−0.68 (−0.86, 1.76)
0.102
0.93 ± 0.68
1.02 ± 1.07
0.09 (−0.65, 1.34)
1.33 (−0.04, 2.71)
0.016
0.31 ± 0.33
0.44 ± 0.15
−0.13 (−0.23, 0.76)
0.15 (0.03, 0.26)
0.010
0.61 ± 0.32
0.29 ± 0.19
-0.47 (−0.58, 0.76)
−0.25 (−0.38, 0.29)
0.002
0.90 ± 0.44
0.39 ± 0.24
−0.51 (0.66, 1.02)
−0.18 (−0.45, 0.29)
0.009
0.37 ± 0.64
0.04 ± 0.01
−0.33 (−0.47, 0.86)
0.20 (−0.35, 0.10)
0.216
0.11 ± 0.08
0.05 ± 0.09
−0.05 (−0.07, 0.13)
0.05 (−0.19, 0.13)
0.007
0.07 ± 0.06
0.05 ± 0.01
−0.02 (−0.04, 0.08)
0.02 (−0.07, 0.04)
0.171
272.6 ± 35.1
298.4 ± 47.5
25.2 (11.3, 37.1)
−22.2 (−37.4, 10.2)
0.004
gas chromatography coupled with a flame-ionization detector
r; NR: non-responder. 1 Difference between R and NR patients
treatment changes was conducted with a covariance model
calculate differences in the SCFAs and zonulin between the
tatistically significant. Table 3. Fecal SCFAs concentrations and serum zonulin levels at baseline (T0) and post-treatment (T3). Table 3. Fecal SCFAs concentrations and serum zonulin levels at baseline (T0) and post-treatment (T3). 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients R Patients
(N = 28)
NR Patients
(N = 12)
Between-Group
Difference 1
p 2
Acetic acid (mg/g)
Baseline
Post-treatment
Change
0.83 ± 0.39
1.04 ± 0.40
0.20 (−0.39, 0.31) *
0.71 ± 0.15
0.77 ± 0.17
0.06 (−0.30, 0.18)
0.26 (−0.03, 0.56)
0.012
Propionic acid (mg/g)
Baseline
Post-treatment
Change
1.40 ± 1.27
1.01 ± 1.10
−0.39 (−0.51, 0.59)
2.02 ± 1.35
1.70 ± 1.52
−0.32 (−0.9, 0.36)
−0.68 (−0.86, 1.76)
0.102
Butyric acid (mg/g)
Baseline
Post-treatment
Change
1.37 ± 0.45
2.36 ± 1.82
0.99 (−1.2, 2.15) *
0.93 ± 0.68
1.02 ± 1.07
0.09 (−0.65, 1.34)
1.33 (−0.04, 2.71)
0.016
Isobutyric acid (mg/g)
Baseline
Post-treatment
Change
0.58 ± 0.33
0.69 ± 0.05
0.11 (0.07, 0.21)
0.31 ± 0.33
0.44 ± 0.15
−0.13 (−0.23, 0.76)
0.15 (0.03, 0.26)
0.010
Valeric acid (mg/g)
Baseline
Post-treatment
Change
0.30 ± 0.16
0.13 ± 0.07
−0.17 (−0.27, 0.39)
0.61 ± 0.32
0.29 ± 0.19
-0.47 (−0.58, 0.76)
−0.25 (−0.38, 0.29)
0.002
Isovaleric acid (mg/g)
Baseline
Post-treatment
Change
0.50 ± 0.49
0.20 ± 0.13
−0.30 (−0.43, 0.31)
0.90 ± 0.44
0.39 ± 0.24
−0.51 (0.66, 1.02)
−0.18 (−0.45, 0.29)
0.009
4-methylvaleric acid (mg/g)
Baseline
Post-treatment
Change
0.13 ± 0.23
0.07 ± 0.10
−0.06 (−0.09, 0.15)
0.37 ± 0.64
0.04 ± 0.01
−0.33 (−0.47, 0.86)
0.20 (−0.35, 0.10)
0.216
Hexanoic acid (mg/g)
Baseline
Post-treatment
Change
0.15 ± 0.20
0.10 ± 0.10
−0.04 (−0.09, 0.10)
0.11 ± 0.08
0.05 ± 0.09
−0.05 (−0.07, 0.13)
0.05 (−0.19, 0.13)
0.007
Heptanoic acid (mg/g)
Baseline
Post-treatment
Change
0.09 ± 0.15
0.06 ± 0.06
−0.03 (−0.06, 0.07)
0.07 ± 0.06
0.05 ± 0.01
−0.02 (−0.04, 0.08)
0.02 (−0.07, 0.04)
0.171
Zonulin (ng/mL)
Baseline
Post-treatment
Change
257.6 ± 65.4
218.1 ± 76.4
−39.3 (−52.2, 23.9)
272.6 ± 35.1
298.4 ± 47.5
25.2 (11.3, 37.1)
−22.2 (−37.4, 10.2)
0.004
Short-chain fatty acids (SCFAs) in fecal samples were analyzed by means of gas chromatography coupled with a flame-ionization detector
(GC-FID). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in the SCFAs and zonulin between the
baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and
post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-
MS/MS). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and
post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Serum polyamine levels were measured by means of ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-
MS/MS). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in polyamines between baseline and
post-treatment in R and NR patients. p < 0.05 was considered statistically significant. SCFAs are bacterial-derived metabolites with important physiological functions in the
host and that have anti-cancer properties. Analysis of the fecal levels of SCFAs revealed
significant differences in the concentrations of acetic, butyric, isobutyric, valeric, isovaleric,
and hexanoic acid between the R and NR study groups at post-treatment time point T3. Moreover, we found several significant differences in the within-group comparison of the
fecal concentrations of acetic and butyric acid, which significantly increased after RCT
treatment in the R group. On the other hand, serum zonulin levels (a circulating marker of
gut permeability) were significantly increased in the NR group (but not in R group) after
RCT treatment (Table 3). Int. J. Mol. Sci. 2021, 22, 9549 15 of 25 vels at baseline (T0) and post-treatment (T3). 3. Discussion In this study, we have demonstrated the existence of a significant association between
the gut microbiota and the anti-cancer response of CRC patients treated with neoadjuvant
RCT. Moreover, we have found that some microbial-derived metabolites such as SCFAs
could be at least partially responsible for the response to RCT in these CRC patients. Finally,
we have identified a baseline consortium of CRC-enriched bacterial species that may
potentially serve as diagnostic bacterial markers of a good or bad response to neoadjuvant
RCT. Where Ruminococcus albus, Bifidobacterium bifidum, and Faecalibacterium prausnitzii,
were overrepresented in R patients and chosen as discriminatory variables in our response-
prediction RF model, Fusobacterium nucleatum and Bacteroides fragilis were overrepresented
in the NR patients. The loss of microbial diversity has been associated with chronic health conditions [25–27]
and cancer [27,28] as well as with poor outcomes to certain forms of cancer therapy [29–31]. Accordingly, recent works have also reported that patients with CRC display a lower
bacterial diversity and richness in fecal samples and the intestinal mucosa compared to
healthy individuals [32,33]. In this study, we found that compared to healthy controls, the
CRC microbiota exhibited a state of dysbiosis with a reduced overall bacterial richness
and diversity. In addition, the analysis of the Bray–Curtis PCoA plot for beta diversity
revealed that the CRC patients were clustered separately to the healthy controls, suggesting
important CRC-mediated microbial changes. Related to gut microbiota composition, several microbes have been found to be differ-
entially represented in fecal samples between both study groups. Thus, the gut microbiota
in the CRC patients was enriched with pro-inflammatory opportunistic pathogens and
was depleted in butyrate-producing bacteria, which have been shown to be essential for
the preservation of intestinal homeostasis [34]. In particular, we have shown that some
bacteria such as Fusobacterium nucleatum, Escherichia coli, and Bacteroides fragilis had high
prevalence in CRC patients in comparison to the healthy controls, whereas genera such as
Roseburia, Faecalibacterium, and Bifidobacterium were depleted, demonstrating that microbial
dysbiosis was already present in CRC at the time of diagnosis. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients (r = 0.547 p = 0.014; r = 0.752 p < 0.001) in the NR patients. Finally, Prevotella copri was
positively associated with the serum zonulin levels in NR patients. 2.7. Changes in the Serum Level of Polyamines and Zonulin and Fecal Levels of SCFAs after RCT
Treatment in CRC Patients Short-chain fatty acids (SCFAs) in fecal samples were analyzed by means of gas chromatography coupled with a flame-ionization detector
(GC-FID). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in the SCFAs and zonulin between the
baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. Short-chain fatty acids (SCFAs) in fecal samples were analyzed by means of gas chromatography coupled with a flame-ionization detector
(GC-FID). Values are expressed as mean ± SD or mean (95% CI). R: responder; NR: non-responder. 1 Difference between R and NR patients
at post-treatment when adjusted for baseline. 2 Comparison among post-treatment changes was conducted with a covariance model
(ANCOVA) adjusted for baseline. * Wilcoxon signed-rank test was used to calculate differences in the SCFAs and zonulin between the
baseline and post-treatment in R and NR patients. p < 0.05 was considered statistically significant. In addition, pairwise comparisons using Spearman rank correlation analysis were
then performed between bacterial species enriched in the gut microbiome of both the R and
NR patients and the fecal SCFAs and serum polyamines and zonulin levels. Interestingly,
we found a statistically significant positive correlation between the fecal levels of butyrate
and the abundance of the Faecalibacterium prausnitzii (r = 0.816 p < 0.001) and Ruminoccocus
albus (r = 0.924 p = 0.008) in the R group and between the concentration of propionic acid
and Bacteroides fragilis in the NR group. In addition, negative associations of Faecalibacterium
prausnitzii with the serum levels of spermine (r = −0.619 p = 0.018) and N1,N12-DiAcSP
(r = −0.793 p = 0.01) in the R patients were described, while there was a positive association
between the abundance of Bacteroides fragilis and Fusobacterium nucleatum with the levels
of N1,N12-DiAcSP (r=0.436 p = 0.043; r = 0.637 p = 0.001, respectively) and N8-AcSPD Int. J. Mol. Sci. 2021, 22, 9549 16 of 25 (r = 0.547 p = 0.014; r = 0.752 p < 0.001) in the NR patients. Finally, Prevotella copri was
positively associated with the serum zonulin levels in NR patients. 3. Discussion On the other hand, we observed that gut microbiota composition was relatively stable
over treatment time following RCT treatment, with the exception of a significant decrease
in the abundance of Fusobacterium, Escherichia, and Klebsiella and a significant increase in
Bifidobacterium (probiotic bacteria) at post-treatment time compared to at baseline, show-
ing the beneficial effect of RCT on the gut microbiome of CRC patients. Klebsiella and
Fusobacterium are pathogens normally found in the human intestine that cause diarrhea
and bloodstream infections and that considerably increase the rates of treatment failure
and death [35]. After treatment, the CRC patients were classified as responders (N) versus non-
responders (NR), based on their good or poor response to the RCT. Interestingly, we
found significant differences in the alpha diversity at the genus level, with an increase in
the diversity (Shannon) and richness (Chao 1) in the R patients compared to in the NR
patients. Similarly, there was a statistically significant difference in B-diversity (Bray–Curtis
dissimilarities and Jaccard index), finding a notable clustering effect by response status in
the gut microbiome of these patients, indicating a difference in the bacterial community
composition between the R and NR patients. At the taxa levels, we found a significant enrichment in probiotic and butyrate
producer-bacteria such as Bifidobacterium bifidum, Ruminoccous albus, Roseburia, and Faecal-
ibacterium prausnitzii in the R patients, while the NR patients showed an enrichment in
unfavorable microbial taxa such as Fusobacterium nucleatum, Bacteroides fragilis, Escherichia
coli, Prevotella copri, and Klebsiella. Several studies have shown that butyrate-producing
bacteria are negatively related to irritable bowel disease and colorectal cancer [36,37]. Int. J. Mol. Sci. 2021, 22, 9549 17 of 25 17 of 25 Additionally, both Fusobacterium and Prevotella have been related to recurrent CRC
after chemotherapy. Given that Fusobacterium nucleatum has been previously correlated
with chemoresistance [17], our results may suggest that the higher load of Fusobacterium
nucleatum present in NR patients could be a potential promoter of CRC chemoresistance
and therefore of a poor response to CRC treatment. Similarly, the enterotoxigenic Bacteroides
fragilis, which was also enriched in the NR patients, is a significant source of chronic inflam-
mation, and it has previously been associated with the development and aggressiveness
of colorectal cancer and poor patient outcome [6,38]. 3. Discussion These data also suggest that the gut
microbiota composition of the R patients shifted towards a microbial profile that has great
similarity to the gut microbiota of the healthy controls. Next, we sought to gain insight into the mechanism through which the gut micro-
biome may influence response to RCT. Regarding the metabolic function of gut microbiota,
in the current study, Picrust analysis showed significant differences between the R and
NR patients. In the NR patients, we have found an increase in the abundance of genes
for lipopolysaccharide biosynthesis as well as for araquidonic acid metabolism, for glu-
tathione metabolism, and for the amino acid metabolism pathways (such as arginine and
proline metabolism) compared to in the R patients. The significant increase of genes for
lipopolysaccharide biosynthesis could be related to the significant increase in the abun-
dance of Gram-negative bacteria such as Escherichia coli in the NR patients; these bacteria
contain specific enzymes that produce LPS, which can induce Toll-like receptor 4 signal-
ing and can promote cell survival and proliferation in CRC patients [39]. Similarly, the
arachidonic acid pathway is important in the development and progression of numerous
malignant diseases, including CRC, due to the fact that araquidonic acid stimulates key
downstream signaling cascades that regulate cell proliferation, apoptosis, angiogenesis, in-
flammation, and immune surveillance [40,41]. With respect to the increase in the genes for
glutathione metabolism in NR patients, some studies have described that the elevated lev-
els of glutathione in tumor cells are able to protect such cells in bone marrow, breast, colon,
larynx, and lung cancers by conferring resistance to several chemotherapeutic drugs [42,43]. Other bacterial functions involving the metabolism of cofactors and vitamins and the en-
ergy production pathways such as oxidative phosphorylation were also increased in NR
patients. These pathways may serve as alternative bioenergetic sources for metabolically
stressed cancer cells [44]. Remarkably, a recent metagenomic analysis reported that the CRC-associated micro-
biome showed an association with the conversion of amino acids into polyamines (e.g., the
biosynthesis of putrescine from the amino acids L-arginine and L-ornithine), indicating
that these metabolites could be particularly important in CRC development and progres-
sion [45]. In our study, significant differences in the serum levels of several polyamines and
acetyl derivatives of polyamines were found between R and NR patients at post-treatment
point. 3. Discussion Moreover, we observed that the abundance of N1,N12-DiAcSP and N8-AcSPD were
positively associated with the increased abundance of Bacteroides fragilis and Fusobacterium
nucleatum in NR patients. p
In fact, Bacteroides spp. and Fusobacterium spp. can synthesize putrescine and
spermidine in vitro and in vivo [46]. Goodwin et al. demonstrated that the purified
Bacteroides fragilis toxin (BFT) up-regulates spermine oxidase in HT29/c1 and T84 colonic
epithelial cells, producing the spermine oxidase-dependent generation of ROS and the
induction of a marker of DNA damage such as γ-H2A.x. [47]. In another study, Johnson
et al. found that antibiotic treatment led to a lower tissue concentration of N1, N12-
diacylspermine and that a disturbed bacterial biofilm was observed in resected CRC tissues
compare to CRC tissues with negative bacterial biofilm, suggesting the implication of gut
microbes in the increase of host generated N1, N12-diacetylspermine [48]. Moreover, the
activation of the amino acid metabolic pathways by the intestinal microbiota of the NR
patients could contribute to the increase in polyamines, which are actively assimilated
by the cells of the intestinal epithelium and induce rapid cell proliferation, favoring the
tumorigenesis [49,50]. Int. J. Mol. Sci. 2021, 22, 9549 18 of 25 18 of 25 On the other hand, several works performed in both cellular and animal models have
demonstrated that CRC is linked to alterations in the metabolism of SCFAs, which have
been shown to exhibit potential anti-carcinogenic effects [51,52]. Here, we have found
that R patients displayed a significant over-representation of genes involved in butanoate
metabolism and a significant increase in the fecal abundance of several SCFAs such as
acetic and butyric acid after RCT treatment. Moreover, there was a positive correlation
between the fecal levels of butyrate and the abundance of Faecalibacterium prausnitzii and
Ruminoccocus albus in these patients. Faecalibacterium praustnitzi is considered important
in health promotion, as it is able to produce butyrate from dietary fibre and possesses
anti-inflammatory properties [53]. A decrease in Faecalibacterium prausnitzii and butyrate
levels defines microbiota dysbiosis in patients suffering inflammatory bowel disease [54]. In addition, Faecalibacterium is able to use the acetate produced by Bifidobacterium (also
increased in N patients) with the subsequent modulation of the intestinal mucus barrier
by the modification of goblet cells and mucin glycosylation [55]. Butyrate is required for
colonic epithelium repair and the production of Treg cells, which regulate the local immune
response and suppress colonic inflammation and carcinogenesis [56]. 3. Discussion Moreover, butyrate
has been described to be able to induce the production of IL-18 by the intestinal epithelial
cells through the activation of the GPR109a receptor, which stimulates mucosal tissue
repair via the regulation of the production and availability of IL-22 [57]. The absence of
IL-18 has been associated with gut microbiota dysbiosis, a dysregulation of the homeostatic
and mucosal repair and alteration of the inflammatory response, producing an increased
susceptibility to carcinogenesis [58]. In addition, after RCT treatment, we found a significant
decrease in the fecal levels of acetic, butyric, isobutyric, and hexanoic acid in the NR study
group compared to in R patients, indicating the exhaustion of butyric acid-producing
microbiota in their colon. In a previous study, hexanoic acid was shown to reduce the
colonization and dysbiotic expansion of potentially pathogenic bacteria in the gut [59]. y
p
p
y p
g
g
Finally, we found that plasma zonulin levels were significantly increased in the NR
patients compared to in the R patients. A higher zonulin level was correlated with the
relaive abundance of Prevotella copri in the R patients. Zonulin is a protein synthesized
in intestinal and liver cells that reversibly modulates the intestinal permeability of the
intestinal epithelial barrier by modulating intercellular tight junctions [60]. Wright et al. found that Prevotella contains key enzymes implicated in mucin degradation, which are able
to disrupt the colonic mucus barrier. A disrupted mucosal barrier may result in increased
intestinal permeability, which allows the diffusion of antigens, toxins, and pathogens
from the luminal environment into the mucosal tissues and circulatory system [55]. As
a consequence, an inflammatory response can be triggered that induces cancer initiation,
progression, and response to anticancer treatment [61]. Then, the significant increase in
Prevotella abundance found in our study could be associated in party with the poor or
non-response to RCT in NR patients. This study has some limitations, such as the relatively small sample size, which could
reduce the power of the study. However, despite the relatively small size of our study, sta-
tistically significant differences were observed, suggesting that the results presented herein
provide solid evidence on the potential contribution of the gut microbiome to RCT out-
comes in CRC patients. 3. Discussion Moreover, our study also has several strengths, such as the careful
design, the well-matched cohorts of CRC patients and controls, a complete definition of the
inclusion and exclusion criteria, and the consideration of lifestyle-associated confounding
factors that may affect the gut microbiota composition, such as dietary pattern. 4.3. DNA Extraction and Gut Microbiota Sequencing The frozen fecal samples were thawed at 4 ◦C to avoid dramatic temperature changes
that might affect bacterial DNA integrity. Afterwards, the fecal samples were manually
homogenized for 30 s with a sterile plastic scoop, and aliquots of 200 mg were used for DNA
extraction using the QIAamp DNA Stool Mini kit following the manufacturer’s instructions
(Qiagen, Hilden, Germany). DNA concentration (A260) and purity (A260/A280 ratio) were
estimated with a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington,
DE, USA). DNA was amplified using the Ion 16S Metagenomics kit (Thermo Fisher Scientific,
Madrid, Spain), which contains a primer pool to amplify multiple variable regions (V2,
3, 4, 6–7, 8 and 9) of the 16S rRNA gene. The Ion PlusTM Fragment Library Kit (Thermo
Fisher Scientific, Madrid, Spain) was used to ligate the barcoded adapters to the generated
amplicons and to create the barcoded libraries, which were pooled and templated on the
automated Ion Chef system (Thermo Fisher Scientific, Madrid, Spain). The sequencing was
done on an Ion S5 platform (Thermo Fisher Scientific, Madrid, Spain). 4.2. Laboratory Measurements Fasting venous blood samples were collected, and serum was separated in aliquots and
was immediately frozen at −80 ◦C. Serum levels of glucose, total cholesterol, triglycerides,
HDL-cholesterol, and LDL-cholesterol were measured in duplicate using a Dimension
autoanalyzer (Dade Behring Inc., Deerfield, IL, USA) using enzymatic methods (Randox
Laboratories Ltd. Ardmore, UK). 4. Materials and Methods
4.1. Study Patients A total of forty patients aged 35–75-years-old who were newly diagnosed with CRC
in stages II–III (T2–T4 and/or N1–N2) from the Radiotherapy Oncology Service at the
Virgen de la Victoria Hospital and with no metastatic lesions detected on imaging were
enrolled in the study and were followed-up with for at least 1 year. All of the CRC patients Int. J. Mol. Sci. 2021, 22, 9549 19 of 25 19 of 25 received only neoadjuvant treatment for 5 weeks with pelvic radiation therapy (50 Gy in
fractions of 2 Gy/session) and oral capecitabine (825 mg/m2/12 h) during radiotherapy
treatment. Patients with a history of colorectal cancer or bowel resection, type 2 dia-
betes, chronic inflammatory bowel disease, severe active infection, or hereditary colorectal
cancer syndromes were excluded from the study. Patients who received pelvic cancer
radiation therapy or anti-tumor treatment in the previous 2 years, who used antibiotics
or immunosuppressants in the previous 2 months, or who regularly used non-steroidal
anti-inflammatory drugs, statins, or probiotics before the study were also excluded. A
pathologist examined surgical specimens and tumor response after neoadjuvant RCT was
determined in surgical specimens according to the tumor regression grades (TRG) system
described by Mandard et al. [62]. We divided the CRC patients into TRG1–2 (patients with
good response or responders (R)) and TRG 3–5 (patients with poor or non-response (NR)). Blood and fecal samples were collected at baseline (T0), 2 and 4 weeks after starting RCT
(T1 and T2, respectively), and 7 weeks after finishing treatment (T3). In the study, we also included fecal samples from 20 healthy patients that were
matched with the CRC patients according to sex, age, and BMI. The healthy controls did
not have gastrointestinal tract disorders or other complications and were not administered
antibiotics or probiotics during the 2 months prior to sample collection. The study protocol was approved by the Medical Ethics Committee at the Virgen de
la Victoria University Hospital and was conducted in accordance with the Declaration of
Helsinki. Written informed consent was provided by all study participants. 4.4. Bioinformatics Analysis Analysis of 16S rRNA amplicons was performed using QIIME (2-2019.4 version). The q-dada2 plugin with the DADA2 pipeline was used for the quality filtering and the
denoised, dereplicated, and chimera filtering of the raw sequence data. The sequence
variants obtained through the DADA2 pipeline were merged into a single feature table
using the q2-feature-table plugin. Using the q2-vsearch plugin with 97% sequence simi-
larity, all amplicon sequence variants from the merged feature table were clustered into
OTU’s using the Open Reference Clustering method against Greengenes version 13_8
with 97% similarity from the OTU reference sequences. The OTUs were aligned with Int. J. Mol. Sci. 2021, 22, 9549 20 of 25 MAFFT (via q2-alignment) and were used to construct a phylogeny with fasttree2 (via
q2-phylogeny). The q2-feature-classifier classify-sklearn naive Bayes taxonomy classifier
was used to assign taxonomy to the OTUs. Alpha diversity metrics (Shannon and Chao1),
beta diversity metrics (Bray–Curtis dissimilarity), and principal coordinate analysis (PCoA)
were estimated using a q2-diversity plugin after the samples were rarefied to 994 sequences
per sample. Alpha diversity significance was estimated with Kruskal–Wallis test, and beta
diversity significance was estimated using the non-parametric ANOSIM test. MAFFT (via q2-alignment) and were used to construct a phylogeny with fasttree2 (via
q2-phylogeny). The q2-feature-classifier classify-sklearn naive Bayes taxonomy classifier
was used to assign taxonomy to the OTUs. Alpha diversity metrics (Shannon and Chao1),
beta diversity metrics (Bray–Curtis dissimilarity), and principal coordinate analysis (PCoA)
were estimated using a q2-diversity plugin after the samples were rarefied to 994 sequences
per sample. Alpha diversity significance was estimated with Kruskal–Wallis test, and beta
diversity significance was estimated using the non-parametric ANOSIM test. 4.5. Analysis of Short-Chain Fatty Acids (SCFAs) in Fecal Samples by Gas Chromatography (GC)
Coupled with a Flame-Ionization Detector The fecal concentrations of SCFAs were measured by GC coupled with a flame-
ionization detector as previously described [63–66] in the Servicios de Apoyo a la Investi-
gación de la Universidad de Extremadura (SAIUEx). Briefly, 20 mg of the fecal samples
were homogenized manually using a spatula in 200 µL of distilled water. Subsequently,
100 µL of homogenized fecal samples were mixed with 40 mg of sodium chloride, 20 mg
of citric acid, 40 µL of 0.1 M hydrochloric acid, and 200 µL of butanol: tetrahydrofuran:
acetonitrile (50:30:20). The samples were then vigorously vortexed for 3 min and were cen-
trifuged at 14,870× g at room temperature for 10 min. 4.4. Bioinformatics Analysis The supernatant was transferred to a
new plastic tube, and 200 µL of a benzyl alcohol–pyridine mixture (3:2) and 100 µL DMSO
were added, and the mixture was vortexed for 5 s. Then, 100 µL of benzyl chloroformate
was added carefully. To release the gases generated by the reaction, the tube lid was kept
open for 1 min. The tube was then closed, and the mixture was vortexed. After derivati-
zation, 200 µL hexane was added to the reaction mixture, and the sample was vortexed
for 5 min followed by a centrifugation step at 21,000× g for 2 min. Subsequently, 100 µL
of derivative extract (upper hexane layer) was transferred to a glass insert, and 5 µL were
injected into the gas chromatograph and were further analyzed using an Agilent 6850 gas
chromatograph coupled with a split/spitless injector and a flame-ionization detector (FID)
(Agilent Technologies, Santa Clara, CA, USA). The temperature of the injector and detector was adjusted to 250 ◦C, and the samples
(5 µL) were injected in a split ratio of 25:1 using a fused-silica capillary DB-23 column
Agilent (60 m × 0.25 mm (internal diameter) coated with a 0.15 µm thick layer of 80.2%
1-methylnaphatalene. Nitrogen was used as the carrier gas at 1 mL/min (hold 4 min),
reduced to 0.8 mL/min (hold 1 min) and then 0.6 mL/min (hold 1 min), and finally
increased to 1 mL/min. The temperature of the FID detector was adjusted and maintained
at 260 ◦C, and the flow rates of H2, the air, and the make-up gas N2 were adjusted to
30 mL/min, 350 mL/min, and 25 mL/min, respectively. The initial oven temperature
was 100 ◦C (hold 2 min), which was increased to 200 ◦C at a rate of 15 ◦C/min, and was
finally maintained at 200 ◦C for 5 min. The identity of the SCFAs detected in the fecal
samples was confirmed through the comparison of their retention times and their mass
spectra with those of the analytical SCFA standards (Sigma–Aldrich, Madrid, Spain). The
standard calibration curves for SCFAs (acetic acid, propionic acid, butyric acid, isobutyric
acid, valeric acid, isovaleric acid, 4- methylvaleric acid, hexanoic acid, and heptanoic acid)
were prepared in triplicate, with a concentration range of 15–1,000 µg/mL. 4.6. Analysis of Serum Polyamine Levels by Ultra-High Performance Liquid Chromatography
Tandem Mass Spectrometry (UHPLC-MS/MS) 4.6. Analysis of Serum Polyamine Levels by Ultra-High Performance Liquid Chromatography
Tandem Mass Spectrometry (UHPLC-MS/MS) For the analysis of the polyamine concentrations, serum samples were processed as
previously described [67]. Briefly, 50 µL of serum (aliquoted in 1.5 mL Eppendorf LoBind
tube) were mixed with 5 µL of internal standard and 167 µL of methanol. The mixture
was vortexed for 1 min, and 334 µL of chloroform was added, vortexed for 1 min, and
centrifuged for 10 min at 15,000 rpm and 4 ◦C. After centrifugation, the upper layer was
collected and was transferred to a new tube, where 100 µL of carbonate–bicarbonate buffer
(pH 9) and 50 µL of dansyl chloride (10 mg/mL in acetone) were added to derivatize the
sample. The mixture was vortexed and was placed in the dark for 1 h at room temperature. A total of two extractions of the compounds were conducted with 250 µL of ethyl acetate, Int. J. Mol. Sci. 2021, 22, 9549 21 of 25 21 of 25 between which 2.5 µL of trifluoroacetic acid were added. A SpeedVac at 45 ◦C was used
to evaporat the combined organic phases, which were stored at −20 ◦C until analysis. An amount of 50 µL of ammonium acetate and 0.2 M acetonitrile (30:70) was used to
reconstitute the samples. Chromatography of the samples was completed with Agilent UHPLC 1290 series
binary pump equipment (Agilent Technologies, Santa Clara, CA, USA), and the separation
was performed on a Kinetex EVO C18 column (2.6 µm particle size, 2.1 mm internal
diameter × 150 mm length) (Phenomenex, Torrance, CA, USA) held at 25 ◦C. A gradient
was established between the water acidified with 0.1% formic acid (A), and acetonitrile
acidified with 0.1% formic acid (B) at a flow rate of 400 µL/min was used as a mobile phase
for elution. The injected amount was 2.5 µL. MS/MS analysis was conducted in an Agilent QqQ 6490 Series mass spectrometer
operating in AJS + ESI. 4.6. Analysis of Serum Polyamine Levels by Ultra-High Performance Liquid Chromatography
Tandem Mass Spectrometry (UHPLC-MS/MS) The optimization of the ionization source parameters was performed
as follows: nebulizer gas (nitrogen) with a pressure of 15 psi, a gas flow of 15 L/min at
200 ◦C, a sheath gas flow of 11 L/min at 350 ◦C, a capillary voltage of 2.5 kV, and a nozzle
voltage of 1000 V in a MassHunter Optimizer (Agilent Technologies, version 6.0) g
p
g
g
An Agilent UHPLC 1290 Infinity II Series coupled to an Agilent QqQ/MS 6490 Series
(Agilent Technologies, Sta. Clara, CA, USA) was used for LC-MS/MS analysis, while
chromatographic separation was performed using a Kinetex EVO C18 analytical column
(2.6 µm; 2.1 mm × 150 mm) (Phenomenex, Torrance, CA, USA) Quantification was completed with the commercial standards ornithine, spermine,
arginine, spermidine, putrescine, N1-acetylspermidine, N8-acetylspermidine, N1-acetylsp-
ermine, N1-acetylputrescine, N1,N8-diacetylspermidine, and N1,N12-diacetylspermine
(Toronto Research Chemicals, North York, ON, Canada). The internal standards of the
amino acids were arginine (13C6, 15N4) and lysine (13C6, 15N2) (Cambridge Isotope
Laboratories), and for the polyamines, the internal stanfdards comprised putrescine-d8,
spermidine-d6, spermine-d20, and N8-acetylspermidine-d3 (Toronto Research Chemicals). 4.7. Intestinal Permeability Analysis Plasma levels of zonulin were measured in duplicate using an ELISA commercial kit
(Immunodiagnostik AG, Bensheim, Germany). Mean values were used for data analysis. Intra- and inter-assay coefficients of variation were between 3–10%, and the detection limit
was 0.22 ng/mL. 4.8. Statistical Analysis The Kruskal–Wallis rank-sum test was performed to compare the bacterial abun-
dance between the study groups, and the false discovery rate (FDR) using the Benjamini–
Hochberg method was applied to correct the significant p-values (q < 0.05). The Kruskal–
Wallis rank-sum test and subsequent post hoc Bonferroni were used to analyze differences
in the clinical and biochemical variables between three study groups, whereas differences
between the two groups were analyzed using the Mann–Whitney U test. Inter-group
comparison among post-treatment changes in fecal SCFAs and plasma zonulin levels
were performed using a covariance model (ANCOVA) adjusted for baseline. A Wilcoxon
signed-rank test was used to calculate differences in fecal SCFAs and plasma zonulin
between baseline and the post-treatment timepoint T3. The Spearman correlation coeffi-
cients were calculated to estimate the correlations between the bacterial taxa and microbial
derived-metabolites (SCFAs and polyamines) and the permeability. Statistical analyses
were conducted with the statistical software package SPSS version 26.0 (SPSS Inc., Chicago,
IL, USA). Random forests (RF) were used to predict baseline bacteria (species-level relative
abundance data) related to the neoadjuvant RCT response using the default parameters of
the R implementation of the algorithm (R package “randomForest”), and bootstrapping (n
= 500) was used to assess the classification accuracy. P values below 0.05 were considered
statistically significant. Int. J. Mol. Sci. 2021, 22, 9549 22 of 25 5. Conclusions In this study, we have demonstrated that the gut microbiota in CRC patients differs in
intestinal microbiota composition in comparison with healthy controls. In CRC patients,
the gut microbiota is characterized by a significantly lower bacterial diversity and richness,
a significant increase in proinflammatory opportunistic pathogens, and a decrease in the
relative abundance of beneficial or commensal butyrate-producing bacteria. In addition, neoadjuvant RCT treatment did not induce significant changes in gut
microbiota diversity and composition, with the exception of a significant decrease in
Fusobacterium, Escherichia, and Klebsiella and a significant increase in Bifidobacterium at
post-treatment time compared to baseline. Nevertheless, after the classification of CRC
patients in the R and NR groups to the neoadjuvant RCT, we observed a significant increase
in the diversity and richness in R patients compared to in the NR patients. Additionally, a
compositional change was shown between both study patient groups, with a significant
enrichment of probiotic and butyrate-producing bacteria in the R patients, accompanied
by an enrichment in unfavorable pro-inflammatory bacteria in the NR patients. Moreover,
the NR patients had significantly higher levels of spermine and some acetyl derivatives
of polyamines and serum zonulin and significantly lower levels of fecal of acetic, butyric,
isobutyric, and hexanoic acids than the R patients. These microbial-derived metabolites
are important factors that connect the intestinal microbiota to CRC and could be respon-
sible for RCT efficiency. Moreover, in the NR patients, the PICRUSt analysis found an
over-representation of genes involved in lipopolysaccharide biosynthesis as well as in
araquidonic acid and glutathione metabolism, genes from pathways associated with bacte-
rial pathogenesis, inflammation, cell survival, proliferation, and therapy response. In addition, we also identified a baseline consortium of CRC-enriched bacterial species
(Ruminococcus albus, Bifidobacterium bifidum, Faecalibacterium prausnitzii, Fusobacterium nu-
cleatum, and Bacteroides fragilis) that potentially could predict cancer treatment outcome,
suggesting that the intestinal composition in CRC patients is important in predicting the
response of the gut microbiome to neoadjuvant RCT. Altogether, our results suggest that a
healthy gut microbiome could be indispensable for an optimum therapeutic response and
that dysbiotic microbiota could be the underlying reason for variable responses to similar
therapeutic strategies in different patients. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22179549/s1. Author Contributions: Conceptualization, M.I.Q.-O. and J.G.-M.; methodology, L.S.-A., A.L.-I.,
B.R.-M., I.P.-A. and A.G.-G.; Investigation, L.S.-A., B.R.-M., I.P.-A., A.L.-I., A.O., R.O. and A.G.-G.;
validation, L.S.-A., A.O., R.O., I.P.-A. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ijms22179549/s1. Conflicts of Interest: The authors declare no conflict of interests. Conflicts of Interest: The authors declare no conflict of interests. Conflicts of Interest: The authors declare no conflict of interests. References 1. Arnold, D.; Lueza, B.; Douillard, J.Y.; Peeters, M.; Lenz, H.J.; Venook, A.; Heinemann, V.; Van Cutsem, E.; Pignon, J.P.; Tabernero,
J.; et al. Prognostic and predictive value of primary tumour side in patients with RAS wild-type metastatic colorectal cancer
treated with chemotherapy and EGFR directed antibodies in six randomized trials. Ann. Oncol. 2017, 28, 1713–1729. [CrossRef] 1. Arnold, D.; Lueza, B.; Douillard, J.Y.; Peeters, M.; Lenz, H.J.; Venook, A.; Heinemann, V.; Van Cutsem, E.; Pignon, J.P.; Tabernero,
J.; et al. Prognostic and predictive value of primary tumour side in patients with RAS wild-type metastatic colorectal cancer
treated with chemotherapy and EGFR directed antibodies in six randomized trials. Ann. Oncol. 2017, 28, 1713–1729. [CrossRef]
2. Liang, Q.; Chiu, J.; Chen, Y.; Huang, Y.; Higashimori, A.; Fang, J.; Brim, H.; Ashktorab, H.; Ng, S.C.; Ng, S.S.M.; et al. Fecal
bacteria act as novel biomarkers for noninvasive diagnosis of colorectal cancer. Clin. Cancer Res. 2017, 23, 2061–2070. [CrossRef]
3. Yazici, C.; Wolf, P.G.; Kim, H.; Cross, T.L.; Vermillion, K.; Carroll, T.; Augustus, G.J.; Mutlu, E.; Tussing-Humphreys, L.;
Brauschweig, C.; et al. Race-dependent association of sulfidogenic bacteria with colorectal cancer. Gut 2017, 66, 1983–1994. [CrossRef] [PubMed] py
2. Liang, Q.; Chiu, J.; Chen, Y.; Huang, Y.; Higashimori, A.; Fang, J.; Brim, H.; Ashktorab, H.; Ng, S.C.; Ng, S.S.M.; et al. Fecal
bacteria act as novel biomarkers for noninvasive diagnosis of colorectal cancer. Clin. Cancer Res. 2017, 23, 2061–2070. [CrossRef] 3. Yazici, C.; Wolf, P.G.; Kim, H.; Cross, T.L.; Vermillion, K.; Carroll, T.; Augustus, G.J.; Mutlu, E.; Tussing-Humphreys, L.;
Brauschweig, C.; et al. Race-dependent association of sulfidogenic bacteria with colorectal cancer. Gut 2017, 66, 1983–1994. [CrossRef] [PubMed] 4. Yu, J.; Feng, Q.; Wong, H.S.; Zhang, D.; Liang, Q.Y.; Qin, Y.; Tang, L.; Zhao, H.; Stenvang, J.; Li, Y.; et al. Metagenomic analysis
of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer. Gut 2017, 66, 70–78. [CrossRef]
[PubMed] [
]
5. Helmink, B.A.; Khan, M.A.W.; Hermann, A.; Gopalakrishnan, V.; Wargo, J.A. The microbiome, cancer, and cancer therapy. Nat. Med. 2019, 25, 377–388. [CrossRef] [PubMed] 6. McQuade, J.L.; Daniel, C.R.; Helmink, B.A.; Wargo, J.A. Modulating the microbiome to improve therapeutic response in cancer. Lancet Oncol. 2019, 20, e77–e91. [CrossRef] 7. Yi, Y.; Shen, L.; Shi, W.; Xia, F.; Zhang, H.; Wang, Y.; Zhang, J.; Wang, Y.; Sun, X.; Zhang, Z.; et al. References Scott, T.A.; Quintaneiro, L.M.; Norvaisas, P.; Lui, P.P.; Wilson, M.P.; Leung, K.Y.; Herrera-Dominguez, L
Clayton, P.T.; et al. Host-microbe co-metabolism dictates cancer drug efficacy in C. elegans. Cell 2017, 1 15. Gonzalez-Sarrias, A.; Tome-Carneiro, J.; Bellesia, A.; Tomas-Barberan, F.A.; Espin, J.C. The ellagic acid-derived gut microbiota
metabolite, urolithin A, potentiates the anticancer effects of 5-fluorouracil chemotherapy on human colon cancer cells. Food Funct. 2015, 6, 1460–1469. [CrossRef] 16. Liu, J.; Liu, C.; Yue, J. Radiotherapy and the gut microbiome: Facts and fiction. Radiat. Oncol. 2021, 16, 9. [CrossRef] [PubMed]
17. Yu, T.; Guo, F.; Yu, Y.; Sun, T.; Ma, D.; Han, J.; Qian, Y.; Kryczek, I.; Sun, D.; Nagarsheth, N.; et al. Fusobacterium nucleatum
promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 2017, 170, 548–563.E16. [CrossRef] [PubMed] 16. Liu, J.; Liu, C.; Yue, J. Radiotherapy and the gut microbiome: Facts and fiction. Radiat. Oncol. 2021, 16, 17. Yu, T.; Guo, F.; Yu, Y.; Sun, T.; Ma, D.; Han, J.; Qian, Y.; Kryczek, I.; Sun, D.; Nagarsheth, N.; et al. Fusobacterium nucleatum
promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 2017, 170, 548–563.E16. [CrossRef] [PubMed] 18. Roy, S.; Trinchieri, G. Microbiota: A key orchestrator of cancer therapy. Nat. Rev. Cancer 2017, 17, 271–285. [CrossRef]
19. Ohara, T.; Suzutani, T. Intake of Bifidobacterium longum (BB536) and Fructo-Oligosaccharides (FOS) prevents colorectal
carcinogenesis. Euroasian J. Hepatogastroenterol. 2018, 8, 11–17. [CrossRef] 18. Roy, S.; Trinchieri, G. Microbiota: A key orchestrator of cancer therapy. Nat. Rev. Cancer 2017, 17, 271–2
19
Oh
T
S
t
i T I t k
f Bifid b
t
i
l
(BB536)
d F
t
Oli
h
id
(F 18. Roy, S.; Trinchieri, G. Microbiota: A key orchestrator of cancer therapy. Nat. Rev. y
y
py
19. Ohara, T.; Suzutani, T. Intake of Bifidobacterium longum (BB536) and Fructo-Oligosaccharides (FOS) prevents colorectal
carcinogenesis. Euroasian J. Hepatogastroenterol. 2018, 8, 11–17. [CrossRef] g
J
p
g
20. Ross, K. Gut microbial short chain fatty acids are associated with pathological complete response (pCR) after neoadjuvant chemother-
apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 October 2020. 21. Coutzac, C.; Jouniaux, J.M.; Paci, A.; Schmidt, J.; Mallardo, D.; Seck, A.; Asvatourian, V.; Cassard, L.; Saulnier, P.; Lacroix, L.; et al. Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat. Commun. 2020, 11, 2168. g
p
g
20. Ross, K. 5. Conclusions and A.G.-G.; writing—original draft preparation, M.I.Q.-O.,
J.G.-M., L.S.-A. and B.R.-M.; writing—review and editing, all authors; funding acquisition, M.I.Q.-O.;
supervision, M.I.Q.-O. and J.G.-M. All authors have read and agreed to the published version of
the manuscript. Author Contributions: Conceptualization, M.I.Q.-O. and J.G.-M.; methodology, L.S.-A., A.L.-I.,
B.R.-M., I.P.-A. and A.G.-G.; Investigation, L.S.-A., B.R.-M., I.P.-A., A.L.-I., A.O., R.O. and A.G.-G.;
validation, L.S.-A., A.O., R.O., I.P.-A. and A.G.-G.; writing—original draft preparation, M.I.Q.-O.,
J.G.-M., L.S.-A. and B.R.-M.; writing—review and editing, all authors; funding acquisition, M.I.Q.-O.;
supervision, M.I.Q.-O. and J.G.-M. All authors have read and agreed to the published version of
the manuscript. Funding: This work was supported by PI15/00256 from the Institute of Health “Carlos III” (ISCIII),
co-funded by the Fondo Europeo de Desarrollo Regional-FEDER. Maria Isabel Queipo-Ortuño was
supported by the “Miguel Servet Type II” program (CPI18/00003, ISCIII, Spain, co-funded by the
Fondo Europeo de Desarrollo Regional-FEDER) and by the “Nicolas Monardes” research program
of the Consejería de Salud (C-0030-2018, Junta de Andalucía, Spain. Bruno Ramos Molina was
supported by the “Miguel Servet Type I” program (CP19/00098, ISCIII, Spain, co-funded by the
Fondo Europeo de Desarrollo Regional-FEDER). Lidia Sanchez-Alcoholado was the recipient of a
predoctoral grant (PE-0106-2019) from the Consejería de Salud y Familia (co-funded by the Fondo
Europeo de Desarrollo Regional-FEDER, Andalucia, Spain). Aurora Laborda-Illanes was the recipient
of a predoctoral grant, PFIS-ISCIII (FI19-00112), co-funded by the Fondo Europeo de Desarrollo
Regional-FEDER, Madrid, Spain. Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and was approved by the Ethics Committee of Virgen de la Victoria
University Hospital (30 October 2015). Int. J. Mol. Sci. 2021, 22, 9549 23 of 25 23 of 25 Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available upon request from the
corresponding author. The data are not publicly available, as they contain information that could
compromise the privacy of research participants. References Gut microbiome components
predict response to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer: A prospective, longitudinal
study. Clin. Cancer Res. 2021, 27, 1329–1340. [CrossRef] [PubMed] y
8. Alexander, J.L.; Wilson, I.D.; Teare, J.; Marchesi, J.R.; Nicholson, J.K.; Kinross, J.M. Gut microbiota modulation of chemotherapy y
8. Alexander, J.L.; Wilson, I.D.; Teare, J.; Marchesi, J.R.; Nicholson, J.K.; Kinross, J.M. Gut microbiota modulation of chemotherapy
efficacy and toxicity Nat Rev Gastroenterol Hepatol 2017 14 356–365 [CrossRef] [PubMed] 8. Alexander, J.L.; Wilson, I.D.; Teare, J.; Marchesi, J.R.; Nicholson, J.K.; Kinross, J.M. Gut microbiota
efficacy and toxicity. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 356–365. [CrossRef] [PubMed] J
J
J
J
J
efficacy and toxicity. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 356–365. [CrossRef] [PubMed] efficacy and toxicity. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 356 365. [CrossRef] [PubMed]
Sauer, R.; Becker, H.; Hohenberger, W.; Rödel, C.; Wittekind, C.; Fietkau, R.; Martus, P.; Tschmelitsch, J.; Hag
Preoperative versus postoperative chemoradiotherapy for rectal cancer N Engl J Med 2004 351 1731 1 y
y
p
9. Sauer, R.; Becker, H.; Hohenberger, W.; Rödel, C.; Wittekind, C.; Fietkau, R.; Martus, P.; Tschmelitsch, J.; Hager, E.; Hess, C.F.; et al. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N. Engl. J. Med. 2004, 351, 1731–1740. [CrossRef] Becker, H.; Hohenberger, W.; Rödel, C.; Wittekind, C.; Fietkau, R.; Martus, P.; Tschmelitsch, J.; Hager, E.; Hes p
p
p
py
g
J
,
,
[
]
10. Ma, B.; Gao, P.; Wang, H.; Xu, Q.; Song, Y.; Huang, X.; Sun, J.; Zhao, J.; Luo, J.; Sun, Y.; et al. What has preoperative
radio(chemo)therapy brought to localized rectal cancer patients in terms of perioperative and long-term outcomes over the past
decades? A systematic review and meta-analysis based on 41,121 patients. Int. J. Cancer 2017, 141, 1052–1065. [CrossRef] y
y
,
p
J
,
,
[
]
11. Pouncey, A.L.; Scott, A.J.; Alexander, J.L.; Marchesi, J.; Kinross, J. Gut microbiota, chemotherapy and the host: The influence of
the gut microbiota on cancer treatment. Ecancermedicalscience 2018, 12, 868. [CrossRef] 12. Koppel, N.; Maini Rekdal, V.; Balskus, E.P. Chemical transformation of xenobiotics by the human gut microbiota. Science 2017,
356, eaag2770. [CrossRef] 13. Parida, S.; Sharma, D. The power of small changes: Comprehensive analyses of microbial dysbiosis in breast cancer. Biochim. Biophys. Acta Rev. Cancer 2019, 1871, 392–405. [CrossRef] p y
14. 22.
Yoshioka, N.; Taniguchi, Y.; Yoshida, A.; Nakata, K.; Nishizawa, T.; Inagawa, H.; Kohchi, C.; Soma, G. Intestinal macrophages
involved in the homeostasis of the intestine have the potential for responding to LPS. Anticancer Res. 2009, 29, 4861–4865.
23.
Childs, A.; Mehta, D.; Gerner, E. Polyamine-dependent gene expression. Cell. Mol. Life Sci. 2003, 60, 1394–1406. [CrossRef] g
p
g
Ross, K. Gut microbial short chain fatty acids are associated with pathological complete response (pCR) after n
apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 References Gut microbial short chain fatty acids are associated with pathological complete response (pCR) after neoadjuvant chemother-
apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 October 2020. ,
y
p
g
p
p
(p
)
j
apy for breast cancer. In Proceedings of the 12th European Breast Cancer Conference, Virtual Conference, 2–3 October 2020. 21. Coutzac, C.; Jouniaux, J.M.; Paci, A.; Schmidt, J.; Mallardo, D.; Seck, A.; Asvatourian, V.; Cassard, L.; Saulnier, P.; Lacroix, L.; et al. Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat. Commun. 2020, 11, 2168. [CrossRef] [PubMed] 21. Coutzac, C.; Jouniaux, J.M.; Paci, A.; Schmidt, J.; Mallardo, D.; Seck, A.; Asvatourian, V.; Cassard, L.; Saulnier, P.; Lacroix, L.; et al. Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer. Nat. Commun. 2020, 11, 2168. [CrossRef] [PubMed] 22. Yoshioka, N.; Taniguchi, Y.; Yoshida, A.; Nakata, K.; Nishizawa, T.; Inagawa, H.; Kohchi, C.; Soma, G. Intestinal macrophages
involved in the homeostasis of the intestine have the potential for responding to LPS. Anticancer Res. 2009, 29, 4861–4865. 23. Childs, A.; Mehta, D.; Gerner, E. Polyamine-dependent gene expression. Cell. Mol. Life Sci. 2003, 60, 1394–1406. [CrossRef] 22. Yoshioka, N.; Taniguchi, Y.; Yoshida, A.; Nakata, K.; Nishizawa, T.; Inagawa, H.; Kohchi, C.; Soma, G. Intestinal macrophages
involved in the homeostasis of the intestine have the potential for responding to LPS. Anticancer Res. 2009, 29, 4861–4865. 23. Childs, A.; Mehta, D.; Gerner, E. Polyamine-dependent gene expression. Cell. Mol. Life Sci. Int. J. Mol. Sci. 2021, 22, 9549 24 of 25 24 of 25 24. Nakayama, Y.; Torigoe, T.; Minagawa, N.; Yamaguchi, K. The clinical usefulness of urinary N(1), N(12)-diacetylspermine
(DiAcSpm) levels as a tumor marker in patients with colorectal cancer. Oncol. Lett. 2012, 3, 970–974. [CrossRef] (DiAcSpm) levels as a tumor marker in patients with colorectal cancer. Oncol. Lett. 2012, 3, 970–974. [CrossRef]
25. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486,
207–214. [CrossRef] p
p
25. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486,
207–214. [CrossRef] [
]
26. Qin, J.; Li, R.; Raes, J.; Arumugam, M.; Burgdorf, K.S.; Manichanh, C.; Nielsen, T.; Pons, N.; Levenez, F.; MetaHIT Consortium;
et al. A human gut microbial gene catalogue established by metagenomic sequencing. References Cell Host Microbe 201 36. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vázquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E.; Knights, D.; Song, S.J.;
Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014, 15, 382–392. [CrossRef]
37
W
N
Y
X
Zh
R
Li J
Xi
X
H
Y Ch
Y Y
F L
N
W
Z
l D
bi
i
i
f f
l 36. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vázquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E.; Knights, D.; Song, S.J.;
Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. Cell Host Microbe 2014, 15, 382–392. [CrossRef]
37. Wu, N.; Yang, X.; Zhang, R.; Li, J.; Xiao, X.; Hu, Y.; Chen, Y.; Yang, F.; Lu, N.; Wang, Z.; et al. Dysbiosis signature of fecal
microbiota in colorectal cancer patients. Microb. Ecol. 2013, 66, 462–470. [CrossRef] 37. Wu, N.; Yang, X.; Zhang, R.; Li, J.; Xiao, X.; Hu, Y.; Chen, Y.; Yang, F.; Lu, N.; Wang, Z.; et al. Dys
microbiota in colorectal cancer patients. Microb. Ecol. 2013, 66, 462–470. [CrossRef] 38. Bao, Y.; Tang, J.; Qian, Y.; Sun, T.; Chen, H.; Chen, Z.; Sun, D.; Zhong, M.; Chen, H.; Hong, J.; et al. Long noncoding RNA BFAL1
mediates enterotoxigenic Bacteroides fragilis-related carcinogenesis in colorectal cancer via the RHEB/mTOR pathway. Cell Death
Dis. 2019, 10, 675. [CrossRef] [PubMed] 39. Hsu, R.Y.; Chan, C.H.; Spicer, J.D.; Rousseau, M.C.; Giannias, B.; Rousseau, S.; Ferri, L.E. LPS-induced TLR4 signaling in human
colorectal cancer cells increases beta1 integrin-mediated cell adhesion and liver metastasis. Cancer Res. 2011, 71, 1989–1998. [CrossRef] [
]
40. Murff, H.J.; Shu, X.O.; Li, H.; Dai, Q.; Kallianpur, A.; Yang, G.; Cai, H.; Wen, W.; Gao, Y.T.; Zheng, W. A prospective study of
dietary polyunsaturated fatty acids and colorectal cancer risk in Chinese women. Cancer Epidemiol. Biomark. Prev. 2009, 18,
2283–2291. [CrossRef] [
]
41. Wang, D.; Mann, J.R.; DuBois, R.N. The role of prostaglandins and other eicosanoids in the gastrointestinal tract. Gastroenterology
2005, 128, 1445–1461. [CrossRef] 42. Bansal, A.; Simon, M.C. Glutathione metabolism in cancer progression and treatment resistance. J. Cell Biol. 2018, 217, 2291–2298. [CrossRef] [PubMed] 43. Lu, S.C. Regulation of glutathione synthesis. Mol. Asp. Med. 2009, 30, 42–59. [CrossRef] [PubMed] 43. Lu, S.C. Regulation of glutathione synthesis. Mol. Asp. Med. 2009, 30, 42–59. [CrossRef] [PubMed]
44. Coffelt, S.B.; Wellenstein, M.D.; de Visser, K.E. References Nature 2010, 464, 59–65. [CrossRef]
27. Garrett, W.S. Cancer and the microbiota. Science 2015, 348, 80–86. [CrossRef] [PubMed] g
g
g
y
g
q
g
27. Garrett, W.S. Cancer and the microbiota. Science 2015, 348, 80–86. [CrossRef] [PubMed] ,
,
,
[
] [
]
28. Drewes, J.L.; Housseau, F.; Sears, C.L. Sporadic colorectal cancer: Microbial contributors to disease prevention, development and
therapy. Br. J. Cancer 2016, 115, 273–280. [CrossRef] [PubMed] 29. Taghinezhad-S, S.; Mohseni, A.H.; Fu, X. Intervention on gut microbiota may change the strategy for management of colorectal
cancer. J. Gastroenterol. Hepatol. 2020, 36, 1508–1517. [CrossRef] [PubMed] 30. Gopalakrishnan, V.; Spencer, C.N.; Nezi, L.; Reuben, A.; Andrews, M.C.; Karpinets, T.V.; Prieto, P.A.; Vicente, D.; Hoffman, K.;
Wei, S.C.; et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science 2018, 359,
97–103. [CrossRef] 31. Heshiki, Y.; Vazquez-Uribe, R.; Li, J.; Ni, Y.; Quainoo, S.; Imamovic, L.; Li, J.; Sørensen, M.; Chow, B.K.C.; Weiss, G.J.; et al. Predictable modulation of cancer treatment outcomes by the gut microbiota. Microbiome 2020, 8, 28. [CrossRef] 32. Chen, W.; Liu, F.; Ling, Z.; Tong, X.; Xiang, C. Human intestinal lumen and mucosa-associated microbiota in patients with
colorectal cancer. PLoS ONE 2012, 7, e39743. [CrossRef] [PubMed] 33. Saffarian, A.; Mulet, C.; Regnault, B.; Amiot, A.; Tran-Van-Nhieu, J.; Ravel, J.; Sobhani, I.; Sansonetti, P.J.; Pédron, T. Crypt-
and mucosa-associated core microbiotas in humans and their alteration in colon cancer patients. mBio 2019, 10, e1315–e1319. [CrossRef] [PubMed] 34. Gao, Z.; Guo, B.; Gao, R.; Zhu, Q.; Qin, H. Microbiota disbiosis is associated with colorectal cancer. Front. Microbiol. 2015, 6, 20. [CrossRef] [PubMed] 35. Yan, X.; Liu, L.; Li, H.; Qin, H.; Sun, Z. Clinical significance of Fusobacterium nucleatum, epithelial-mes
cancer stem cell markers in stage III/IV colorectal cancer patients OncoTargets Ther 2017 10 5031–504 35. Yan, X.; Liu, L.; Li, H.; Qin, H.; Sun, Z. Clinical significance of Fusobacterium nucleatum, epithelial-mesenchymal transition, and
cancer stem cell markers in stage III/IV colorectal cancer patients. OncoTargets Ther. 2017, 10, 5031–5046. [CrossRef] ,
;
,
;
,
; Q
,
;
,
g
, p
y
,
cancer stem cell markers in stage III/IV colorectal cancer patients. OncoTargets Ther. 2017, 10, 5031–5046. [CrossRef] g
p
g
36. Gevers, D.; Kugathasan, S.; Denson, L.A.; Vázquez-Baeza, Y.; Van Treuren, W.; Ren, B.; Schwager, E
Yassour, M.; et al. The treatment-naive microbiome in new-onset Crohn’s disease. References Neutrophils in cancer: Neutral no more. Nat. Rev. Cancer 2016, 16, 431–446. [CrossRef] 44. Coffelt, S.B.; Wellenstein, M.D.; de Visser, K.E. Neutrophils in cancer: Neutral no more. Nat. Rev. Cancer 2016, 16, 431–446. [CrossRef] 44. Coffelt, S.B.; Wellenstein, M.D.; de Visser, K.E. Neutrophils in cancer: Neutral no more. Nat. R
[CrossRef] [
]
45. Thomas, A.M.; Manghi, P.; Asnicar, F.; Pasolli, E.; Armanini, F.; Zolfo, M.; Beghini, F.; Manara, S.; Karcher, N.; Pozzi, C.; et al. Metagenomic analysis of colorectal cancer datasets identifies cross-cohort microbial diagnostic signatures and a enlace with
choline degradation. Nat. Med. 2019, 25, 667–678. [CrossRef] [PubMed] g
46. Noack, J.; Dongowski, G.; Hartmann, L.; Blaut, M. The human gut bacteria Bacteroides thetaiotaomicron and Fusobacterium
varium produce putrescine and spermidine in cecum of pectin-fed gnotobiotic rats. J. Nutr. 2000, 130, 1225–1231. [CrossRef] 47. Goodwin, A.C.; Destefano Shields, C.E.; Wu, S.; Huso, D.L.; Wu, X.; Murray-Stewart, T.R.; Hacker-Prietz, A.; Rabizadeh, S.; Woster,
P.M.; Sears, C.L.; et al. Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis. Proc. Natl. Acad. Sci. USA 2011, 108, 15354–15359. [CrossRef] 48. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen-
bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef] 48. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen-
bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef]
49. Mendez, R.; Kesh, K.; Arora, N.; Di Martino, L.; McAllister, F.; Merchant, N.; Banerjee, S.; Banerjee, S. Microbial dysbiosis and 48. Johnson, C.H.; Dejea, C.M.; Edler, D.; Hoang, L.T.; Santidrian, A.F.; Felding, B.H.; Ivanisevic, J.; Cho, K.; Wick, E.C.; Hechen
bleikner, E.M.; et al. Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab. 2015, 21, 891–897. [CrossRef]
49. Mendez, R.; Kesh, K.; Arora, N.; Di Martino, L.; McAllister, F.; Merchant, N.; Banerjee, S.; Banerjee, S. Microbial dysbiosis and
polyamine metabolism as predictive markers for early detection of pancreatic cancer. Carcinogenesis 2020, 41, 561–570. [CrossRef]
[PubMed] 49. Mendez, R.; Kesh, K.; Arora, N.; Di Martino, L.; McAllister, F.; Merchant, N.; Banerjee, S.; Banerjee, S. Microbial dysbiosis and
polyamine metabolism as predictive markers for early detection of pancreatic cancer. Carcinogenesis 2020, 41, 561–570. [CrossRef]
[PubMed] Int. J. Mol. Sci. 2021, 22, 9549 25 of 25 50. References Arruabarrena-Aristorena, A.; Zabala-Letona, A.; Carracedo, A. Oil for the cancer engine: The cross-talk between oncogenic
signaling and polyamine metabolism. Sci. Adv. 2018, 4, eaar2606. [CrossRef] 51. Bhat, M.I.; Kapila, R. Dietary metabolites derived from gut microbiota: Critical modulators of epigenetic changes in mammals. Nutr. Rev. 2017, 75, 374–389. [CrossRef] 52. Jahani-Sherafat, S.; Alebouyeh, M.; Moghim, S.; Amoli, H.A.; Safaei, H.G. Role of gut microbiota in the pathogenesis of colorectal
cancer; a review article. Gastroenterol. Hepatol. Bed Bench. 2018, 11, 101–109. p
53. Sokol, H.; Seksik, P.; Furet, J.P.; Firmesse, O.; Nion-Larmurier, I.; Beaugerie, L.; Cosnes, J.; Corthier, G.; Marteau, P.; Doré., J. Low
counts of Faecalibacterium prausnitzii in colitis microbiota. Inflamm. Bowel Dis. 2009, 15, 1183–1189. [CrossRef] 54. Lopez-Siles, M.; Martinez-Medina, M.; Abellà, C.; Busquets, D.; Sabat-Mir, M.; Duncan, S.H.; Aldeguer, X.; Flint, H.J.; Garcia-Gil,
L.J. Mucosa-associated Faecalibacterium prausnitzii phylotype richness is reduced in patients with inflammatory bowel disease. Appl. Environ. Microb. 2015, 81, 7584–7592. [CrossRef] 55. Rios-Covian, D.; Gueimonde, M.; Duncan, S.H.; Flint, H.J.; de los Reyes-Gavilan, C.G. Enhanced butyrate formation by cross-
feeding between Faecalibacterium prausnitzii and Bifidobacterium adolescentis. FEMS Microbiol. Lett. 2015, 362, fnv176. [CrossRef] 56. Singh, N.; Gurav, A.; Sivaprakasam, S.; Brady, E.; Padia, R.; Shi, H.; Thangaraju, M.; Prasad, P.D.; Manicassamy, S.; Munn, D.H.;
et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and
carcinogenesis. Immunity 2014, 40, 128–139. [CrossRef] g
y
57. Salcedo, R.; Worschech, A.; Cardone, M.; Jones, Y.; Gyulai, Z.; Dai, R.M.; Wang, E.; Ma, W.; Haines, D.; O’hUigin, C.; et al. MyD88-mediated signaling prevents development of adenocarcinomas of the colon: Role of interleukin 18. J. Exp. Med. 2010, 207,
1625–1636. [CrossRef] 58. Vaishnava, S.; Behrendt, C.L.; Ismail, A.S.; Eckmann, L.; Hooper, L.V. Paneth cells directly sense gu
homeostasis at the intestinal host-microbial interface. Proc. Natl. Acad. Sci. USA 2008, 105, 20858–2 59. Van Immerseel, F.; De Buck, J.; Boyen, F.; Bohez, L.; Pasmans, F.; Volf, J.; Sevcik, M.; Rychlik, I.; Haesebrouck, F.; Ducatelle, R. Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with
Salmonella enterica serovar Enteritidis. Appl. Environ. Microbiol. 2004, 70, 3582–3587. [CrossRef] pp
60. Sturgeon, C.; Fasano, A. Zonulin, a regulator of epithelial and endothelial barri
inflammatory diseases. Tissue Barriers 2016, 4, e1251384. [CrossRef] [PubMed] pp
60. Sturgeon, C.; Fasano, A. References Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic
inflammatory diseases. Tissue Barriers 2016, 4, e1251384. [CrossRef] [PubMed] 61. Wright, D.P.; Rosendale, D.; Roberton, A.M. Prevotella enzymes involved in mucin oligosaccharide degr
a small operon of genes expressed during growth on mucin. FEMS Microbiol. Lett. 2000, 190, 73–79. [C Rosendale, D.; Roberton, A.M. Prevotella enzymes involved in mucin oligosaccharide degradation and evide
f
d d
i
th
i
FEMS Microbiol Lett 2000 190 73 79 [C
R f] [P bM d 62. Mandard, A.M.; Dalibard, F.; Mandard, J.C.; Marnay, J.; Henry-Amar, M.; Petiot, J.F.; Roussel, A.; Jacob, J.H.; Segol, P.; Samama, G. Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic
correlations. Cancer 1994, 73, 2680–2686. [CrossRef] 63. Ribeiro, W.R.; Vinolo, M.A.R.; Calixto, L.A.; Ferreira, C.M. Use of gas chromatography to quantify
serum, colonic luminal content and feces of mice. Bio-Protocol 2018, 8, e3089. [CrossRef] o, M.A.R.; Calixto, L.A.; Ferreira, C.M. Use of gas chromatography to quantify short chain fatty acids in the
nal content and feces of mice. Bio-Protocol 2018, 8, e3089. [CrossRef] 64. Mendes, E.; Acetturi, B.G.; Thomas, A.M.; Martins, F.; Crisma, A.R.; Murata, G.; Braga, T.T.; Camâra, N.; Franco, A.; Setubal,
J.C.; et al. Prophylactic supplementation of Bifidobacterium longum 51A protects mice from ovariectomy-induced exacerbated 64. Mendes, E.; Acetturi, B.G.; Thomas, A.M.; Martins, F.; Crisma, A.R.; Murata, G.; Braga, T.T.; Camâra, N.; Franco, A.; Setubal,
J.C.; et al. Prophylactic supplementation of Bifidobacterium longum 51A protects mice from ovariectomy-induced exacerbated
allergic airway inflammation and airway hyperresponsiveness. Front. Microbiol. 2017, 8, 1732. [CrossRef] [PubMed] allergic airway inflammation and airway hyperresponsiveness. Front. Microbiol. 2017, 8, 1732. [CrossRef] [PubMed] 65. Fellows, R.; Denizot, J.; Stellato, C.; Cuomo, A.; Jain, P.; Stoyanova, E.; Balázsi, S.; Hajnády, Z.; Liebert, A.; Kazakevych, J.; et al. Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases. Nat. Commun. 2018, 9, 105. [CrossRef] 66. Liang, J. A sensitive method for the quantification of short-chain fatty acids by benzyl chloroformate derivatization combined
with GC-MS. Analyst 2020, 145, 2692–2700. [CrossRef] 67. Samarra, I.; Ramos-Molina, B.; Queipo-Ortuño, M.I.; Tinahones, F.J.; Arola, L.; Delpino-Rius, A.; Herrero, P.; Canela, N. Gender-
related differences on polyamine metabolome in liquid biopsies by a simple and sensitive two-step liquid-liquid extraction and
LC-MS/MS. Biomolecules 2019, 9, 779. [CrossRef]
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Temporal dynamics of faculty hiring in mathematics
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ARTICLE
https://doi.org/10.1057/s41599-023-01708-9
OPEN
Temporal dynamics of faculty hiring in
mathematics
1234567890():,;
Cody FitzGerald
1,2 ✉,
Yitong Huang2,3 ✉, Katelyn Plaisier Leisman1 & Chad M. Topaz4,5,6
University faculty hiring networks are known to be hierarchical and to exacerbate various
types of inequity. Still, a detailed, historical understanding of hiring dynamics lacks in many
academic fields. We focus on the field of mathematics, analyzing over 120,000 records from
150 institutions over seven decades to elucidate the temporal dynamics of hiring doctoralgranting (DG) faculty at the individual and departmental levels. We demonstrate that the
disparity between the number of mathematics Ph.D.s awarded and the number of DG faculty
positions filled has grown over time. Even institutions with the best records of DG faculty
placement have experienced a temporal decline in the probability of their graduates obtaining
a DG faculty position. By quantifying the mathematical prestige of each department with a
network statistic, authority centrality, we find an approximately linear relationship between
the log of the prestige of one’s Ph.D. institution and the log of the probability of obtaining a
faculty position. Moreover, we observe associations suggesting that the probability of DG
faculty placement has decreased over time and is smaller for women than for men. On the
departmental level, a group of 14 elite departments dominated the authority centrality of the
entire network between 1950 and 2019. Strikingly, one department within this elite group
increased its centrality scores consistently, which hints at the possibility for a department to
improve its prestige. This analysis highlights the challenges of transitioning from Ph.D. holder
to faculty member in mathematics.
1 Department of Engineering Sciences and Applied Mathematics, Northwestern University, Evanston, IL, USA. 2 NSF-Simons Center for Quantitative Biology,
Evanston, IL, USA. 3 Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA. 4 Institute for the Quantitative Study of Inclusion,
Diversity, and Equity, Williamstown, MA, USA. 5 Williams College, Williamstown, MA, USA. 6 University of Colorado–Boulder, Boulder, CO, USA.
✉email: cody.fitzgerald@northwestern.edu; yitong.huang@northwestern.edu
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A
HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9
Introduction
n estimated two thousand mathematics Ph.D.s are awarded annually in the United States (Reys et al., 2022). At
the same time, only around 300 tenure-track positions in
doctoral degree-granting mathematics departments are under
recruitment each cycle. Moreover, upwards of 70% of these
positions are filled by faculty who are not new Ph.D.s (Jahan
et al., 2019). The hiring process is not just competitive but is
opaque and poorly understood by applicants (Fernandes et al.,
2020). The result of a search is eventually known publicly, but the
list of applicants and the selection criteria used are not revealed.
Success in acquiring a faculty position may not only depend on
an individual scholar’s research productivity and academic credentials (Fernandes et al., 2020), but also on demographic factors
such as race, gender, and childhood socioeconomic status
(Clauset et al., 2015; Morgan et al., 2022; Wapman et al., 2022;
White-Lewis, 2020).
In recent years there has been growing interest in quantifying
the faculty hiring process in various fields (Barnett et al., 2010;
Clauset et al., 2015; Cowan and Rossello, 2018; Fernandes et al.,
2020; Fowler et al., 2007; Hanneman, 2001; Lee et al., 2021; Mai
et al., 2015; Wapman et al., 2022; Zuo et al., 2019). For instance,
Clauset et al. (2015) used a network science approach to study the
trajectories of 19,000 faculty members in business, computer
science, and history. They found that faculty hiring networks have
a particularly hierarchical structure. They also found that doctoral
program prestige is a strong predictor of faculty placement and
that there exists a bias towards men as compared to women with
the same training, especially in the fields of business and computer science (Clauset et al., 2015). A follow-up study in 2021,
which used the same data set, utilized adaptive rewiring network
models to test mechanisms that give rise to the hierarchical
nature of the faculty hiring observed in the 2015 study (Lee et al.,
2021). It concludes that a mixture of two mechanisms, total
faculty production and local homophily, likely drives the
dynamics of real-world faculty hiring. Another recent study
combined a survey of tenure-track faculty members in eight
fields, US census data, and the NSF Survey of Earned Doctorates
to study the impact of socioeconomic status on faculty position
acquisition and found that faculty members tended to grow up in
wealthier homes and were 25 times more likely to have a parent
who held a Ph.D., as compared to the general US population
(Morgan et al., 2022). An extensive study of faculty hiring
between 2011 and 2020 encompassed 295,089 US faculty members in 10,612 departments across 107 fields and eight domains
(including mathematics and computing) using data from the
Academic Analytics Research Center (Wapman et al., 2022). This
study confirmed the hierarchical structure observed in previous
studies extends across many fields of academic hiring. The study
also uncovered populations of US faculty members with high
attrition rates: US faculty members trained outside the US,
Canada, and the UK; faculty members that are trained and hired
by the same university; and faculty members that are trained at
universities from which relatively few graduates acquire faculty
positions. Finally, this study found that recent changes in the
gender parity of faculty members are explained mainly by the
retirement of older faculty members, who tend to be men
(Wapman et al., 2022). A survey-based statistical analysis of
academic job seekers primarily in the life sciences identified significant associations between receiving an academic job offer and
the number of job applications filed, winning a career transition
award, total citation count, authorship of high-profile papers, and
postdoctoral fellowships (Fernandes et al., 2020). The same study
also found significant negative associations between receiving an
academic job offer and the number of years an applicant is on the
job market and joint industry and academic job searches
2
(Fernandes et al., 2020). Despite these efforts spent investigating
academic hiring, a comprehensive understanding of the evolution
of the mathematics faculty hiring process over many decades is
lacking. Our analysis moves towards filling this gap in
understanding.
We complement the existing work on faculty hiring by
studying doctoral-granting (DG) faculty hiring in mathematics
over the past 70 years at the individual and departmental scale
using the Mathematics Genealogy Project (MGP, http://www.
genealogy.ams.org), which is an extensive database of graduate
advisor–advisee relationships. We consider academic hiring as a
complex system at two different scales: the individual level and
the department level. At the individual level, we examine over
120,000 records from MGP to uncover characteristics that lead to
successful DG faculty placements in mathematics. Here, we set
out to understand which factors, academic or otherwise, allow a
mathematics graduate degree holder to transition to a DG faculty
member in mathematics and how generally difficult this transition is to make. We will refer to this transition as the graduate-tofaculty transition (GFT) throughout the paper. We begin our
investigation into the GFT by studying 150 US math departments
and show that the GFT is decreasing over time. This finding even
holds for historically “well-placing” departments, which annually
graduated at least one student who eventually acquired a DG
faculty position in mathematics between 1950 and 2015. We also
find statistically significant academic-based and gender-based
factors that influence the GFT.
In addition to our individual-level examination of the GFT, we
use network analysis methods to investigate the department level.
Myers et al. (2011) analyzed the MGP database between 1973 and
2011 and showed that department authority scores correlate with
departmental rankings from US News & World Reports and the
National Research Council. Hub and authority centrality were
originally developed in the context of ranking web pages
(Kleinberg, 1999). We join Myers et al. (2011) in using these
centralities as proxies for departmental prestige. Similar to the
definitions given in Myers et al. (2011), we define a department
with a high authority value as one where Ph.D. recipients go on to
become DG faculty at prestigious schools and a department with
a high hub value as one containing many DG faculty who
received their Ph.D. at prestigious schools. Expanding on Myers
et al. (2011), we conduct a fine-grained analysis that explores
gains and losses in the hub and authority centrality of US math
departments between 1950 and 2019.
In our analysis, we find that a subset of 14 “elite” departments
holds approximately 70% of the authority centrality of the entire
network. Moreover, the total centrality held by these elite
departments remains relatively constant between 1950 and 2019.
However, the share of centrality held individually by each of these
elite departments varies over that time frame. Strikingly, we find
that one of the 14 elite departments dramatically increased its
share of both hub and authority centrality.
In summary, our investigations into the GFT and the temporal
dynamics of centrality at the departmental scale make three
contributions:
●
●
●
We perform an analysis of the evolution of academic hiring
within the field of mathematics between 1950 and 2019.
We begin to uncover academic and demographic factors
associated with successfully acquiring a DG faculty position
at the individual level in the field of mathematics.
We find that an elite group of departments comprise a large
portion of network centrality between 1950 and 2019.
Furthermore, the portion of centrality held by the elite
group remains relatively stable throughout that time span.
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However, we find one math department that has
dramatically and consistently increased its share of both
hub and authority centrality, the latter of which is a proxy
for rank.
Data collection
We begin with a sample of 150 Ph.D.-granting institutions based
on the U.S. News Graduate Schools Top Mathematics Programs
rankings. The union of three archived U.S. News rankings from
1998, 2010, and 2018 comprise a final sample of 150 institutions
that we analyze.
We then proceed to gather data from the Mathematics Genealogy Project (MGP, http://www.genealogy.ams.org). We collected all data on October 4, 2022. For each of the 150 schools, we
search MGP for all individuals who ever obtained a Ph.D. from
that school. From the search results, we retain each person’s
name, the year of their Ph.D., and a hyperlink to their full record
within MGP. We acquire 121,521 records of graduates from
150 schools. Of these, 466 records are missing a year, accounting
for 0.4% of the data. For the remaining records, the year ranges
from 1792 to 2022, with a median year of 1997 and a mean year
of approximately 1993.
Next, we gather information about which individuals have
served as DG faculty. From MGP data, the only means we have to
infer whether the target individual became a DG faculty member
is to check whether they advised Ph.D. students who also appear
in MGP. For each person in our database, we see if there are any
students listed on their individual MGP record. If there are no
students listed, we assume the individual did not serve as DG
faculty. If there are students listed, we list all the schools at which
the students received their Ph.D.s and take the mode as the primary school at which the advisor was DG faculty. For example, if
an individual advised 8 Ph.D. students at the University of Iowa
and 2 at the University of Indiana, we would take the University
of Iowa as the institution where the individual was DG faculty. If
the mode is not unique, we record the first (in chronology)
institution. It is a limitation of our approach that we cannot
identify faculty in departments that do not grant Ph.D.’s, nor DG
faculty who do not have any Ph.D. students appearing in MGP,
either through omission of data or through not having served as
advisor. Overall, we identify a DG faculty institution for 24,928
individuals. Finally, in our construction of hiring networks, we
restrict attention to those who became DG faculty at one of the
150 US News-ranked schools, which results in a data set of 19,372
individuals.
Next, if available, we gather information about the school
where each individual’s advisor received a Ph.D., as well as how
many students the advisor is listed on MGP as having mentored.
Finally, for each record, we extract the individual’s first name
and run it through the genderize.io algorithm (Demografix
ApS, 2022) to infer the individual’s gender. From the outset, it is
important to recognize the limitations of this approach. Most
critically, inferred gender is not the same as actual gender. The
only accurate source for information on an individual’s gender is
the individual themself. However, many of the MGP records
correspond to deceased individuals, and even for living individuals, a survey methodology is unlikely to produce much data.
Moreover, an additional severe limitation of our approach is the
algorithm’s unfortunate use of binary gender. Overall, it would be
strongly preferable to have self-identified gender information, and
information unrestricted to a binary scale. Lacking this data, we
proceed with using the gender inference algorithm, keeping in
mind its serious shortcomings.
For each name, the algorithm outputs an inferred binary
gender based on its internal database of names with self-identified
ARTICLE
gender as scraped from online sources. For example, consider the
first name Kelsey. Suppose that the genderize.io database
contains 900 instances of this name, 765 coming from selfidentified women, and 135 coming from self-identified men.
Then the algorithm would infer the gender of “woman” and
would report a probability of 0.85, based on the frequency 765/
900. An additional limitation of the algorithm is now apparent:
there is no way to know the extent to which the underlying data
might be biased. For instance, if we had access to self-identified
gender information for all individuals in the world with the name
Kelsey, would the observed frequency of women be close to the
0.85 reported by the algorithm?
Keeping all of the limitations in mind, we proceed by choosing
a gender inference probability below which we are unwilling to
accept the algorithm’s inference. We initially experimented with a
high threshold, p = 0.95, but manual examination of the excluded
data suggested that an overwhelming majority of discarded names
were of East or South Asian origin. Discarding so many of these
names would result in the erasure of those groups from our study.
Thus, to achieve more inclusion, we seek a cutoff probability
threshold p* such that the frequencies of inferred men and
women in the excluded data are equal to the average probabilities
of being a man and being a woman in that same excluded data;
that is to say, we strive for aggregate internal consistency in the
excluded data. We find a single value of p* satisfying this criterion, namely p* ≈ 0.6, and we exclude data where p < p*. We
find 93,882 inferred men (77.2%) and 22,410 inferred women
(18.4%). For 5229 individuals (4.3%), our cutoff for probability
precluded an inference.
Results
Focusing on individuals: the graduate to faculty transition rate.
We begin our analysis by computing the probability that a
mathematics Ph.D. holder becomes a faculty member who advises
one or more mathematics Ph.D. students to graduation. For this
analysis, we use all 121,055 records in our dataset that have a
graduation year listed. Figure 1 shows the total number of
mathematics Ph.D. graduates each year from 1900 to 2019, and
the number of those Ph.D. graduates who are listed on MGP as
having eventually advised students. Both the number of
Fig. 1 The number of mathematics Ph.D. degrees awarded from 150 US
math departments (green solid curve) and the number of people who
later became doctoral-granting (DG) faculty members from this pool
(blue dotted curve) between the years 1900 and 2019. The steep decline
beginning in 2010 is simply an indication of the necessary time lag between
receiving one’s Ph.D. and graduating one’s first Ph.D. student.
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Fig. 2 The combined graduate to doctoral-granting (DG) faculty
transition (GFT) rate for 150 DG US math departments from 1900 to
2019. The GFT rate decays nonlinearly over time.
mathematics Ph.D. degrees awarded and the number of people
who receive a Ph.D. and go on to become a DG faculty member
in mathematics increase between 1900 and 1970, but after
roughly 1970 the number of new DG faculty members saturates,
and even declines slightly, while total annual graduates continue
to rise. Most of the steep decline evident in eventual advisors after
about 2010 is due to the fact that many of those who graduated
more recently may not have advised students yet (but perhaps
will eventually).
By dividing the number of people who receive a Ph.D. and go
on to become a DG faculty member in mathematics (blue dotted
curve in Fig. 1) by the total number of mathematics Ph.D. degrees
awarded (green solid curve in Fig. 1), we obtain the graduate to
DG faculty transition (GFT) rate.
The annual GFT rate for all records, shown in Fig. 2, can be
interpreted as a global measure of the relative ease of acquiring a
DG faculty position in mathematics. The GFT rate decays
nonlinearly from 1900 through 2019. In the past 30 or so years,
the GFT rate has trended closer to zero than it previously had
over the time scale we considered, though we again note that the
data is incomplete for recent graduates who may eventually train
future students but have not yet done so.
To further investigate the GFT rate, we look at what happens
when we restrict the data to graduates from a list of “well-placing”
schools between the years of 1950 and 2015. We choose these
schools to determine if a subset of math departments were
immune from the global behavior of the GFT rate over this time
span. We choose these years because the data are less complete
before 1950, and graduates after 2015 may not have had enough
time to acquire a DG faculty position and advise their first
trainee. We define a well-placing school as a school that between
these years annually graduated at least one mathematics Ph.D.
student who eventually mentored a student at one of the 150
departments we included in our analysis. Ten schools fall into this
category, including Harvard University, Massachusetts Institute
of Technology, Princeton University, Stanford University, University of Chicago, University of North Carolina–Chapel Hill,
University of California–Berkeley, University of Michigan,
University
of
Pennsylvania,
and
University
of
Wisconsin–Madison. The minimum, median, and maximum
GFT rates for schools in this group are shown in Fig. 3. We can
see that the transition rate of “well-placing” mathematics
departments declines in a similar manner to the global trend
4
Fig. 3 The minimum, median, and maximum graduate-to-faculty
transition (GFT) rates for 10 schools that have annually graduated at
least one mathematics Ph.D. student who became a doctoral-granting
(DG) faculty member in a department in one of the 150 departments we
considered between 1950 and 2015. All three measures decrease, but the
maximum GFT rate most sharply declines between the years of 1950-2015.
seen in Fig. 2. Note the spread that exists in Fig. 3 in the 1950s
and 1960s dies off over time as the minimum, median, and
maximum GFT rates each trend downward over time.
Centrality scores: hubs and authorities. Here, we apply the
network science approach described in Myers et al. (2011) to a
larger data set and incorporate temporal dynamics into the centrality analysis. Myers et al. constructed a doctoral-granting
faculty hiring network based on data from the MGP database
between the years 1973 and 2011. In their network, the nodes
represent US math departments and the edges are representative
of DG faculty hiring. The edges are weighted by the number of
people who were trained as graduate students in a department
and acquire DG faculty positions in another department. The
edges point from the DG faculty department towards the graduate training department, and the graduate training and DG
faculty departments can be the same if the person was trained and
hired by the same department. As an example, a professor in the
Purdue University Department of Mathematics who received a
Ph.D. from the University of Wisconsin–Madison Department of
Mathematics is represented as an edge pointing from the Purdue
University Department of Mathematics node to the University of
Wisconsin-Madison Department of Mathematics node.
In Myers et al. (2011), network centrality was analyzed for 58
math departments using hub and authority centrality scores. Hub
and authority centrality scores were originally developed in the
context of ranking web pages on the internet (Kleinberg, 1999)
and were used in the search engine for Ask.com (Newman, 2018).
Mathematically, centrality authority and centrality hub vectors x
and y are the left and right singular vectors corresponding to the
largest singular value of the adjacency matrix, A, scaled so their
elements each sum to 1. Thus, they satisfy
x ¼ αAy;
and
y ¼ βAT x;
where constants α and β satisfy αβ = 1/σ2 and σ is the largest
singular value of A (Newman, 2018).
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Fig. 4 Graduate-to-faculty transition (GFT) rate by average authority
score of graduate training department between the years of 1950 and
2019 on a log–log plot. The dashed line represents a least-squares fit of the
data, yielding an r2 value of 0.7 on the log-log axes.
Fig. 5 The fraction of doctoral-granting (DG) faculty members who
acquire a faculty position in a DG department with a higher authority
score than the department where they completed their graduate studies
versus the year of Ph.D. graduation. The fraction hovers between 15% and
25% and is relatively stable over seven decades.
In Myers et al. (2011), departments with high authority scores
are defined as “a strong source of prestigious Ph.D. students,” and
departments with high hub scores are defined as “a strong
destination.” We refine their definitions for the reader’s clarity.
We take a department with a high authority score to mean an
institution from which Ph.D. recipients go on to become DG
faculty at prestigious schools, and we define a department with a
high hub score as a department that hires many DG faculty who
received a Ph.D. from a prestigious school. Next, we compute the
GFT rate as a function of authority score for each of the
150 schools in our set that graduated students between the years
of 1950 and 2019 (see Fig. 4).
As expected, higher authority scores, suggesting higher
prestige, correlate with higher GFT rates. The data appear linear
on a log–log plot of the GFT rate versus the authority score of the
Ph.D. training departments (r2 = 0.7 via least-squares fitting on
the log-log axes). It is worth pointing out that San Diego State
University (GFT rate = 0.5, authority score ≈ 1.36 × 10−5)
appears to be an outlier in the group because only two graduates
in our dataset obtained a Ph.D. from San Diego State University,
and one of them eventually became a DG faculty member.
Furthermore, we look at the probability of moving up (higher
authority score) when transitioning from Ph.D. holder to DG
faculty. Of the graduates who eventually go on to advise students
at one of the 150 schools of interest, we compute the fraction of
those who have advised students at an institution with a greater
authority score than that of their Ph.D. granting institution. This
fraction is shown in Fig. 5. It remains relatively stable between
1950 and 2019, largely hovering between 15% and 25%. Note that
graduates from 2019 may not have enough time to advise
students yet, and in fact, our data only contains two individuals
who graduated in 2019 and already have advisees listed.
Ph.D.; their inferred gender; prestige measures of their Ph.D.
granting institution and their advisor’s Ph.D. granting institution;
and finally, the number of Ph.D. students overseen by their
advisor. We now explain these choices in detail.
Since the academic job market is tighter in some years and less
so in others, we include the year of Ph.D. receipt as an
explanatory variable. To address differential outcomes depending
on gender, we include inferred gender in the model. We also
include the interaction of the inferred gender with the year of
Ph.D. receipt in order to allow for the possibility of a shifting
hiring landscape for women over time.
To account for the prestige of the target individual’s Ph.D.
granting institution, we (initially) include its hub and authority
scores as computed over a 10-year time period up to but not
including the Ph.D. year. For that same time period, to account
for the influence of the target’s Ph.D. advisor as part of the job
search process, we include the hub and authority scores of that
person’s Ph.D. granting institution. Finally, to account for the
target’s Ph.D. advisor’s advising patterns, albeit in a crude
manner, we include the total number of students who received
Ph.D.s from the target’s advisor over the same 10-year window. It
is important to recall that the target Ph.D. and advisor Ph.D. hub
and authority scores are all calculated from the same network and
thus may be correlated. In fact, the correlation between hub and
authority for the target is r ≈ 0.66 and for the advisor is r ≈ 0.51.
Among the four network scores, the two least correlated are the
target’s authority score and the advisor’s hub score (r ≈ 0.16), so
we include these in our model. While including the other two
network scores might increase model fit, it could be at the
expense of interpretability, especially if estimates for the effects of
strongly correlated variables have opposite signs.
Concretely, the model described above is
Logistic model: probability of faculty placement. To identify
factors associated with a target individual obtaining a doctoralgranting (DG) faculty position, we construct a logistic regression
model. The dichotomous outcome variable is whether or not the
target individual became a DG faculty. As discussed previously,
we infer DG faculty status from the individual’s record of student
advising as listed in MGP, should it exist. We include several
explanatory variables in our model: the target’s year of receipt of
Log Odds β0 þ β1 ðYearÞ þ β2 ðInferred Gender WomanÞ
þ β3 ðYear Inferred Gender WomanÞ
þ β4 ðTarget0 s Ph:D: AuthorityÞ þ β5 ðAdvisor0 s Ph:D: HubÞ
þ β6 ðNumber of Students Advised by AdvisorÞ;
ð1Þ
where since we use a logistic regression framework, Log Odds is
the log odds of obtaining a DG faculty position.
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Table 1 Estimates obtained from fitting model (1), which describes the log odds of an individual becoming a DG faculty member,
based on N = 81, 691 records (see the section “Logistic model: probability of faculty placement” for details, including important
limitations of the modeling approach).
Coefficient of
Estimate
Standard error
z
Significant
(Intercept)
Year
Inferred gender woman
Year ⋅ inferred gender woman
Target’s Ph.D. authority
Advisor’s Ph.D. hub
Students advised by advisor
72.9
−0.0377
−13.9
0.00688
1.37
0.0714
−0.0479
1.28
0.000644
3.81
0.00192
0.0310
0.0521
0.00229
57.1
−58.6
−3.65
3.59
44.3
1.37
−20.9
***
***
***
***
***
***
Significance column is coded as *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 6 The number of departments that comprise 50% and 90% of the
hub and authority centrality between the years 1950 and 2019. Centrality
scores are computed using a rolling window of 10 years, indicated on the xaxis by the first year of the decade. Black and blue curves correspond to
50% and 90% of centrality, respectively. Solid and dashed curves
correspond to hub and authority centrality, respectively. This analysis
includes 106 schools.
Before fitting the regression, we perform several data filtering
steps. First, to be consistent with the time period we have studied
previously, we filter out target individuals who received their
Ph.D. prior to 1950 or after 2015, resulting in a data set of 104,674
records. Second, to be consistent with whom we include in our
network, we further restrict attention to individuals whose
advisor received their Ph.D. at one of the 150 schools we study,
reducing the data set to 85,323. While we have not formally coded
the geography of the advisor Ph.D. institutions for the excluded
records, inspection suggests that the vast majority are schools
outside of the United States. Third, we are forced to eliminate any
records missing a gender inference. Removing these 3,569 records
leaves 81,754 remaining. Fourth and last, in 63 cases (constituting
0.1% of the remaining data) we are unable to compute prestige
scores because no one from the target individual’s Ph.D. granting
institution became DG faculty during the 10-year time window
prior to the year of Ph.D. receipt. We must eliminate these
records, resulting in a final data set of 81,691 records.
Next, to again be consistent with our previous decisions, we
take the outcome variable to be not merely whether the target
individual became a DG faculty member, but whether they
became a DG faculty member at one of the 150 schools we study.
There are 4032 individuals in the data set who did become DG
faculty but not at one of the 150 schools, and so along with 64,381
6
individuals who did not become DG faculty at any school in our
data set, these individuals have an outcome variable coded
as false.
In summary, our logistic model investigates whether individuals who received a Ph.D. at one of the 150 schools and whose
advisor received their Ph.D. at one of those same schools ended
up becoming DG faculty at still one of those same schools. We
have 81,691 records, and the observed frequency of a positive
outcome (becoming DG faculty at one of the 150 schools) is 0.16.
We fit the model using the standard glm command in the
RStudio statistical computing environment. In terms of evaluating our model, we steer clear of measures of predictive accuracy
because our intention is not to build a classifier. Indeed, with such
a limited set of explanatory variables, we have no expectation that
our model could be used in this way. However, we can still hope
to assess if there are important associations between the outcome
variable and the explanatory variables that we do have. Loosely,
our situation could be compared to a linear regression that
produces a low coefficient of determination but statistically
significant coefficients. Thus, to diagnose our model, we set a
relatively low bar and compare it to a null model. A likelihood
ratio test of our model results in p < 0.001, suggesting that the
model is preferable to the null one. Table 1 provides estimates
from our model.
The signs of the significant coefficients have the following
interpretations. The negative coefficient on year means that
overall, there is an association between time and a decreased
probability of obtaining a DG faculty position. Similarly, the
negative coefficient on inferred gender woman indicates an
association between being a woman and a decreased probability
of obtaining a DG faculty position. The positive coefficient on the
interaction of year and inferred gender woman suggests that the
decreased probability of hiring for inferred women is becoming
less severe over time. The positive coefficient on the authority
score of the individual’s Ph.D. granting institution shows an
association between that institution’s prestige and its students
being hired as DG faculty. This result is perhaps not surprising
given the way the authority score is constructed. There is one
additional significant coefficient, namely, the negative coefficient
on the number of students advised by the individual’s Ph.D.
advisor. This coefficient indicates an association that is less
intuitive to us and could perhaps be a target for further research.
We could hypothesize that when an advisor advises more
students, they are able to provide less individualized attention
to each student. This reduced attention might decrease the
probability of a DG faculty placement, either because the student
receives insufficient guidance or because the situation discourages
the student from wanting to be in academia. Regardless, these are
merely conjectures, and ethnographic data might shed further
light on plausible explanations.
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Temporal dynamics of US math departments
To begin our department-level analysis, we compute the hub and
authority centrality scores of a subset of 106 of the US mathematics departments we considered, starting in 1950 and going
through 2019, using a rolling window of 10 years for each network construction, thereby extending the analysis presented in
Myers et al. (2011). We determine this subset by computing the
centrality scores of our entire set of 150 departments for each
rolling decade starting with 1950 and ending with 2019. Any
school that does not appear as an institution with DG faculty
during each rolling 10-year period returns an authority (hub)
score of zero. We omit any school which returned a zero centrality score for any of the rolling decades and re-compute the
centrality scores for the remaining 106 departments. From this
network analysis, we find that a minority of departments hold the
majority of the hub and authority centrality. Figure 6 shows that
between five and seven departments hold 50% of the authority
centrality and between 11 and 19 departments hold 50% of the
hub centrality between 1950 and 2019, calculated over rolling
decades, and plotted by the first year of each decade. Roughly
one-third of departments hold 90% of the authority centrality and
two-thirds of departments hold 90% of the hub centrality between
the years of 1950 and 2019.
Furthermore, we explore the temporal dynamics of hub and
authority centrality associated with elite math departments
between the years of 1950 and 2019. To select elite departments to
study, we compute the departments with the seven highest
authority scores across seven network constructions spanning
1950–1959, 1960–1969, 1970–1979, 1980–1989, 1990–1999,
2000–2009, and 2010–2019 and take the union of the department
sets. As a result of this computation, the following departments
meet our definition of elite: California Institute of Technology,
Carnegie Mellon University, Columbia University, Cornell University, Harvard University, Massachusetts Institute of Technology, Princeton University, Stanford University, University of
Chicago, University of California–Berkeley, University of Michigan,
University
of
Washington,
University
of
Wisconsin–Madison, and Yale University. Together this group of
departments consistently holds approximately 72% of the
authority centrality and approximately 43% of the hub centrality
between 1950 and 2019 (see Fig. 7).
While the share of hub and authority centrality held by this
group of elite departments remains relatively constant between
1950 and 2019, the individual share of hub and authority centrality held by each elite department changes more dramatically
over time. We compute the Kendall rank correlation coefficient
(Kendall, 1948) of authority and hub centrality time series
spanning decades beginning in 1950 and ending in 2019 for pairs
of elite departments. In this context, a Kendall rank correlation
coefficient close to 1 would indicate that the centrality score
trajectories of two departments between 1950 and 2019 move in
the same direction. Similarly, a Kendall rank correlation coefficient close to −1 would indicate that the centrality trajectories of
two departments between 1950 and 2019 move in opposite
directions. Large and significant Kendall rank correlation coefficients associated with pairs of elite departments’ hub and
authority centrality time series are summarized in Fig. 8.
Strikingly, we find consistent gains between 1950 and 2019 in
both hub and authority centrality by the Carnegie Mellon University Departments of Mathematics while the Massachusetts
Institute of Technology Department of Mathematics and the Yale
University Department of Mathematics fall in both measures of
centrality over the same time course, as shown in Figs. 8 and 9.
To understand what caused the gains in hub and authority
centrality between 1950 and 2019, we plot the rolling averages
(using a 10-year window) of graduates from Carnegie Mellon
ARTICLE
Fig. 7 The fraction of hub (green solid curve) and authority (orange
dotted curve) centrality held in total by California Institute of
Technology, Carnegie Mellon University, Columbia University, Cornell
University, Harvard University, Massachusetts Institute of Technology,
Princeton University, Stanford University, University of Chicago,
University of California–Berkeley, University of Michigan, University of
Washington, University of Wisconsin–Madison, and Yale University
between the years of 1950 and 2019. Centrality scores are computed
using a rolling window of 10 years, indicated on the x-axis by the first year
of the decade. To select elite departments to study, we compute the
departments with the seven highest authority scores across seven network
constructions spanning 1950–1959, 1960–1969, 1970–1979, 1980–1989,
1990–1999, 2000–2009, and 2010–2019 and take the union of the
department sets. Both the hub and authority centrality held by this group of
elite departments remains relatively stable over this time frame.
University, Massachusetts Institute of Technology, and Yale
University who eventually became a doctoral-granting (DG) math
faculty, as well as the number of DG math faculty hires at each
university, starting with the year 1950 and ending with the year
2019 (see Fig. 10). For Carnegie Mellon University, we observe
that the average number of graduates that eventually became a
DG faculty and the number of DG faculty hires both initially rise,
and then remain somewhat constant. In contrast, for Yale University, we observe an initial rise in the average number of
graduates that eventually became DG faculty followed by a
decline starting slightly after 1980. We also see a decline in the
average number of DG faculty hires at Yale University over time.
For 10-year windows near the end of the range considered, such
as 2010 to 2019, the graduate student and DG faculty averages are
slightly lower, likely due to the fact that graduate students may
not have secured a DG faculty position yet, and similarly for
faculty members who have been hired but have not yet had a
student graduate.
Discussion
In this study, we analyze the MGP database to understand how
academic faculty selection operates in the field of mathematics. In
particular, we seek to elucidate the factors that influence the
transition from mathematics graduate degree holder to doctoralgranting (DG) faculty member and understand temporal trends
related to this transition. We first show a growing disparity
between the number of mathematics Ph.D.s awarded and the
number of DG faculty positions acquired over time. This disparity is most extreme after 1970. The GFT rate has reached its
lowest point during the last 30 years, and even historically wellplacing departments exhibit the same trend. Additionally, we find
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Fig. 8 Significant (p ≤ 0.05) and large (∣τ∣ > 0.5) Kendall rank correlation coefficients for time series of the hub (left) and authority (right) scores of
elite math departments. We focus mainly on the relationships between the Carnegie Mellon University, Massachusetts Institute of Technology, and Yale
University Departments of Mathematics.
Fig. 9 Time series of hub and authority centrality scores associated with 106 math departments between the years 1950 and 2019. Centrality scores
are computed using a rolling window of 10 years, indicated on the x-axis by the first year of the decade. The hub and authority centrality score time series
for Carnegie Melon University, Massachusetts Institute of Technology, and Yale University math departments are highlighted in red, yellow, purple,
respectively. The geometric mean of all 106 departments’ temporal hub and authority score is plotted as a dashed blue curve. Other elite departments are
plotted in dark gray and all other departments are plotted in light gray. The hub and authority centrality time series for the Carnegie Melon University
Department of Mathematics increases as the hub and authority centrality time series associated with the Massachusetts Institute of Technology and Yale
University Departments of Mathematics decrease in a significant manner.
Fig. 10 Averaged DG faculty hires and averaged graduate production at the Carnegie Mellon University, Massachusetts Institute of Technology, and
Yale University Departments of Mathematics over time. (Left): Rolling averages using a 10-year window of DG math faculty hires at Carnegie Mellon
University, Massachusetts Institute of Technology, and Yale University, by the year of their first graduated student. (Right): Rolling averages using a
10-year window of graduates from Carnegie Mellon University, Massachusetts Institute of Technology, and Yale University who eventually became a
doctoral-granting (DG) math faculty, by year of graduation. Both subplots start with the year 1950 and end with the year 2019.
8
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that the log of the GFT rate correlates with the log of the
authority score of the graduate training institution. A logistic
regression model reveals that both time and the authority score of
an individual’s Ph.D. granting department are significantly
associated with the probability of obtaining a DG faculty position
in a department. Moreover, individuals inferred to be women
appear to be disadvantaged as compared to men, though the
disadvantage narrows over time.
At the department level, we find that the Carnegie Mellon
University Department of Mathematics increased its hub and
authority centrality consistently over time, while the Massachusetts Institute of Technology and Yale University Departments of
Mathematics fell in both measures over the same time frame.
These gains and losses in centrality correspond to the temporal
dynamics of the average number of graduate students produced
(who eventually become a DG math faculty) and averaged DG
math faculty hires at Carnegie Mellon University and Yale University, respectively. Increasing graduate student output (specifically those that become DG faculty members), and DG faculty
hiring, would have resulted in increasing the authority and hub
scores, respectively. However, the underlying details of what
made these universities more or less attractive to graduate students and faculty remain unclear but may involve some combination of department culture, department research interests, and
its relevance to funding initiatives over this period, amongst a
suite of other unknown factors.
Our study has several limitations regarding the data we used.
We do not have information about the number of job applications individuals filed, career transition award recipient status,
total citation counts, high-impact journal authorship records,
postdoctoral fellowship recipient status, the length of time an
individual spends on the job market, or if individuals conducted
joint academic-industry job searches. It was previously shown
that these factors are significantly associated with receiving an
academic job offer in a recent survey analysis (Fernandes et al.,
2020). We also do not have information pertaining to an individual’s desire and decision-making process regarding pursuing
an academic career versus another type of employment. Additionally, we are only studying the transition from mathematics
Ph.D. graduate to DG faculty member in mathematics. It is
possible that mathematics departments have hired DG faculty
members who are not listed in the MGP database due to their
graduate training field. Our study also does not include DG
faculty members who do not advise graduate students, for
example, mathematics Ph.D. holders who teach at liberal arts
colleges, community colleges, or even in non-mathematics
departments.
Our study also has important limitations regarding the centrality metric we used. Our method for computing temporal
trajectories of network centrality is rather straightforward, and
other more sophisticated frameworks have been designed for this
purpose (see, e.g., Taylor et al., 2017, 2019, 2021; Kawakatsu et al.,
2021). In our study, we used hub and authority centrality, which
are eigenvector-based centrality metrics, to quantify the importance of math departments in our faculty hiring network. Following Myers et al. (2011), we used hubs and authorities because
of their interpretable meaning in the context of math departments. However, eigenvector-based centralities can exhibit a
phenomenon known as “localization,” in which centrality is
concentrated in a handful of nodes relative to the rest of the
network (Martin et al., 2014). This property is known to be
related to the structure of the network and “is particularly visible
in networks with high-degree hubs1 or power-law degree distributions” (Martin et al., 2014). To address this challenge, the
recent work of Taylor et al. (2017) has proposed a generalization
of eigenvector centrality for temporal networks. They find that
ARTICLE
“the strength of the coupling between layers is important for
determining multiscale properties of centrality, such as localization phenomenon.” They tested their method on the MGP
database in the weak and strong coupling limits and noted, “We
believe this weak-coupling regime to be inappropriate for the
MGP Ph.D. exchange network, as mathematics department
prestige should not fluctuate wildly from one year to the next.”
They also comment that “For scenarios in which exploring various [couplings] is not computationally feasible, we highlight that
restricting one’s attention to the [strong coupling] limit can still
yield very informative results.” While we do not use the exact
method in Taylor et al. (2017), we do expect the layers of our
temporal network to be relatively strongly coupled due to our use
of a 10-year rolling window for each network construction. For
example, the network that spans 1950–1959 shares much of the
same information with the network that spans 1951–1960.
In summary, we find that the Graduate Faculty Transition rate,
an imperfect indicator of the relative ease of acquiring a DG
faculty position in mathematics, is decaying over time, even for
historically well-placing departments. We also uncover statistically significant factors (academic and otherwise) that influence
the transition, including gender, year, target’s Ph.D. authority,
and the number of students advised by Ph.D. advisor. At the
department level, we find that the Carnegie Mellon University
Department of Mathematics consistently gained both hub and
authority centrality between 1950 and 2019, a rare behavior in
our network analysis. Taken together, we find that acquiring a
DG faculty position is becoming more difficult and overall the
rankings of departments remain relatively constant, though it is
not impossible for departments to improve their ranking
over time.
Data availability
The code and an anonymized version of the Mathematics Genealogy Project dataset (https://mathgenealogy.org/index.php) we
analyzed is posted here https://github.com/cefitzg/MGP_plots.
Received: 17 October 2022; Accepted: 13 April 2023;
Note
1 Martin and coworkers state the definition of “hubs” as “nodes of unusually high
degree,” not to be confused with the technical definition of hubs and authority
centralities in the section “Centrality scores: hubs and authorities”.
References
Barnett GA, Danowski JA, Feeley TH, Stalker J (2010) Measuring quality in
communication doctoral education using network analysis of faculty-hiring
patterns. J Commun 60(2):388–411
Clauset A, Arbesman S, Larremore DB (2015) Systematic inequality and hierarchy
in faculty hiring networks. Sci Adv 1(1):e1400005
Cowan R, Rossello G (2018) Emergent structures in faculty hiring networks, and
the effects of mobility on academic performance. Scientometrics 117:527–562
Demografix ApS. https://genderize.io/. Accessed 8 Oct 2022
Fernandes JD, Sarabipour S, Smite C et al. (2020) A survey-based analysis of the
academic job market. eLife 9:1–30
Fowler JH, Grofman B, Masuoka N (2007) Social networks in political science:
hiring and placement of Ph. Ds, 1960–2002. Political Sci Politics 40:729–739
Hanneman RA (2001) The prestige of Ph.D. granting departments of sociology: a
simple network approach. Connections 24(1):68–77
Jahan N, Barr TH, Rose CA, Macias VP (2022) Academic recruitment, hiring, and
attrition during 2018–2019. Not Am Math Soc. https://www.ams.org/
profession/data/annual-survey/advance_release_annsurv-recruitmenthiring_report2019.pdf
Katakatsu M, Chodrow PS, Eikmeier N, Larremore DB (2021) Emergence of
hierarchy in networked endorsement dynamics. Proc Natl Acad Sci USA
118(16):e2015188118
HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | (2023)10:247 | https://doi.org/10.1057/s41599-023-01708-9
9
ARTICLE
HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS | https://doi.org/10.1057/s41599-023-01708-9
Kendall MG (1948) Rank correlation methods. Griffin
Kleinberg JM (1999) Authoritative sources in a hyperlinked environment. J ACM
46(5):604–632
Lee E, Clauset A, Larremore DB (2021) The dynamics of faculty hiring networks.
EPJ Data Sci 10(1):48
Mai B, Liu J, González-Bailón S (2015) Network effects in the academic market:
Mechanisms for hiring and placing PhDs in communication (2007–2014). J
Commun 65(3):558–583
Martin T, Zhang X, Newman MEJ (2014) Localization and centrality in networks.
Phys Rev E 90(5):2808
Morgan AC, LaBerge N, Larremore DB et al. (2022) Socioeconomic roots of academic faculty. Nat Hum Behav. https://doi.org/10.1038/s41562-022-01425-4
Myers SA, Mucha PJ, Porter MA (2011) Mathematical genealogy and department
prestige. Chaos 21(4):041104
Newman M (2018) Networks. Oxford University Press
Reys R, Reys B, Shih J (2022) Some patterns of PhDs in mathematics awarded
annually by institutions of higher education in the United States over the last
two decades. Not Am Math Soc 69(1):96–107
Taylor D, Myers S, Clauset A, Porter M, Mucha P (2017) Eigenvector-based
centrality measures for temporal networks. Multiscale Model Simul
15(1):537–574
Taylor D, Porter MA, Mucha PJ (2019) Supracentrality analysis of temporal networks with directed interlayer coupling. In: Holme P, Saramäki J (eds)
Temporal network theory. Computational social sciences. Springer, Cham
Taylor D, Porter M, Mucha P (2021) Tunable eigenvector-based centralities for
multiplex an temporal networks. Multiscale Model Simul 19(1):113–147
Wapman KH, Zhang S, Clauset A, Larremore DB (2022) Quantifying hierarchy
and dynamics in US faculty hiring and retention. Nature 610:120–127
White-Lewis DK (2020) The facade of fit in faculty search processes J High Educ
91(6):833–857
Zuo Z, Zhao K, Ni C (2019) Standing on the shoulders of giants?—Faculty hiring in
information schools. J Informetr 13:341–353
Acknowledgements
CF was supported by the NSF-Simons Center for Quantitative Biology at Northwestern
University (NSF: 1764421 and Simons Foundation/SFARI 597491-RWC) and James S.
McDonnell Foundation Postdoctoral Fellowship Award in Complex Systems (https://doi.
org/10.37717/2020-1591).
10
Competing interests
The authors declare no competing interests.
Ethical approval
This article does not contain any studies with human participants performed by any of
the authors.
Informed consent
This article does not contain any studies with human participants performed by any of
the authors.
Additional information
Correspondence and requests for materials should be addressed to Cody FitzGerald or
Yitong Huang.
Reprints and permission information is available at http://www.nature.com/reprints
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New types of the partnership agreement in the context of collective use of goods and services (sharing economy)
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© Подузова Е. Б., 2019
* Подузова Екатерина Борисовна, кандидат юридических наук, доцент кафедры гражданского права,
доцент кафедры нотариата Московского государственного юридического университета имени О.Е. Ку-
тафина (МГЮА)
ekaterinak7785@yandex.ru
125993, Россия, г. Москва, ул. Садовая-Кудринская, д. 9 ГРАЖДАНСКОЕ И СЕМЕЙНОЕ ПРАВО Е. Б. Подузова* Новые виды договора простого товарищества
в контексте коллективного использования
товаров и услуг (sharing economy)1 Аннотация. Современный социально-экономический контекст совместного потребления
(sharing economy) ставит новые цели и задачи использования конструкций организации сов
местной деятельности. В статье представлены основные теоретические и практические проблемы видов договора
простого товарищества как организационного договора, формы организации и ведения сов
местной деятельности. Материал статьи подготовлен с учетом реформы договорного права Российской Федерации,
новых тенденций в науке гражданского права и в правоприменительной практике. Также была
учтена актуальная судебная практика, в которой содержатся новые подходы к толкованию
обязательства и договора. В связи с этим особое значение имеют постановления Пленума
Верховного Суда РФ (например, от 24 марта 2016 г. № 7 «О применении судами некоторых
положений Гражданского кодекса Российской Федерации об ответственности за нарушение
обязательств», от 22 ноября 2016 г. № 54 «О некоторых вопросах применения общих положе-
ний Гражданского кодекса Российской Федерации об обязательствах и их исполнении»). Особое внимание было уделено правовой природе, конститутивным признакам отдельных Особое внимание было уделено правовой природе, конститутивным признакам отдельных
видов договора простого товарищества, их проявлению в современном гражданском законо-
дательстве и практике его применения. Ключевые слова: коллективное использование, договор простого товарищества, сделка,
разновидности договора, правовая природа, организация, организующий договор, договор об
организации совместной деятельности, имущественный элемент, встречное предоставление,
инвестиционное товарищество, горное товарищество. Актуальные проблемы российского права. 2019. № 8 (105) август DOI: 10.17803/1994-1471.2019.105.8.086-094 1
Исследование выполнено при финансовой поддержке РФФИ в рамках научного проекта № 18-29-16054
«Концептуальные основы правового регулирования отношений по коллективному использованию то-
варов и услуг (sharing economy) в условиях развития цифровых технологий». Актуальные проблемы российского права. 2019. № 8 (105) август 86 Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) Подузова Е. Б. Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) предполагается, что агрегаторы осуществляют
подбор заказчику конкретного исполнителя,
устанавливают цены на услуги исполнителей. Во
втором случае лишь предоставляется удобный
сервис для поиска исполнителя8. З
а последнее десятилетие популярность
коллективного использования товаров
и услуг возросла в значительной мере. В первую очередь большой спрос приобрела
бизнес-модель Uber, построенная по прин-
ципу экономики совместного потребления —
sharing economy firm2 (ride-sharing company3,
transportation network companies4, online-
enabled car transportation service5). В бизнес-сре-
де и вне ее активно используется модель work-
sharing6, известная со времен Великой депрес-
сии в США в 1920-е гг. Данная модель предпо-
лагает вывод ряда работ и услуг за пределы
персонала компании либо обмен знаниями,
поиск лиц, выполняющих работы и оказываю-
щих услуги в конкретной бытовой сфере. Такая
модель представлена, например, в сервисах
YouDo, profi.ru, а также в сервисах взаимного
изучения языков посредством общения с их
носителями7. З
а Каждая из правовых форм реализации сов
местного потребления представляет собой
совместную деятельность. Использование
платформ-агрегаторов не предполагает стро-
гих требований к правовой форме данных от-
ношений. В судебной практике отмечается, что
несоблюдение требований к форме договора
при достижении сторонами соглашения по всем
его существенным условиям не свидетельствует
о том, что договор не был заключен9. Возникает
вопрос о возможности применения конструкции
договора простого товарищества для правового
оформления отношений по коллективному ис-
пользованию товаров и услуг. В рамках гражданско-правовой реформы
произошли существенные изменения граждан-
ского законодательства и практики его приме- В литературе выделяются две разновидности
таких компаний-агрегаторов. В первом варианте 4
См.: Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition
Policy International. 2015. № LS 15-11 ; № 15-29. 5
См.: Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy
International, Forthcoming. November 24, 2015. P. 1. 6
What is work-sharing // URL: https://www.wisegeek.com/what-is-work-sharing.htm (дата обращения: 20 фев-
раля 2019 г.). 7
См., например: URL: https://www.rosettastone.com/lp/sbsr/livemocha/?prid=livemocha_com (дата обра-
щения: 20 февраля 2019 г.). 8
См. об этом подробнее: Савельев А. И. м.: Rogers B. The Social Costs of Uber // University of Chicago Law Review Dialogue. 2015. № 28. P. 85. 2
См.: Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation of
the Sharing Economy // George Mason Law & Economics Research Paper. 2015. № 15-01. P. 1. DOI: 10.17803/1994-1471.2019.105.8.086-094 Электронная коммерция в России и за рубежом: правовое регули-
рование. М. : Статут, 2016 ; Бычков А. Посредничество при оказании юридических услуг: практика, риски,
перспективы // Новая бухгалтерия. 2016. № 9. С. 130—143 ; Иванов А. А. Бизнес-агрегаторы и право //
Закон. 2017. № 5. С. 145—157 ; Молотников А. Е., Архипов Е. В. Социальные сети и компании-агрегаторы:
правовые аспекты деятельности // Предпринимательское право. 2017. № 4 ; Чесалина О. В. Работа на
основе интернет-платформ (crowdwork и work on demand via apps) как вызов трудовому и социальному
праву // Трудовое право в России и за рубежом. 2017. № 1 ; De Stefano V. The rise of the «just-in-time
workforce»: On-demand work, crowdwork and labour protection in the «gig-economy» // Conditions of work
and employment, Series No. 71. Geneva : ILO, 2016. P. 1. URL: http://www.ilo.org/wcmsp5/groups/public/-
--ed_protect/---protrav/---travail/documents/publication/wcms_443267.pdf (дата обращения: 31 января
2019 г.). 9
Постановление Пленума Верховного Суда РФ от 25 декабря 2018 г. № 49 «О некоторых вопросах при-
менения общих положений Гражданского кодекса Российской Федерации о заключении и толковании
договора». П. 3 // СПС «КонсультантПлюс». 9
Постановление Пленума Верховного Суда РФ от 25 декабря 2018 г. № 49 «О некоторых вопросах при-
менения общих положений Гражданского кодекса Российской Федерации о заключении и толковании
договора». П. 3 // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 87 Гражданское и семейное право жет быть признан недействительным в судеб-
ном порядке по требованию любого другого
участника данного договора с возложением на
управляющего товарища обязанности по воз-
мещению всем участникам данного договора
причиненных им в связи с этим убытков. нения10, изменились традиционные теорети-
ко-практические подходы к понятию и призна-
кам договора простого товарищества, к выявле-
нию его новых разновидностей. Среди новых разновидностей этого догово-
ра можно назвать в первую очередь договор
инвестиционного товарищества11. Базируясь на
классической модели договора простого товари-
щества, договор инвестиционного товарищества
имеет специфические черты, характерные для
товариществ — юридических лиц. Предусмотрены квалифицированные поря-
док и форма заключения договора инвестицион-
ного товарищества. В частности, договор считает-
ся заключенным, а внесенные в него изменения
считаются вступившими в силу со дня нотари-
ального удостоверения данного договора или
внесенных в него изменений. Договор инвести-
ционного товарищества с новым его участником
считается заключенным со дня нотариального
удостоверения соглашения о присоединении. DOI: 10.17803/1994-1471.2019.105.8.086-094 К таким чертам относятся: особая цель —
осуществление совместной инвестиционной
деятельности исключительно для извлечения
прибыли; управляющие товарищи осуществля-
ют от имени всех товарищей ведение общих
дел и несут солидарную ответственность за по-
следствия таких действий (бездействия); общее
число участников договора инвестиционного
товарищества не должно быть более 15, в со-
став общего имущества товарищей вносится
имущество, являющееся собственностью това-
рища, либо имущество, находящееся в его дове-
рительном управлении. В судебной практике от-
носительно распределения прибыли в данном
договоре указывается, что прежде чем требо-
вать распределения прибыли, товарищ обязан
внести вклад в общее дело12. В судебной практике существенные усло-
вия договора инвестиционного товарищества
включают в себя условия общей конструкции
(договора простого товарищества): условия о со-
вместной деятельности участников договора, об
их общей цели и об объединении ими вкладов,
предметом договора простого товарищества
является совместная деятельность по достиже-
нию общей цели13. К числу существенных усло-
вий договора инвестиционного товарищества
также относятся размер и порядок выплаты
вознаграждения управляющего товарища (ст. 5
Федерального закона «Об инвестиционном то-
вариществе»), политика ведения общих дел
(инвестиционная декларация) — ст. 8 данного
Федерального закона. Законом установлены ограничения для уча-
стия управляющего товарища в договорах: он не
вправе участвовать одновременно в двух и бо-
лее договорах инвестиционного товарищества,
если хотя бы один из них содержит запрет на
такое участие, в противном случае договор мо- Однозначно признаваемой судебной практи-
кой14 разновидностью договора простого това- 10 См.: постановление Пленума Верховного Суда РФ от 24 марта 2016 г. № 7 «О применении судами не-
которых положений Гражданского кодекса Российской Федерации об ответственности за нарушение
обязательств» ; постановление Пленума Верховного Суда РФ от 22 ноября 2016 г. № 54 «О некоторых
вопросах применения общих положений Гражданского кодекса Российской Федерации об обязательствах
и их исполнении» // СПС «КонсультантПлюс». 10 См.: постановление Пленума Верховного Суда РФ от 24 марта 2016 г. № 7 «О применении судами не-
которых положений Гражданского кодекса Российской Федерации об ответственности за нарушение
обязательств» ; постановление Пленума Верховного Суда РФ от 22 ноября 2016 г. № 54 «О некоторых
вопросах применения общих положений Гражданского кодекса Российской Федерации об обязательствах
и их исполнении» // СПС «КонсультантПлюс». едеральный закон от 28 ноября 2011 г. № 335‑ФЗ «Об инвестиционном товариществе» // СПС «Ко
льтантПлюс». 11 Федеральный закон от 28 ноября 2011 г. № 335‑ФЗ «Об инвестиционном товариществе» // СПС «Кон-
сультантПлюс». 12 См.: постановление Арбитражного суда Поволжского округа от 28 мая 2015 г. 10 См.: постановление Пленума Верховного Суда РФ от 24 марта 2016 г. № 7 «О применении судами не-
которых положений Гражданского кодекса Российской Федерации об ответственности за нарушение
обязательств» ; постановление Пленума Верховного Суда РФ от 22 ноября 2016 г. № 54 «О некоторых
вопросах применения общих положений Гражданского кодекса Российской Федерации об обязательствах
и их исполнении» // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август DOI: 10.17803/1994-1471.2019.105.8.086-094 № Ф06-22892/2015 по делу м.: постановление Арбитражного суда Поволжского округа от 28 мая 2015 г. № Ф06-22892/2015 по дел
А12-29061/2013 // СПС «КонсультантПлюс». м.: постановление Четырнадцатого арбитражного апелляционного суда от 27 января 2016 г. по дел
А05-6313/2015 // СПС «КонсультантПлюс». 14 См.: постановление ФАС Московского округа от 13 февраля 2014 г. № Ф05-17103/2013 по делу № А40-
24572/13-25-124 // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 88 Подузова Е. Б. Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) ду
Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) движимыми и недвижимыми вещами; 3) дело-
вая репутация и деловые связи. рищества (помимо договора инвестиционного
товарищества) являются страховые (перестрахо-
вочные) пулы (ст. 14.1 Закона РФ от 27 ноября
1992 г. № 4015-1 «Об организации страхового
дела в Российской Федерации»). Данные дого-
воры простого товарищества имеют специаль-
ные цели создания и деятельности — обеспече-
ние финансовой устойчивости его участников,
исполнение ими обязательств по страховым
выплатам, размер которых может превысить
собственные средства (капитал) одной страхо-
вой организации, — и действуют на принципах
сострахования или перестрахования. Существенными условиями договора горного
товарищества являются: данные, позволяющие
определить участок недр, право пользования
которым предоставлено или будет предоставле-
но пользователю недр в соответствии с законо-
дательством Российской Федерации о недрах
и по поводу деятельности на котором заключа-
ется договор; доли или порядок определения
долей сторон в распределяемой между сторо-
нами доходной части добытых при исполнении
обязательств по договору горного товарищества
углеводородного сырья и попутно извлекаемых
ресурсов или в распределяемых между сторона-
ми доходах от их реализации. Договор горного
товарищества должен быть заключен в простой
письменной форме. На стадии законопроекта15 предлагается
введение еще двух разновидностей договора
простого товарищества со специальной сферой
применения и субъектным составом. Так, по договору горного товарищества сто-
роны обязуются совместно осуществлять де-
ятельность по разработке на определенном
участке (участках) недр в соответствии с ус-
ловиями лицензии на пользование недрами,
которая получена или будет получена одной
из них в соответствии с законодательством
Российской Федерации о недрах, и распре-
делять между собой добытые сырье, ресур-
сы или доходы от их реализации. Содержание
данного договора всегда открыто для третьих
лиц. Стороны договора горного товарищества:
лицо — пользователь недр и оператор. Субъ-
ектный состав этого договора: российские юри-
дические лица, являющиеся коммерческими
организациями, а также иностранные юриди-
ческие лица. 15 Проект федерального закона «Об особенностях совместной деятельности в сфере недропользования
и о внесении изменений в отдельные законодательные акты Российской Федерации» (подготовлен
Минприроды России) (не внесен в ГД ФС РФ, текст по состоянию на 21 декабря 2016 г.) // СПС «Консуль-
тантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август DOI: 10.17803/1994-1471.2019.105.8.086-094 В то же
время положения Гражданского кодекса РФ не
оставляют сомнений в том, что сделка является
юридическим фактом (см., например, п. 1 ст. 8,
153, п. 2 ст. 307 ГК РФ), порождающим граждан-
ское правоотношение, включая обязательство. Как представляется, следует различать су-
ществование правоотношения, существование
прав, обязанностей и реализацию прав, испол-
нение обязанностей, то есть стадию реализации
содержания правоотношения. Правоотношение
возникает из факта совершения сделки либо из
факта наступления отлагательного условия сдел-
ки (ст. 157 ГК РФ). Срок исполнения обязатель-
ства не оказывает влияния на его существова-
ние (ст. 314 ГК РФ), срок (периоды) реализации
прав также не оказывает влияния на существо-
вание вещного правоотношения. В противном
случае, если обязательственное правоотно-
шение не существует до момента исполнения
обязательства, то и вещное правоотношение
не существует в те моменты, когда не проис-
ходит его реализации посредством активных К мнению о таком существе договора про-
стого товарищества затруднительно присоеди-
ниться. Исходя из позиции автора сделка соз-
дает возможность возникновения у лица, ее
совершившего, обязанностей и утраты им прав
помимо его воли, то есть сделка порождает не
правоотношение, а возможность его возник-
новения, отрицается волевой элемент сделки. В таком случае сделка не может быть квали-
фицирована в качестве юридического факта,
поскольку конститутивным признаком любого
юридического факта является создание, изме-
нение либо прекращение правоотношения17. Из
факта совершения сделки возникает правовая
связь, и эта связь обладает волевой составля-
ющей, субъектным составом, содержанием,
направлена на определенный объект. В то же
время положения Гражданского кодекса РФ не
оставляют сомнений в том, что сделка является
юридическим фактом (см., например, п. 1 ст. 8,
153, п. 2 ст. 307 ГК РФ), порождающим граждан-
ское правоотношение, включая обязательство. В том же законопроекте представлена двух-
ступенчатая модель организационных отно-
шений, реализуемая посредством заключения
и исполнения двух договоров об организации
совместной деятельности. Следует отметить не-
корректность применения термина «договор
совместного инвестирования» в свете наличия
легализованной и применяемой на практике
конструкции договора инвестиционного това-
рищества, имеющей свои особенности и сферу
применения. Субъектный состав договора совместного ин-
вестирования по общему правилу не ограничен. Кроме того, все стороны договора совместного
инвестирования могут выступать на стороне
оператора по договору горного товарищества,
управляющий товарищ по договору совмест-
ного инвестирования выполняет функции от-
ветственного оператора по договору горного
товарищества. 16 Скловский К. И. Сделка и ее действие. Комментарий главы 9 ГК РФ (понятие, виды и форма сделок. Не-
действительность сделок). М. : Статут, 2015.
17 См., например: Российское гражданское право / отв. ред. Е. А. Суханов. М. : Статут, 2011. Т. 1. (автор
главы — В. С. Ем). DOI: 10.17803/1994-1471.2019.105.8.086-094 Вторым предлагаемым к введению дого-
вором является договор совместного инвести-
рования в деятельность по разработке недр
(договор совместного инвестирования). Его
стороны обязуются определять в пределах,
установленных договором, порядок осущест-
вления одной из сторон договора от своего име-
ни и в общих интересах прав и обязанностей
оператора по договору горного товарищества
(далее — управляющий товарищ) в целях по-
следующего распределения между сторонами
договора совместного инвестирования получен-
ных управляющим товарищем углеводородно-
го сырья и попутно извлекаемых ресурсов или
доходов от их реализации, а также совместно
осуществлять финансирование участия управ-
ляющего товарища в договоре горного товари-
щества в качестве оператора. Таким образом,
договор совместного инвестирования привязан
к договору горного товарищества (если договор
горного товарищества, для целей участия управ-
ляющего товарища в котором заключается дого-
вор совместного инвестирования, не заключен,
договор совместного инвестирования вступает
в силу с момента заключения договора горного Вкладами сторон договора горного товари-
щества в совместную деятельность признается
все, что они вносят в общее дело, включая: 1) их
действия, направленные на достижение общих
целей, включая действия по предоставлению
друг другу необходимой информации, а также
взаимодействие с третьими лицами; 2) имуще-
ство, в том числе имущественные права, вклю-
чая исключительные права и право пользования 15 Проект федерального закона «Об особенностях совместной деятельности в сфере недропользования
и о внесении изменений в отдельные законодательные акты Российской Федерации» (подготовлен
Минприроды России) (не внесен в ГД ФС РФ, текст по состоянию на 21 декабря 2016 г.) // СПС «Консуль-
тантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 89 Гражданское и семейное право товарищества на тех условиях, которые прини-
мались во внимание сторонами договора со-
вместного инвестирования при его заключении,
кроме случаев, предусмотренных договором)
и заключается для его обслуживания. туре указывается, что он не имеет обязательств
и не порождает их, а все товарищи не выступают
как кредиторы или должники по отношению
друг к другу16. друг к другу . К мнению о таком существе договора про-
стого товарищества затруднительно присоеди-
ниться. Исходя из позиции автора сделка соз-
дает возможность возникновения у лица, ее
совершившего, обязанностей и утраты им прав
помимо его воли, то есть сделка порождает не
правоотношение, а возможность его возник-
новения, отрицается волевой элемент сделки. В таком случае сделка не может быть квали-
фицирована в качестве юридического факта,
поскольку конститутивным признаком любого
юридического факта является создание, изме-
нение либо прекращение правоотношения17. Из
факта совершения сделки возникает правовая
связь, и эта связь обладает волевой составля-
ющей, субъектным составом, содержанием,
направлена на определенный объект. DOI: 10.17803/1994-1471.2019.105.8.086-094 Существенными условиями договора со-
вместного инвестирования являются: данные,
позволяющие определить участок недр, по по-
воду деятельности на котором заключен дого-
вор; сведения о договоре горного товарище-
ства, для целей участия управляющего товарища
в котором заключается договор совместного
инвестирования, если такой договор горного
товарищества заключен; наименование сторо-
ны — управляющего товарища; доли сторон
в распределяемой между ними доходной ча-
сти добытых углеводородного сырья и попутно
извлекаемых ресурсов или в распределяемых
между сторонами доходах от их реализации. Договор совместного инвестирования должен
быть заключен в простой письменной форме. Как представляется, следует различать су-
ществование правоотношения, существование
прав, обязанностей и реализацию прав, испол-
нение обязанностей, то есть стадию реализации
содержания правоотношения. Правоотношение
возникает из факта совершения сделки либо из
факта наступления отлагательного условия сдел-
ки (ст. 157 ГК РФ). Срок исполнения обязатель-
ства не оказывает влияния на его существова-
ние (ст. 314 ГК РФ), срок (периоды) реализации
прав также не оказывает влияния на существо-
вание вещного правоотношения. В противном
случае, если обязательственное правоотно-
шение не существует до момента исполнения
обязательства, то и вещное правоотношение
не существует в те моменты, когда не проис-
ходит его реализации посредством активных Применительно к общей категории договора
простого товарищества в юридической литера- Актуальные проблемы российского права. 2019. № 8 (105) август 90 Подузова Е. Б. Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) ду
Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) правомерности подобного состава и правового
режима общего имущества товарищей выступа-
ет лишь специальное правовое регулирование
соответствующих общественных отношений. действий. Если вещь, например, украдена в та-
кой момент, то нет оснований для применения
гражданско-правовых способов защиты (ст. 12,
301—303 ГК РФ), поскольку в этот момент не
было и самого правоотношения собственности. Договор простого товарищества, будучи од-
ним из видов организующих сделок, обладает
ее конститутивными признаками — неимуще-
ственной направленностью, организационным
основанием. Договор простого товарищества
носит невзаимный характер, в нем отсутствует
встречное предоставление, он порождает ор-
ганизационное обязательство. Имущественный
элемент в этом договоре выступает как основа
совместной деятельности и не влияет на об-
щую организующую направленность. Договор
простого товарищества порождает внутренние
и внешние правоотношения. Внутренние пра-
воотношения — организационные, существуют
между сторонами этого договора. Внешние от-
ношения возникают на базе организационных
между стороной (сторонами) этого договора
и третьими лицами и носят как имущественный,
так и неимущественный характер. Внутренние
правоотношения организуют внешние, посред-
ством которых образуется прибыль (убытки) то-
варищей. 18 Определение Высшего Арбитражного Суда РФ от 20 августа 2013 г. № ВАС-8174/13 по делу № А70-
3394/2012 ; постановление Арбитражного суда Волго-Вятского округа от 9 декабря 2014 г. № Ф01-
4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс». 20 См.: Companies Act, 2006. д
у
у
19 Companies Act, 2006 // URL: http://www.legislation.gov.uk/ukpga/2006/46/section/29 (дата обращения:
28 января 2019 г.). 3394/2012 ; постановление Арбитражного суда Волго Вятского округа от 9 декабря 2014 г. № Ф01
4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс».
19 Companies Act, 2006 // URL: http://www.legislation.gov.uk/ukpga/2006/46/section/29 (дата обращения:
28 января 2019 г.). 18 Определение Высшего Арбитражного Суда РФ от 20 августа 2013 г. № ВАС-8174/13 по делу № А70-
3394/2012 ; постановление Арбитражного суда Волго-Вятского округа от 9 декабря 2014 г. № Ф01-
4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс».
19 Companies Act, 2006 // URL: http://www.legislation.gov.uk/ukpga/2006/46/section/29 (дата обращения:
28 января 2019 г.).
20 См : Companies Act 2006 DOI: 10.17803/1994-1471.2019.105.8.086-094 В судебной практике подтверждается
организующая природа договора простого то-
варищества18. Полагаем, что договор простого товарище-
ства — это юридический факт, порождающий
правоотношение. Согласно ст. 1041 ГК РФ совер-
шение действий по достижению общей цели со-
вместной деятельности является обязанностью
по договору простого товарищества. Данная
обязанность может быть исполнена принуди-
тельно при применении такого способа защиты
гражданских прав, как присуждение к испол-
нению обязанности в натуре (ст. 12 ГК РФ). До-
говор простого товарищества порождает два
вида правоотношений — обязательственное
и вещное, поскольку согласно п. 1 ст. 1043 ГК РФ
внесенное товарищами имущество, которым
они обладали на праве собственности, а также
произведенная в результате совместной дея-
тельности продукция и полученные от такой
деятельности плоды и доходы признаются их
общей долевой собственностью, если иное не
установлено законом или договором простого
товарищества либо не вытекает из существа обя-
зательства. Имущество товарищей — сложный
объект гражданских прав, включающий в себя
деньги, иное имущество, профессиональные
и иные знания, навыки и умения, а также дело-
вую репутацию и деловые связи (п. 1 ст. 1042
ГК РФ). Общее имущество товарищей состоит из
ряда объектов, не относящихся к числу объектов
гражданских прав, а также к числу непереда-
ваемых объектов гражданских прав (деловая
репутация, ст. 150 ГК РФ). В рамках договора
простого товарищества возникает вещное право
на исключительное право, если оно входит в со-
став общего имущества товарищей, что также
вступает в противоречие с положениями ч. IV
Гражданского кодекса РФ. Аргументом в пользу Следует обратить внимание, что в системе
общего права существуют договоры (соглаше-
ния) об организации совместной деятельности
(ст. 8 английского Акта о компаниях 2006 г.19,
в английской бизнес-сфере используется кон-
струкция соглашения о партнерстве20). Дан-
ные соглашения относятся к числу договоров,
которые возможно принудительно исполнить
посредством обращения в суд, в понимании
английской доктрины и практики эти договоры
обладают встречным предоставлением. Разновидности договора простого товарище-
ства, как и сам договор простого товарищества, 18 Определение Высшего Арбитражного Суда РФ от 20 августа 2013 г. № ВАС-8174/13 по делу № А70-
3394/2012 ; постановление Арбитражного суда Волго-Вятского округа от 9 декабря 2014 г. № Ф01-
4991/2014 по делу № А43-17526/2013 // СПС «КонсультантПлюс». Актуальные проблемы российского права. 2019. № 8 (105) август 91 Гражданское и семейное право относятся по своей правовой природе к числу
организующих сделок с имущественными эле-
ментами, выступающими основой совместной
деятельности. Изменение конструкции договора
простого товарищества посредством введения
и предложения новых его разновидностей явля-
ется важным достижением реформы граждан-
ского законодательства. DOI: 10.17803/1994-1471.2019.105.8.086-094 Однако современные
конструкции этого договора и его разновидно-
стей, предлагаемые к введению, не учитывают
организующую правовую природу отношений
товарищества, особую судьбу имущественной
основы деятельности товарищей, взаимосвязь
внутренних и внешних правоотношений. Зако-
нодательство о договорах простого товарище-
ства нуждается в дальнейшем изменении. дукт (товар, услуга), подлежащий совместному
потреблению. Для заключения договора про-
стого товарищества и его видов используется
классическая модель организации контрактных
связей. Напротив, для заключения соглашений
о совместном использовании товаров и услуг
во многих случаях применяются агрегаторы как
посредники между сторонами. Договор про-
стого товарищества порождает два вида пра-
воотношений — обязательственное и вещное,
поскольку согласно п. 1 ст. 1043 ГК РФ внесенное
товарищами имущество, которым они облада-
ли на праве собственности, а также произве-
денная в результате совместной деятельности
продукция и полученные от такой деятельности
плоды и доходы признаются их общей долевой
собственностью. Соглашения о совместном ис-
пользовании товаров и услуг отношений соб-
ственности не порождают. Возникает вопрос в целесообразности адап-
тации законодательства о договорах простого
товарищества к соглашениям о совместном ис-
пользовании товаров и услуг. Договор простого
товарищества не тождественен данным согла-
шениям, поскольку в соглашениях о совмест-
ном использовании товаров и услуг отсутствует
единая совместная общая цель, к достижению
которой стремятся все участники любого вида
договора простого товарищества. В свою оче-
редь, в договоре простого товарищества изна-
чально отсутствует единый материальный про- Общей чертой договоров простого товарище-
ства и соглашений о совместном использовании
товаров и услуг является совместная деятель-
ность их сторон. В связи с этим при разработке
норм, регулирующих отношения по совмест-
ному использованию товаров и услуг, возмож-
но учитывать субсидиарное применение норм
о договорах простого товарищества к таким от-
ношениям. БИБЛИОГРАФИЯ ду
Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy) 9. De Stefano V. The rise of the «just-in-time workforce»: On-demand work, crowdwork and labour protection
in the «gig-economy» // Conditions of work and employment. Series No. 71. — Geneva : ILO, 2016. — URL:
http://www.ilo.org/wcmsp5/groups/public/—-ed_protect/—-protrav/—-travail/documents/publication/
wcms_443267.pdf (дата обращения: 31 января 2019 г.). 10. Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy
International, Forthcoming. — November 24, 2015. 10. Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy
International, Forthcoming. — November 24, 2015. // 11. Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition
Policy International. — 2015. — № LS 15-11; № 15—29. Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation
the Sharing Economy // George Mason Law & Economics Research Paper. — 2015. — № 15—01. 13. Rogers B. The Social Costs of Uber // University of Chicago Law Review Dialogue. — 2015. — № 2 13. Rogers B. The Social Costs of Uber // University of Chicago Law Review Dialogue. — 2015. — № 28. Материал поступил в редакцию 20 февраля 2019 г. БИБЛИОГРАФИЯ 1. Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая
бухгалтерия. — 2016. — № 9. — С. 130—143. 1. Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая
бухгалтерия. — 2016. — № 9. — С. 130—143. 1. Бычков А. Посредничество при оказании юридических услуг: практика, риски, перспективы // Новая
бухгалтерия. — 2016. — № 9. — С. 130—143. у
р
2. Иванов А. А. Бизнес-агрегаторы и право // Закон. — 2017. — № 5. — С. 145—157. 3. Молотников А. Е., Архипов Е. В. Социальные сети и компании-агрегаторы: правовые аспекты дея-
тельности // Предпринимательское право. — 2017. — № 4. 3. Молотников А. Е., Архипов Е. В. Социальные сети и компании-агрегаторы: правовые аспекты дея-
тельности // Предпринимательское право. — 2017. — № 4. 4. Российское гражданское право / отв. ред. Е. А. Суханов. — М. : Статут, 2011. — Т. 1. 4. Российское гражданское право / отв. ред. Е. А. Суханов. — М. : Статут, 2011. — Т. 1. 5. Савельев А. И. Электронная коммерция в России и за рубежом: правовое регулирование. — М. : С
2016. 5. Савельев А. И. Электронная коммерция в России и за рубежом: правовое регулирование. — М. : Статут,
2016. Скловский К. И. Сделка и ее действие. Комментарий главы 9 ГК РФ (понятие, виды и форма сдело
Недействительность сделок). — М. : Статут, 2015. 7. Челышев М. Ю. Вопросы межотраслевых связей гражданского права в Концепции развития граж-
данского законодательства и проекте Гражданского кодекса Российской Федерации // Гражданское
право. — 2011. — № 1. — С. 3—7. 7. Челышев М. Ю. Вопросы межотраслевых связей гражданского права в Концепции развития граж-
данского законодательства и проекте Гражданского кодекса Российской Федерации // Гражданское
право. — 2011. — № 1. — С. 3—7. 8. Чесалина О. В. Работа на основе интернет-платформ (crowdwork и work on demand via apps) как вызов
трудовому и социальному праву // Трудовое право в России и за рубежом. — 2017. — № 1. 8. Чесалина О. В. Работа на основе интернет-платформ (crowdwork и work on demand via apps) как вызов
трудовому и социальному праву // Трудовое право в России и за рубежом. — 2017. — № 1. Актуальные проблемы российского права. 2019. № 8 (105) август 92 Подузова Е. Б. Новые виды договора простого товарищества в контексте коллективного использования товаров и услуг (sharing economy Подузова Е. Б. 21 The research has been carried out with the financial support of RFBR within the framework of scientific project
№ 18-29-16054 “Conceptual bases of legal regulation of relations of sharing economy in the era of digital
technologies.” Актуальные проблемы российского права. 2019. № 8 (105) август Подузова Е. Б. NEW TYPES OF THE PARTNERSHIP AGREEMENT IN THE CONTEXT
OF COLLECTIVE USE OF GOODS AND SERVICES (SHARING ECONOMY)21 PODUZOVA Ekaterina Borisovna, PhD in Law, Associate Professor of the Department of Civil Law,
Associate Professor of the Department of Notaries of the Kutafin Moscow State Law University
(MSAL)
ekaterinak7785@yandex.ru
125993, Russia, Moscow, ul. Sadovaya-Kudrinskaya, d. 9 PODUZOVA Ekaterina Borisovna, PhD in Law, Associate Professor of the Department of Civil Law,
Associate Professor of the Department of Notaries of the Kutafin Moscow State Law University
(MSAL) Abstract. The modern socio-economic context of shared consumption (sharing economy) sets new goals and
objectives of using joint activities. The article presents the main theoretical and practical problems of types of the partnership agreement as an
organizational agreement, forms of organization and conduct of joint activity. The article is prepared with due regard to the reform of contract law of the Russian Federation, new trends in the
science of civil law and in law enforcement practice. Also, the article takes into account the jurisprudence that
contains new approaches to the interpretation of an obligation and agreement under consideration. In this regard,
the decisions of the Plenum of the Supreme Court of the Russian Federation (for example, the Decision dated
March 24, 2016 No. 7 “On Application by Courts of Certain Provisions of the Civil Code of the Russian Federation
Concerning the Breach of the Obligations,» 22 November 2016 No. 54 “On Certain Issues of Application of General
Provisions of the Civil Code of the Russian Federation on Obligations and Their Performance”) are subjected to
thorough examination. Particular attention is paid to the legal nature, constitutional features of certain types of the partnership agreement,
their manifestations in modern civil legislation and practice of application. Keywords: collective use, partnership agreement, transaction, types of contract, legal nature, organization,
organizing agreement, agreement on organization of joint activities, property element, consideration, investment
partnership, mining partnership. 21 The research has been carried out with the financial support of RFBR within the framework of scientific project
№ 18-29-16054 “Conceptual bases of legal regulation of relations of sharing economy in the era of digital
technologies.” 93 Гражданское и семейное право Актуальные проблемы российского права. 2019. № 8 (105) август REFERENCES (TRANSLITERATION) 1. Bychkov A. Posrednichestvo pri okazanii yuridicheskih uslug: praktika, riski, perspektivy // Novaya
buhgalteriya. — 2016. — № 9. — S. 130—143. 2. Ivanov A. A. Biznes-agregatory i pravo // Zakon. — 2017. — № 5. — S. 145—157. 3. Molotnikov A. E., Arhipov E. V. Social’nye seti i kompanii-agregatory: pravovye aspekty deyatel’nosti //
Predprinimatel’skoe pravo. — 2017. — № 4. 4. Rossijskoe grazhdanskoe pravo / otv. red. E. A. Suhanov. — M. : Statut, 2011. — T. 1. 5. Savel’ev A. I. Elektronnaya kommerciya v Rossii i za rubezhom: pravovoe regulirovanie. — M. : Statut, 5. Savel ev A. I. Elektronnaya kommerciya v Rossii i za rubezhom: pravovoe regulirovanie. M. : Statut, 2016. 6. Sklovskij K. I. Sdelka i ee dejstvie. Kommentarij glavy 9 GK RF (ponyatie, vidy i forma sdelok. Nedejstvitel’nost’
sdelok). — M. : Statut, 2015. 7. Chelyshev M. Yu. Voprosy mezhotraslevyh svyazej grazhdanskogo prava v Koncepcii razvitiya grazhdanskogo
zakonodatel’stva i proekte Grazhdanskogo kodeksa Rossijskoj Federacii // Grazhdanskoe pravo. — 2011. —
№ 1. — S. 3—7. 8. Chesalina O. V. Rabota na osnove internet-platform (crowdwork i work on demand via apps) kak vyzov
trudovomu i social’nomu pravu // Trudovoe pravo v Rossii i za rubezhom. — 2017. — № 1. 9. De Stefano V. The rise of the «just-in-time workforce»: On-demand work, crowdwork and labour protection
in the «gig-economy» // Conditions of work and employment. Series No. 71. — Geneva : ILO, 2016. — URL:
http://www.ilo.org/wcmsp5/groups/public/—-ed_protect/—-protrav/—-travail/documents/publication/
wcms_443267.pdf (data obrashcheniya: 31 yanvarya 2019 g.). 10. Edelman B. G. Whither Uber? Competitive Dynamics in Transportation Networks // Competition Policy
International, Forthcoming. — November 24, 2015. 11. Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition
Policy International. — 2015. — № LS 15-11; № 15—29. 11. Geradin D. Should Uber Be Allowed to Compete in Europe? And If so How? // Forthcoming in Competition
Policy International. — 2015. — № LS 15-11; № 15—29. 12. Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation of
the Sharing Economy // George Mason Law & Economics Research Paper. — 2015. — № 15—01. 12. Rauch D. E., Schleicher D. Like Uber, but for Local Governmental Policy: The Future of Local Regulation of
the Sharing Economy // George Mason Law & Economics Research Paper. — 2015. — № 15—01. 13. Rogers B. REFERENCES (TRANSLITERATION) The Social Costs of Uber // University of Chicago Law Review Dialogue. — 2015. — № 28. Актуальные проблемы российского права. 2019. № 8 (105) август 94
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Triangulating Moderate Impact of Social Media Marketing Communication Between Performance Expectancy, Effort Expectancy and Social Influence on Business Performance in Cashew Industry in Sri Lanka
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Triangulating Moderate Impact of Social Media Marketing Communication
Between Performance Expectancy, Effort Expectancy and Social Influence on
Business Performance in Cashew Industry in Sri Lanka Triangulating Moderate Impact of Social Media Marketing Communication
Between Performance Expectancy, Effort Expectancy and Social Influence on
Business Performance in Cashew Industry in Sri Lanka Sandunima Kaluarachchi
(
chamodisandunima@gmail.com
) SLIIT
https://orcid.org/0009-0008-4673-4591 1. Introduction With the exponential growth of internet- enabled social media subscribers, advancements in digital media platform have created new value positions in the
cashew industry. The multiple benefits of social media marketing (e.g. ubiquity, mobility, internet access convenience, personalization, flexibility and
information distribution) enable new marketing services that can address previously unmet retail needs (Yang, 2010, M. Kang, 2014, Duffett, 2017). Social
media marketing services can provide e-commerce activities in a different way than traditional shopping services. Unlike the traditional market, SMMC enables
salespeople to send personalized information and pinpoint user location services to customers in real-time interactions via an online platform. Users can create personalized online business pages, communicate and interact with customers, and exchange content created by themselves (user-generated
content) or information from other cashew business related sources on social media platform such as Facebook, Twitter, LinkedIn, YouTube, WhatsApp,
Instagram and Google+ (Duffett, 2017, Tarsakoo and Charoensukmongkol, 2020). Traditional media such as television, radio, newspapers, banners, leaflets
and magazines have traditionally communicated business conduct and how sellers think, but in the 21st century, social media has begun to replace traditional
media’s enduring and significant role in the business sector. Zhang and Du (2020) stated, from the perspective of entrepreneurs this shift in behavior provides
both an opportunity and challenge. Marketers are increasingly relying on social media to market and promote their cashew items among customers. Furthermore, incorporating content that is both amusing and relevant would encourage customers to interact and share the information with their friends (Raji
et al., 2019). This important aspect, often known as “word of mouth” (WOM), might be seen as the future of SMMC. According to the suggestion of Chen and Qasim (2021), through seamless communication social media marketing channels can provide the benefits of an
optimal e-business experience. For example, majority of famous sports arenas, restaurants, bars, theaters, and catering services in the United States (US) as
well as all major supermarkets provide cashew nut-based products. In addition, they serve as bulk supplies for producers of confectionery such as chocolate,
ice cream, cakes, and others. Moreover, Feni, a locally created alcoholic beverage made from cashew apples and only accessible in Goa, has a 45 percent
higher strength. In Malaysia, Forager cashew milk yogurt was also introduced. The product is available for purchase online. Singh et al. 1. Introduction (2021) study, prove in
India, dried fruit sales increased by 20% after they adapted to an online marketing platform and their traditional sales decreased by 9%. Therefore, researchers
demonstrated that cashew nut social media marketing can reach a large audience and boost business success. Though these services are promising, consumer adoption of social media marketing channels in Sri Lanka is unclear because rural districts are less likely to
use a variety of digital marketing platforms than sellers in India and European countries. Statistics data shows cashew contributes 1% of the national GDP
(Gross domestic product) (INC, 2021). Further Kapurubandara (2009) reported that, the majority of Sri Lankan sellers in rural areas are unwilling to use social
media for business purposes. This could be the reason the social media marketing platform was developed and positioned without a thorough grasp of rural
sellers. Moreover, DCSSL (2020) statistics data proves, the total digital literacy rate is 50.1% in Sri Lanka, with urban 66.3%, rural 48.1%, and estate 25.6%, and
also given the rural area representation in the statistics, sellers in the Kajugama district study area should not expect much awareness of digital marketing
platforms. Therefore, lack of digital awareness is a big issue in the social media marketing context in Sri Lanka. According to Chambers et al. (2020) E-
marketing is another highly affordable and practical strategy for Small & medium enterprises (SME), yet the majority of SMEs lack technological expertise. h
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k Though these services are promising, consumer adoption of social media marketing channels in Sri Lanka is unclear because rural districts are less likely to
use a variety of digital marketing platforms than sellers in India and European countries. Statistics data shows cashew contributes 1% of the national GDP
(Gross domestic product) (INC, 2021). Further Kapurubandara (2009) reported that, the majority of Sri Lankan sellers in rural areas are unwilling to use social
media for business purposes. This could be the reason the social media marketing platform was developed and positioned without a thorough grasp of rural
sellers. 1. Introduction Moreover, DCSSL (2020) statistics data proves, the total digital literacy rate is 50.1% in Sri Lanka, with urban 66.3%, rural 48.1%, and estate 25.6%, and
also given the rural area representation in the statistics, sellers in the Kajugama district study area should not expect much awareness of digital marketing
platforms. Therefore, lack of digital awareness is a big issue in the social media marketing context in Sri Lanka. According to Chambers et al. (2020) E-
marketing is another highly affordable and practical strategy for Small & medium enterprises (SME), yet the majority of SMEs lack technological expertise. Furthermore, since the cashew nut sector is linked to the micro cultivation industry, it is also. Studies showing that the majority of business owners lack proper
knowledge of social media and how to use it for their business reveal an empirical gap in the literature. However, there has been less advancement in cashew
nut social media marketing, and there has been lack of study on the subject in Sri Lanka or other countries. Thus, it is timely to examine the underlying drivers
of Sri Lankan sellers’ intention to use digital marketing platforms. Based on sellers' perceptions of online business capabilities, this study will provide
implications for business and social media marketing in building e-business services. marketing is another highly affordable and practical strategy for Small & medium enterprises (SME), yet the majority of SMEs lack technological expertise. Furthermore, since the cashew nut sector is linked to the micro cultivation industry, it is also. Studies showing that the majority of business owners lack proper
knowledge of social media and how to use it for their business reveal an empirical gap in the literature. However, there has been less advancement in cashew
nut social media marketing, and there has been lack of study on the subject in Sri Lanka or other countries. Thus, it is timely to examine the underlying drivers
of Sri Lankan sellers’ intention to use digital marketing platforms. Based on sellers' perceptions of online business capabilities, this study will provide
implications for business and social media marketing in building e-business services. The rest of this paper is structured as follows. Section 2 examines the literature on industry business performance and technology adaptation as well as the
primary antecedent variables. A conceptual model and associated hypotheses are advanced from this point of view. section 3 presents the research
methodology. Research Article Keywords: Social media marketing communication, Business performance, Performance expectancy, Effort expectancy, Social influence
Posted Date: April 20th, 2023 Keywords: Social media marketing communication, Business performance, Performance expectancy, Effort expectancy, Social influence DOI: https://doi.org/10.21203/rs.3.rs-2838965/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/14 Abstract The theoretical triangulation (TT) in social media marketing communication and business performance is rare to observe. The purpose of this paper is to
present a critical review of the contributions of multiple theories of performance expectancy theory (PET), effort expectancy theory (EET), and social influence
theory (SIT) and explain whether social media marketing communication (SMMC) moderates the relationship between those theories and business
performance (BP). A sample of 218 cashew salespeople from Sri Lanka were surveyed, and the data were analysed using structural equation modelling (SEM)
through AMOS 26.0. The finding of the SEM analysis indicated that PE, EE and SI have significant positive relationship with potential sellers’ business
performance. The moderation effect results showed that SMMC was a poor moderator of the relation between the EE and BP, as well as the SI and BP,
nevertheless, the result showed that SMMC is a partial moderator of the relation between the PE and BP. The current study method necessitates caution in
generalizing the findings. More variables may be introduced in future studies to explore the moderation effect between the predictor and BP of the unified
theory of acceptance and use of technology (UTAUT) framework. The current study helps cashew sellers widen their horizons while evaluating the practical
results of academic research. The findings of this study not only demonstrate that the TT appears to be acceptable in explaining predictors and BP in the
cashew industry, but also indicate the resilience of the TT’s ability to predictors and BP in a different research context. Through SEM analysis, this study
attempts to add SMMC as a moderator in the UTAUT framework and investigate the moderating effect between predictors and BP. In order to that this study
aims to bridge the gap between these moderating effects and how salespeople in SMMC impact BP. 1. Introduction Section 4 presents the data analysis and results. Section 5 contains an in-depth discussion of the results. The various causal factors are
highlighted and paper close by discussing some implications for future researchers. 2. Literature Review Page 2/14 Page 2/14 This study contends that there is a relationship between predictor variables and business performance and that there is no moderating impact between social
media marketing communication comprehension and predictor variables on business performance. However, researchers provide evidence that theoretical
triangulation happened in this study. The literature addressed various variables from the UTAUT model in order to suggest technological aspects of cashew
sellers to improve business performance and profit. A type of online marketing that uses social media apps as a marketing tool is knows as social media
marketing communication (Duffett, 2017, Raji et al., 2019, Samoggia et al., 2019). For the purposes of this study, the moderating impact of social media
marketing communication activities refers to the expectations of sellers who do cashew business through the online. Cashew sellers and business performance Business performance (BP), which is closely related to commercial effectiveness, is determined by an entrepreneur's capacity to put best practices into
practice in order to deliver a good or service that meets customers' expectations. As a result, the cashew nut industry is working hard to develop the
competencies and skills necessary to satisfy the customers by anticipating and meeting their needs. According to Peterson and Altounian (2019) BP is the
ability of a particular business to carry out a plan in order to achieve business goals. Therefore, it was considered an essential tool for merchants and
entrepreneurs to evaluate the effectiveness of their business. BP can measure a combination of financial and non-financial measurements. Financial
performance can be measured based on sales volume, monthly profit, and total assets; non-financial performance can also be measured based on customer
retention rate, business reputation, and product and service innovation. Moreover, the role of cashew salespeople in the convenience and quality cashew marketing and distribution system is a widely debated at the global level. Sellers are crucial players in the promotion of marketing and more sustainable business patterns. They have the ability to influence cashew producers,
manufacturers and consumers, as well as guide customers to buy or recommend the product to others. Cashew sales are currently carried out at the retail and
grocery level (Priyashantha, 2022). Previous studies have investigated this trend to better understand whether sellers’ marketing activities impact business
performance (Liu et al., 2022, Peterson and Altounian, 2019). According to recent statistics Priyashantha (2022), the cashew business’s performance has
declined steadily over the last five years. 2. Literature Review Every business’s reputation is important, and they frequently strive to create a trustworthy, responsible and carrying
image in the minds of their customers (Ambroise and Prim-Allaz, 2017, Nguyen and Adomako, 2021). Therefore, the presence, innovations, communication
patterns, physical location and marketing platform of various product categories might influence customer preferences during purchasing and consumption
processes. Furthermore, the previous studies demonstrated, the cashew business sector can create job opportunities and more revenue. However, Sri Lanka still only
earns a small portion of the cashew nut value chain. Moreover, Araujo et al. (2021) study proved it should be mentioned that although though some
professions appear to be reserved for males, women nonetheless make significant contributions to these fields by managing households, processing cashews,
and performing other jobs that generate income. Because the majority of small cashew business owners are women, this sector can see more female
employees than male. Many small and medium cashew businesses in the country are still struggling to survive. Small businesses to close their doors due to a
lack of raw materials, high production costs, and lack of innovation in comparison to other countries. Unified theory of acceptance and use of technology UTAUT is a well-known theory developed by Venkatesh et al. (2003). This model is used to analyze individuals’ intentions to use technology. The model seeks
to comprehend information system use and intention to use as a function of contracts drawn from a variety of theories including performance and effort
expectancy (expectancy theory), social influence (social cognitive theory) (Reyes-Mercado, 2018). Social media marketing communication moderates the
effects of these variables on use and intention to use technology in present study. UTAUT was created in response to the needs of rural enterprises. Voluntary
use in this context refers to the optional use of the e-WOM system, but UTAUT has also been applied to salespeople. Furthermore, the information systems
literature has extended and updated the UTAUT model to include factors from marketing process (awareness, motivation and knowledge). This paper employs
UTAUT for the sake of technology awareness and because rural business owners in developing countries have a low rate of adaptability to new technology. F
i
th h
th Framing the hypotheses 3. Research Methodology As per Sridharan (2021), triangulation, first used in the social sciences and psychology, is now used in a number of management studies to solve issues with
theory formulation and interpretation. According to Modell (2015), there are five basic types of triangulation. First, by using “data triangulation”, several data
sources can be used in a single study. Second, “methodological triangulation” entails examining the program using a variety of qualitative and/or quantitative
techniques. Third, utilizing numerous separate "investigators" in the analysis process is known as "investigator triangulation." Fourth, using several viewpoints
or disciplines to interpret a single set of data is known as “theory triangulation”. Fifth, “Environmental Triangulation” uses a variety of settings, locations, and
other crucial aspects of the environment where the study was conducted, such the time of day or season. As a result, this study contributes the fourth element,
which is applied to studying more than one theory. Based on conceptual framework those are the performance expectancy, effort expectancy and social
influence. Furthermore, many scholars who are interested in social media marketing and business performance have discovered that the employment of
multiple theories, also referred to as "theoretical triangulation" or "theoretical pluralism," is useful (Hoque et al., 2015). Therefore, theoretical triangulation has
this as one of its espoused benefits. The entire quantitative method is utilized in this study to collect data. According to Mustafa et al. (2020), approaches to quantitative data that use numerical
measurement are known as quantitative methods. Researchers use a set of questionnaires built to randomly distribute to cashew sellers in Kajugama district. A total of 218 sets of questionnaires distributed to small and large cashew sellers throughout Kajugama district in Sri Lanka. For analysis, 216 sets of fully
filled out questionnaires were received. This amount allows for a 99% response rate to be recorded. Based on the suggestion of over 80% from Mustafa et al. (2020), this response rate is acceptable. The structural equation modeling (SEM) method was used to analyze the questionnaire set. The second generation of
multivariate analysis used in the study is called SEM (Pim-Wusu et al., 2022). In order to analyze the data collected by questionnaires, academics and
researchers both use this strategy extremely frequently. The usage of AMOS software to adapt this SEM method is highly appropriate because the outcome of
the analysis will be more precise. Performance expectancy According to the above arguments SI is hypothesized to be: H3: Social influence has a positive relationship with seller’s business performance. Triangulating the three theories with social media marketing communication Researcher focus on the triangulation theory: the performance expectancy, effort expectancy, and the social influence. In order to that researchers try to
understand the moderate impact of triangulating the three theories with business performance through the social media marketing communication. According
to Rahi and Abd.Ghani (2019) the extent to which adopting a social media marketing communication (SMMC) as a business platform will assist the business
growth and contribute to reach large audience is referred to as PE. Results Chua et al. (2018) proved that as it supports sales targets and maximizes revenue,
PE plays a crucial role in influencing sellers. Rahi et al. (2019) proved that PE is the most powerful predictor of behavioral intention to use social media
marketing tools. A similar variable to EE is perceived ease of use, which refers to the cognitive effort associated with adapting social media platform. Reyes-
Mercado (2018) and Abu Afifa et al. (2022) find that one of the more critical antecedents shaping users’ perceptions toward BP and SMMC is perceived ease
of use. One of the primary characteristics of social media is that it allows users to market products to one another while conducting physical business, and it
allows users to monitor, reviewing and share business details on the go via online services. There is value in expanding the business to measure it. However,
Wu and Ho (2022) claimed that, EE has a less significant impact on the behavioral intentions of SMMC users in Taiwan, as it is insufficient to attract business
owners solely through EE factors. People are more likely to use the SMMC platform to communicate and exchange information with their reference groups,
such as friends, family, and coworkers. SI indicates how strongly people believe significant others, such as family and friends, believe they should use an
SMMC (Zhang and Gong, 2021a, Zhang and Gong, 2021b, Rahi and Abd.Ghani, 2019). They have a tendency to affect people's decisions to adopt or use
social media. Chua et al. (2018) proposed in the research of the SMMC, SI is significant in determining users' behavioral intentions. As a result, the authors
arrive at the following hypothesis: H4: Social media marketing communication has the moderating impact between three theories and business pe Performance expectancy Performance expectancy The degree to which salespeople believe that adapting technology will boost their individual performance is referred to as performance expectancy (PE) (Abu
Afifa et al., 2022). According to the original UTAUT formulation, PE is the most powerful predictor of behavioral intention (Guggemos et al., 2020, Garone et al.,
2019). UTAUT models are congruent with studies in the domain of information technology applied to BP. Rahi and Abd.Ghani (2019) analyze the adoption of
electronic word of mouth (e-WOM) marketing system, in developing countries is examined, and it is discovered that PE has significant positive relationship
with BP. González Bravo et al. (2020); Bui and Fowler (2022) and Burau and Andersen (2014) combine UTAUT with theoretical triangulation to analyze the
intent to use technology. They discover that, when technology provides users with credible information, PE influencer user intention to use. As a result, it is
reasonable to believe that higher PE will result in a more favorable desire to do business online. In light of the facts presented, the following hypothesis is
proposed: H1: Performance expectancy has a positive relationship with seller’s business performance. Effort expectancy Effort expectancy Effort expectancy (EE) is related to the user’s expectation of ease. Authors like Rahi et al. (2019) demonstrated that, when users believe that social media
marketing is easy to use and does not require much effort, they are more likely to adopt online business. Joa and Magsamen-Conrad (2022) and Mensah and Page 3/14 Onyancha (2021) argued that users are more likely to adopt online marketing if they perceive is simple. Previous research has found a relationship between EE
and user intent to engage in online business (Abu Afifa et al., 2022, Aytekin et al., 2022, Reyes-Mercado, 2018). As a result, EE is hypothesized to be: H2: Effort expectancy has a positive relationship with seller’s business performance. The relationship between social influence (SI) and BP is a hotly disputed topic. The level of societal pressure imposed on individuals to adopt new
technologies is characterized as SI (Abu Afifa et al., 2022, Rahi and Abd.Ghani, 2019). According to Chen et al. (2021), SI will positively affect user intent to
adopt online business. previous research has found that SI has a significant relationship on online business performance (Reyes-Mercado, 2018, Chan et al.,
2022, Wu and Ho, 2022). Demographic characteristics of the sample Demographic characteristics of the sample The demographic characteristics of the sample are shown in Table 1. The study sample’s sellers were mostly female (30-40 years old: 75.2%). This overall age
group included the most dynamic sellers portion of all respondents (Akbarov, 2022). Furthermore, by studying seller’s online marketing knowledge and
perceptions in female groups, one may be able to predict future trends in their society’s female entrepreneur’s behavior (Akbarov, 2022, Potrich et al., 2016). The sample included 70.6% small business owners and 29.4% large business owners. The majority (88.1%) belonged to the group that promoted their
business using social media platforms. Table I provides more detailed information on the sample’s demographic characteristics. Table I: Demographic characteristics
Variables
Categories
N
(%)
Gender
Male
Female
66
150
30.3
69.7
Age
Less than 20
20-30
30-40
Above 40
-
-
162
54
-
-
75.2
24.8
Business type
Small
Large
152
64
70.6
29.4
Social media usage
Yes
No
190
26
88.1
11.9
Total
216
100% Table I: Demographic characteristics Table I: Demographic characteristics
Variables
Categories
N
(%)
Gender
Male
Female
66
150
30.3
69.7
Age
Less than 20
20-30
30-40
Above 40
-
-
162
54
-
-
75.2
24.8
Business type
Small
Large
152
64
70.6
29.4
Social media usage
Yes
No
190
26
88.1
11.9
Total
216
100% Variables
Categories
N
(%)
Gender
Male
Female
66
150
30.3
69.7
Age
Less than 20
20-30
30-40
Above 40
-
-
162
54
-
-
75.2
24.8
Business type
Small
Large
152
64
70.6
29.4
Social media usage
Yes
No
190
26
88.1
11.9
Total
216
100% Reliability and validity test A quantitative research investigation must have validity and reliability (Rajeh et al., 2015). The scales of the items used to measure each construct are tested
for reliability prior to data analysis using SEM and to confirm the constructions internal consistency (Alzadjal et al., 2022, Rajeh et al., 2015, Arain et al., 2020). Researchers Rajeh et al. (2015), indicated that consistency of study findings provided by data collection techniques is dependent on reliability. To examine the
inter-correlation and reliability of the constructs, Cronbach’s value was measured using SPSS 26.0. Cronbach’s alpha assesses the accuracy with which a set
of observed variables measures a single unidimensional latent construct (Reyes-Mercado, 2018). According to Rahi et al. (2019), for a set of observed items, a
Cronbach’s α coefficient greater than 0.7 is considered acceptable reliability. 3. Research Methodology In the present study, the IBM SPSS 26.0 and AMOS 26.0 programs was utilized to investigate the hypothesized relationship
and validate the measurement model using structural equation modeling. CFA tests were conducted for each factor to determine their compatibility. The loading factor value, which must be positive and must be greater than 0.50, but
not greater than 1.00 is the main criterion for assessing this compatibility. The three fitness validity criteria that have been proposed for this fit are fitness
index, convergent validity and construct validity. The RMSEA value for fitness index should be less than 0.08 (Agrawal et al., 2021, Senthilkumar and Arulraj,
2011, Al-Fadhali, 2022), while the GFI, CFI and TLI values should be greater than 0.90 (Mustafa et al., 2020, Agrawal et al., 2021). The relative/ normed chi-
square value should be approximately less than 5.0 Mustafa et al. (2020), convergent validity ( Average variance extracted or AVE ) and construct validity
measurements. According to Anser et al. (2022), a minimum of 0.50 is required for AVE to be able to determine compatibility, while Mustafa et al. (2020), Page 4/14 Page 4/14 suggested that the AVE value is more than 0.5 and complies with the convergent validity measurement standards. The criteria for reliability measurement are
next. A composite reliability value of greater than 0.7 is necessary for this criterion. suggested that the AVE value is more than 0.5 and complies with the convergent validity measurement standards. The criteria for reliability measurement are
next. A composite reliability value of greater than 0.7 is necessary for this criterion. Demographic characteristics of the sample The present study analysis reveals that all Cronbach’s coefficients obtained are greater than 0.7, implying that the set of observed variables are good
measures of single unidimensional latent construct (Rajeh et al., 2015, Wu and Ho, 2022). The test produced alpha values ranging from 0.947 to 0.973,
indicating a measurement equipment with an acceptable level of reliability. As depicted in results all constructs have Cronbach's alpha values that are
significantly higher than 0.7, demonstrating the validity of the scales. For a good model fit, all factors loading of the measurement items should be more than
0.5 (Ts, 2022). As suggested by, Rahi and Abd.Ghani (2019) factor loading levels had to be bigger than 0.6. However, the present study, Kaiser-Meyer-Olkin
(KMO) value represented a range of 0.836 to 0.877. Therefore, following construct reliability and validity testing, the measurement and structural model are
validated using confirmatory factor analysis. The model identification, the relative value of x2, and the goodness-of-fit indices are all examined. Multicollinearity test In a multiple regression analysis, multicollinearity is defined as the degree to which two or more independent variables are strongly linearly connected to each
other. Ts (2022) stated, the model’s precision decreases as multicollinearity increase. According to Rajeh et al. (2015), VIF reflects the degree of
multicollinearity; if VIF is greater than 4, it indicates an problem with multicollinearity. Moreover, VIF was calculated for the suggested five components; it was discovered that VIF varies between 1.005 to 1.015, it falls in the acceptable region,
also, VIF is less than 2, which indicates that the proposed five dimensions are free of multicollinearity. Descriptive statistics Descriptive statistics The mean values of the study’s variables are more than the crucial value of 2.99, indicating that the correlation is upright (Sakaya, 2023). As a result, data
analysis was required to determine whether they fit the characteristics of a normal distribution and to test skewness and kurtosis. The skewness values for
five variables were < 3.00 within the range of (-1 to +1) (Nomran and Haron, 2022, Pattnaik and Pattnaik, 2021), and the kurtosis values are within the range of
(-1 to +1), which is appropriate for SEM research (Sakaya, 2023). The common method bias (CBM) was investigated by following the procedures indicated
by Sakaya (2023), which included using multiple scales to measure the study’s proposed variables. Confirmatory factor analysis Page 5/14 Page 5/14 Confirmatory factor analysis (CFA) was used to evaluate the constructs item loading, validity and reliability. CFA’s first attempt did not engender good model
fit. Due to low factor loadings, the items PE5, EE1, EE2, SI1, SMMC1, BP1 and BP5 were dropped. Therefore, second CFA attempt generated a good model fit
(Table II). The result of x2/ df = 2.365 indicates a good model fit because the recommended value for model fit is less than 3 (Anwar et al., 2018, Sakaya,
2023). According to previous research, GFI = 0.908, AGFI = 0.890, CFI = 0.967, TLI = 0.960 and NFI = 0.944 imply good model fitness (Rajeh et al., 2015,
Bangwal and Tiwari, 2019, Nomran and Haron, 2022). RMR = 0.009 and RMSEA = 0.078 also generated good model fit (Rajeh et al., 2015, Anwar et al., 2018,
Ts, 2022, Sakaya, 2023). PCLOSE greater than 0.05 indicates that the model is well fitted. All of the factor loadings were significantly loaded (P < 0.001), and
almost all of the item standardized regression weights (Figure I) were greater than 0.70, which was considered acceptable (Nanjundeswaraswamy et al., 2022,
Singh and Srivastava, 2019). As a result, all of the model fit criteria were met. However, before testing the hypothesis, it is required to evaluate the
measurement model’s validity and reliability. Notes: * There are no uniform criteria for model fitness. This study relied on Anwar et al. (2018) and Sakaya (2023) because they were
commonly utilized in previous studies, model fitness criteria. Descriptive statistics Page 6/14 Notes: β: standardized beta coefficients, S.E.: standard error, C.R.: critical ratio, *P < 0.05, **P < 0.01, ***P < 0.001 Notes: β: standardized beta coefficients, S.E.: standard error, C.R.: critical ratio, *P < 0.05, **P < 0.01, ***P < 0.001 Examining the moderation In answering the proposed H4, Tables V, VI and VII summarized the findings of the hypothesis testing of the moderating effect of the seller’s online business
performance in rural area. As prescribed by Alzadjal et al. (2021), interaction effect approach was applied in this study. Interaction terms are generated by
multiplying the independent variables by the moderator to examine the moderating effects. According to the previous findings performance expectancy, effort
expectancy and social influence were all significant predictors of the intention to deal with business performance, consequently the interaction impact should
be tested further. The section that follows outline the outcome of the social media marketing communication moderating effect. The moderating effect of social media marketing communication The moderating effect of social media marketing communication The results show that (Table V) social media marketing communication has a positive direct effect on business performance, with a path coefficient value of
0.033 and a critical ratio of 0.752 and the p- value is insignificant, the value is 0.452 (P- value < 0.05). According to Alzadjal et al. (2021) study, if first
hypothesis is significant, then the partial moderation can occur. This suggest that there was a partial moderating effect of social media marketing
communication between the link between performance expectancy and business performance. Figure III depicts structural model’s moderating awareness
relations. The results indicate that (Table VI) social media marketing communication has a direct positive effect on business performance with a path coefficient value
of 0.378 and a critical ratio of 11.473 and p-value is significant the value is *** (p-value < 0.001). with a path coefficient of 0.428 and a critical ratio of 12.912,
the direct effect of effort expectancy on business performance is significant, and the significant value is *** (p-value < 0.001). With a negative path coefficient value of -0.032 and a critical ratio of 1.120 the direct effect of the interaction term (EE*SMMC) is not significant, and the
insignificant value is 0.268 (p-value < 0.05). This suggests that the exist between the potential sellers EE and BP’s connection. Descriptive statistics Table II: Model fits
Model fit criteria
First order estimate
Second order estimate revised
Acceptable range
X2/ df
7.48
2.365
1-3
GFI
0.926
0.908
> 0.90
AGFI
0.757
0.890
> 0.80
CFI
0.926
0.967
> 0.95
TLI
0.907
0.960
> 0.90
NFI
0.916
0.944
> 0.90
RMR
0.060
0.009
< 0.09
RMSEA
0.142
0.078
< 0.08
PCLOSE
0.97
0.94
> 0.05 Notes: * There are no uniform criteria for model fitness. This study relied on Anwar et al. (2018) and Sakaya (2023) because they we
commonly utilized in previous studies, model fitness criteria. Correlation coefficients AMOS was used to measure correlation coefficients, and the findings are shown in Table III. It gives early support for the study’s proposed hypothesis. The
findings show that there is a significant positive relationship between performance expectancy and business performance (r = 0.722, P < 0.01), a significant
positive relationship between effort expectancy and business performance (r = 0.204, P < 0.01), and a significant positive relationship between social influence
and business performance (r = 0.561, P < 0.01). Therefore, these findings supported H1, H2, and H3 in this study. Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Notes: correlation is significant at 0.01 level (2- tailed); *correlation is significant at 0.05 level (2- tailed) Multiple liner regression Table IV and Figure II shows the regression outcome. The analysis reveals that the two variables have positive impact and are significant, with the relationship
between performance expectancy and business performance (β = 0.771 and P = 0.000), when PE goes up by 1 standard deviation the dependent variable BP
goes up by 0.771 standard deviation, also social media marketing communication with business performance (β = 0.332 and P = 0.000), when SMMC goes up
by 1 standard deviation the dependent variable BP goes up by 0.332 standard deviation. However, effort expectancy and social influence not having
significant impact on business performance (β = -0.027 and P = 0.561) (β = 0.039 and P = 0.401) respectively. 5. Discussion In this study, researchers provided a forum for vies on the application of theoretical triangulation and quantitative research methods by using AMOS to
analyze business performance and social media marketing communication actions among sellers in rural areas. To test the relationship between performance
expectancy, effort expectancy, and social influence theories with business performance, as well as the moderating impact of social media marketing
communication in those theories on business performance, researchers used performance expectancy, effort expectancy, and social influence theories with
business performance to demonstrate how triangulation can extend across many competing theories. As a result, scholars propose theoretical triangulation
and diversified research methodologies in order to use multiple theories with competing epistemologies in harmony, in accordance with the premise that
different theories should be considered as complimentary rather than adversaries. A reoccurring point that binds this paper is that theoretical triangulation and
varied research methodologies have the ability to produce a synergy of being mutually informative, allowing for a deeper portrayal of business reality in rural
areas, revealing distinctive online business difficulties or dynamics. In line with Hoque et al. (2015), authors argue that researchers must consider how multiple
theories and methods might be synthesized or integrated. The incorporation of several views would link theories into a coherent and intelligible online
business performance discourse and practice. Here Hoque et al. (2013) argued that, analysing business ambiguities and conflicts is a significant opportunity
provided by field research methods, as they allow for the examination of suggestive themes and counterpoints, interpretations and counter-interpretations, and
various voices surrounding the social construction of e-business performance in rural areas. As a results, the findings indicate that three theories concerning business performance among salespeople in rural areas significantly enhance relationship. This results are in line with the study’s conceptual framework, which assumes a direct relationship between performance expectancy, effort expectancy, social
influence and business performance (Chua et al., 2018). This is also true in the case of cashew sellers, even if salespeople are selling cashew products
through the online their e-marketing awareness effort is critical to increasing their business performance. It also appears that cashew demand, motivates
salespeople to expand their market, increasing their profit and leading to improved business performance. This result supports the findings of Wu and Ho
(2022) and Reyes-Mercado (2018). Therefore, the authors proved that the proposed H1, H2, and H3 (Table III) supported the study. Descriptive statistics This indicates that, even in the
presence of a moderator, both constructs have a strong relationship. Figure IV illustrates the structural model’s moderating awareness relations. Furthermore, in this case, the moderating effect is weak. The weak moderating effect has no effect on the modify of the relationship between the dependent
variable (BP) and the predictor variable (EE). As a result, “SMMC” can be considered as a poor moderator of the relationship between EE and BP. Page 7/14 Page 7/14 Page 7/14 Page 7/14 The regression model outputs are detailed in Table VII. The results show that social media marketing communication has a positive direct effect on business
performance, with a path coefficient value of 0.552 and a critical ratio of 16.482 and p-value of *** (p-value < 0.001). with a path coefficient value of 0.138 and
a critical ratio of 4.001, the direct effect of social influence on business performance is significant, and the significant value is *** (p-value < 0.001). The direct effect of the interaction term (SMMC*SI) is not significant, with a negative path coefficient value of -0.043 and a critical ratio of -1.276, and the
significant value is 0.201 (p-value < 0.05). this suggests that the moderating effect of SMMC did not exist between the potential sellers SI and BP’s connection. Figure V depicts the moderating relationships of awareness in the structural model. Furthermore, in this case, the moderating effect is weak. The weak moderating effect has no effect on the modify of the relationship between the dependent
variable (BP) and the predictor variable (SI). Thus, “SMMC” is a poor moderator because it had no effect on the relationship between SI and BP. The interaction
term effect of the rural area sellers SMMC on the relationship of the PE, EE and SI on the BP is summarized in Table VIII. Limitations and future research direction This study does have numerous limitations. First, the current study uncounted the problem of not knowing the population of the chosen unit of analysis, which
is the number of cashew sellers in Sri Lanka. Certain data has been erased, because cashew corporation lacks a good recording system and relies solely on a
manual system. Based on information from the cashew corporation’s research department, the authors determined the population. As a result, researchers
cannot find cashew salespeople across the entire country. Second, the technology and business performance in the cashew industry were evaluated using
subjective measurement. According to Aghajari and Amat Senin (2014) the limitations of psychological biases affect subjective measures. Therefore, to test
the validity and generalizability of these findings and to throw fresh light on the postulated causal relationships, future research that replicate these measures
and use various measurement techniques are advised. Third, the cashew industry was the focus of the current investigation. The findings may differ
depending on the industry. Therefore, the conclusions from this study may not be extrapolated to other industries, such as manufacturing or banks. Fourth, the
study solely considered the business owner's perspective, marketing techniques, information value, usability, and expectation fulfillment. By including small
factors in a context relevant to an application, the research aims to broaden the scope of the existing theoretical evaluation framework. It also aims to develop
more realistic time-based versions by revisiting and re-examining the existing framework. Consequently, it significantly affects how well researcher understand the complex topic of how online business performance affects entrepreneurs. Future
researchers should focus on qualitative or mixed-method research because it has a big impact on discovering the in-depth analysis regarding the performance
of online businesses. The E-UTAUT model is kindly applied in this study to new online business performance, particularly in Sri Lanka. However, just the three
elements PE, EE, and SI are applied based on UTAUT. Consequently, there can be some generalizability problems with the performance of emerging online
businesses. Future studies should therefore employ all of the UTAUT model's components in order to further the significant discoveries regarding e-business. This increases the legitimacy of the profit growth, making it more beneficial for SMEs and the nation's emerging economic growth. Other exogenous factors
can have an impact on the connection that was investigated. Limitations and future research direction Future studies should concentrate on numerous other significant factors that affect sellers'
expectations, such as marketing strategies and high-performance network systems. Despite these limitations, the authors believe that this study will not only benefit cashew sellers by providing answers to exploratory research questions, but
will also contribute to the body of knowledge in terms of theoretical enrichment to behavioral studies and theoretical triangulation. Findings from this paper
are critical because they pertain to the adaptation of social media marketing communication and business performance studies. The literature revealed that
there are a limited number of studies that extended the theoretical triangulation to examine cashew sellers in rural areas. Another important point is that the
findings of this study not only demonstrate that theoretical triangulation appears to be acceptable in explaining the relationship between predictors and BP in
the field of online business, but also support the robustness of theoretical triangulation's ability to predict variables and BP in a different research context. As a
result, the current study indirectly contributes to expanding the existing body of knowledge and behavioral literature. References Abu Afifa, M. M., Vo Van, H. & Le Hoang Van, T. 2022. Blockchain adoption in accounting by an extended UTAUT model: empirical evidence from an emerging
economy. Journal of Financial Reporting and Accounting, 17 (1), 1-7. Available at:https://doi.org/10.1108/JFRA-12-2021-0434 Aghajari, N. & Amat Senin, A. 2014. Strategic orientation and dual innovative operation strategies. Asia-Pacific Journal of Business Administration, 6 (2), 127-
147. Available at:https://doi.org/10.1108/APJBA-07-2013-0075 Agrawal, S., Singh, V. & Upadhyay, Y. 2021. Structural model of information quality framework to e-agri supply chain. Journal of Advances in Management
Research, 18 (4), 609-634. Available at:https://doi.org/10.1108/JAMR-06-2020-0113 Akbarov, S. 2022. Consumer ethnocentrism and purchasing behavior: moderating effect of demographics. Journal of Islamic Marketing, 13 (4), 898-932. Available at:https://doi.org/10.1108/JIMA-02-2020-0047 Akbarov, S. 2022. Consumer ethnocentrism and purchasing behavior: moderating effect of demographics. Journal of Islamic Marketing, 13 (4), 898-932. Available at:https://doi.org/10.1108/JIMA-02-2020-0047 Akbarov, S. 2022. Consumer ethnocentrism and purchasing behavior: moderating effect of demographics. Journal of Islamic Marketing, 13 (4), 898-932. Available at:https://doi.org/10.1108/JIMA-02-2020-0047 Al-Fadhali, N. 2022. An AMOS-SEM approach to evaluating stakeholders' influence on construction project delivery performance. Engineering, Construction
and Architectural Management, 11 (1), 1-17. Available at:https://doi.org/10.1108/ECAM-09-2021-0780 Al-Fadhali, N. 2022. An AMOS-SEM approach to evaluating stakeholders' influence on construction project delivery performance. Engineering, Construction
and Architectural Management, 11 (1), 1-17. Available at:https://doi.org/10.1108/ECAM-09-2021-0780 Al-Fadhali, N. 2022. An AMOS-SEM approach to evaluating stakeholders' influence on construction project delivery performance. Engineering, Construction
and Architectural Management, 11 (1), 1-17. Available at:https://doi.org/10.1108/ECAM-09-2021-0780 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2021. Moderating the role of religiosity on potential customer intention to deal with
Islamic banks in Oman. Journal of Islamic Marketing, 15 (1), 1-25. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2021. Moderating the role of religiosity on potential customer intention to deal with
Islamic banks in Oman. Journal of Islamic Marketing, 15 (1), 1-25. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2021. Moderating the role of religiosity on potential customer intention to deal with
Islamic banks in Oman. Journal of Islamic Marketing, 15 (1), 1-25. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 Alzadjal, M. A. J., Abu-Hussin, M. F., Md Husin, M. & Mohd Hussin, M. Y. 2022. Moderating the role of religiosity on potential customer intention to deal with
Islamic banks in Oman. Journal of Islamic Marketing, 13 (11), 2378-2402. Available at:https://doi.org/10.1108/JIMA-05-2020-0150 & Prim-Allaz, I. 2017. 5. Discussion According to the empirical results, the suggested model provides a comprehensive comprehension of the elements that drive business performance. PE, EE
and SI explain approximately 61% of the total variance in potential sellers in rural area. The current study extended the theoretical triangulation with a
moderator to examine the interaction effect of SMMC on the relationship between predictors and BP at the theoretical contribution level. The most important Page 8/14 Page 8/14 practical finding of this study is that potential sellers have a poor inclination to deal with social media. These findings should be noted as evidence of a partial
moderating effect in PE theory (Table V). But researchers considered there to be no moderating impact between these predictors and BP. Because the other two
theories (EE and SI) have no moderating effect (Table VI and VII). As a result, H4 is opposed to the study and the author accept the null hypothesis (Table VIII). However, theoretical triangulation still happens with PE, EE, SI and BP, also there is no moderating effect in this study. practical finding of this study is that potential sellers have a poor inclination to deal with social media. These findings should be noted as evidence of a partial
moderating effect in PE theory (Table V). But researchers considered there to be no moderating impact between these predictors and BP. Because the other two
theories (EE and SI) have no moderating effect (Table VI and VII). As a result, H4 is opposed to the study and the author accept the null hypothesis (Table VIII). However, theoretical triangulation still happens with PE, EE, SI and BP, also there is no moderating effect in this study. References Reputation Risk: Anticipation and Management of Reputation Failure. Risk Management. Ambroise, L. & Prim-Allaz, I. 2017. Reputation Risk: Anticipation and Management of Reputation Failure. Risk Management. Anser, M. K., Yousaf, Z., Yasir, M., Sharif, M., Nasir, M. H., Rasheed, M. I., Waheed, J., Hussain, H. & Majid, A. 2022. How to unleash innovative work behavior of
SMEs' workers through knowledge sharing? Accessing functional flexibility as a mediator. European Journal of Innovation Management, 25 (1), 233-248. Available at:https://doi.org/10.1108/EJIM-11-2019-0332 Page 9/14 Anwar, M., Shah, S. Z. A. & Khan, S. Z. 2018. The role of personality in SMEs internationalization: empirical evidence. Review of International Business and
Strategy, 28 (2), 258-282. Available at:https://doi.org/10.1108/RIBS-12-2017-0113 Anwar, M., Shah, S. Z. A. & Khan, S. Z. 2018. The role of personality in SMEs internationalization: empirical evidence. Review of International Business and
Strategy, 28 (2), 258-282. Available at:https://doi.org/10.1108/RIBS-12-2017-0113 Arain, G. A., Bhatti, Z. A., Hameed, I. & Fang, Y.-H. 2020. Top-down knowledge hiding and innovative work behavior (IWB): a three-way moderated-mediation
analysis of self-efficacy and local/foreign status. Journal of Knowledge Management, 24 (2), 127-149. Available at:https://doi.org/10.1108/JKM-11-2018-
0687 Arain, G. A., Bhatti, Z. A., Hameed, I. & Fang, Y.-H. 2020. Top-down knowledge hiding and innovative work behavior (IWB): a three-way moderated-mediation
analysis of self-efficacy and local/foreign status. Journal of Knowledge Management, 24 (2), 127-149. Available at:https://doi.org/10.1108/JKM-11-2018-
0687 Araujo, T. D. S., Da Costa, J. M. A. R., De Oliveira Silva Ribeiro, F., De Jesus Oliveira, A. C., Do Nascimento Dias, J., De Araujo, A. R., Barros, A. B., Da Paixão Brito,
M., De Oliveira, T. M., De Almeida, M. P., De Carvalho Castro, K. N., Dos Santos Fogaça, F. H., Da Silva, D. A. & De Souza, B. W. S. 2021. Nanoemulsion of
cashew gum and clove essential oil (Ocimum gratissimum Linn) potentiating antioxidant and antimicrobial activity. International Journal of Biological
Macromolecules, 193 (1), 100-108. Available at:https://www.sciencedirect.com/science/article/pii/S0141813021021243 Aytekin, A., Özköse, H. & Ayaz, A. 2022. Unified theory of acceptance and use of technology (UTAUT) in mobile learning adoption : Systematic literature review
and bibliometric analysis. COLLNET Journal of Scientometrics and Information Management, 16 (1), 75-116. Available
at:https://doi.org/10.1080/09737766.2021.2007037 Bangwal, D. & Tiwari, P. 2019. Workplace environment, employee satisfaction and intent to stay. International Journal of Contemporary Hospitality
Management, 31 (1), 268-284. Available at:https://doi.org/10.1108/IJCHM-04-2017-0230 Bui, B. & Fowler, C. 2022. Carbon controls in a New Zealand electricity utility: An application of theoretical triangulation. Accounting & Finance, 62 (4), 4423-
4451. References Available at:https://doi.org/10.1111/acfi.12942 Burau, V. & Andersen, L. B. 2014. Professions and Professionals: Capturing the Changing Role of Expertise Through Theoretical Triangulation. The American
Journal of Economics and Sociology, 73 (1), 264-293. Available at:https://doi.org/10.1111/ajes.12062 Chambers, M., Bliss, K. & Rambur, B. 2020. Recruiting Research Participants via Traditional Snowball vs Facebook Advertisements and a Website. Western
Journal of Nursing Research, 42 (10), 846-851. Available at:https://doi.org/10.1177/0193945920904445 Chan, R., Troshani, I., Rao Hill, S. & Hoffmann, A. 2022. Towards an understanding of consumers’ FinTech adoption: the case of Open Banking. International
Journal of Bank Marketing, 40 (4), 886-917. Available at:https://doi.org/10.1108/IJBM-08-2021-0397 Chen, L., Rashidin, M. S., Song, F., Wang, Y., Javed, S. & Wang, J. 2021. Determinants of Consumer’s Purchase Intention on Fresh E-Commerce Platform:
Perspective of UTAUT Model. SAGE Open, 11 (2), 1-14. Available at:https://doi.org/10.1177/21582440211027875 Chen, X. & Qasim, H. 2021. Does E-Brand experience matter in the consumer market? Explaining the impact of social media marketing activities on consumer-
based brand equity and love. Journal of Consumer Behaviour, 20 (5), 1065-1077. Available at:https://doi.org/10.1002/cb.1915 Chua, P. Y., Rezaei, S., Gu, M.-L., Oh, Y. & Jambulingam, M. 2018. Elucidating social networking apps decisions. Nankai Business Review International, 9 (2),
118-142. Available at:https://doi.org/10.1108/NBRI-01-2017-0003 DCSSL. 2020. Computer Literacy Statistics. Sri Lanka LANKA, D. O. C. A. S. S. Available CSSL. 2020. Computer Literacy Statistics. Sri Lanka LANKA, D. O. C. A. S. S. Available DCSSL. 2020. Computer Literacy Statistics. Sri Lanka LANKA, D. O. C. A. S. S. Available
at:http://www.statistics.gov.lk/Resource/en/ComputerLiteracy/Bulletins/AnnualBuletinComputerLiteracy-2020. at:http://www.statistics.gov.lk/Resource/en/ComputerLiteracy/Bulletins/AnnualBuletinComputerLiteracy-2020. Duffett, R. G. 2017. Influence of social media marketing communications on young consumers’ attitudes. Young Consumers, 18 (1), 19-39. Available
at:https://doi.org/10.1108/YC-07-2016-00622 Garone, A., Pynoo, B., Tondeur, J., Cocquyt, C., Vanslambrouck, S., Bruggeman, B. & Struyven, K. 2019. Clustering university teaching staff through UTAUT:
Implications for the acceptance of a new learning management system. British Journal of Educational Technology, 50 (5), 2466-2483. Available
at:https://doi.org/10.1111/bjet.12867 González Bravo, L., Fernández Sagredo, M., Torres Martínez, P., Barrios Penna, C., Fonseca Molina, J., Stanciu, I. D. & Nistor, N. 2020. Psychometric analysis of
a measure of acceptance of new technologies (UTAUT), applied to the use of haptic virtual simulators in dental students. European Journal of Dental
Education, 24 (4), 706-714. Available at:https://doi.org/10.1111/eje.12559 Guggemos, J., Seufert, S. & Sonderegger, S. 2020. Humanoid robots in higher education: Evaluating the acceptance of Pepper in the context of an academic
writing course using the UTAUT. British Journal of Educational Technology, 51 (5), 1864-1883. References Available at:https://doi.org/10.1111/bjet.13006 Hoque, Z., A. Covaleski, M. & N. Gooneratne, T. 2013. Theoretical triangulation and pluralism in research methods in organizational and accounting research. Accounting, Auditing & Accountability Journal, 26 (7), 1170-1198. Available at:https://doi.org/10.1108/AAAJ-May-2012-01024 Hoque, Z., Covaleski, M. A. & Gooneratne, T. N. 2015. A response to “theoretical triangulation and pluralism in accounting research: a critical realist critique”. Accounting, Auditing & Accountability Journal, 28 7, 1151-1159. Available at:https://doi.org/10.1108/AAAJ-05-2015-2058 INC. 2021. The nuts & dried fruits statistical yearbook report USA. FRUIT, I. N. A. D. Available
at:https://www.nutfruit.org/files/tech/1651579968_Statistical_Yearbook_2021-2022.pdf. Page 10/14 Page 10/14 Joa, C. Y. & Magsamen-Conrad, K. 2022. Social influence and UTAUT in predicting digital immigrants’ technology use. Behaviour & Information Technology, 41
(8), 1620-1638. Available at:https://doi.org/10.1080/0144929X.2021.1892192 Joa, C. Y. & Magsamen-Conrad, K. 2022. Social influence and UTAUT in predicting digital immigrants’ technology use. Behaviour & Information Technology, 41
(8), 1620-1638. Available at:https://doi.org/10.1080/0144929X.2021.1892192 Kapurubandara, M. 2009. A Framework to e-Transform sMEs in developing countries. THE ELECTRONIC JOURNAL OF INFORMATION SYSTEMS IN
DEVELOPING COUNTRIES, 39 (1), 1-24. Available at:https://doi.org/10.1002/j.1681-4835.2009.tb00277.x Liu, Y., Dilanchiev, A., Xu, K. & Hajiyeva, A. M. 2022. Financing SMEs and business development as new post Covid-19 economic recovery determinants. Economic Analysis and Policy, 76 (1), 554-567. Available at:https://www.sciencedirect.com/science/article/pii/S0313592622001485 M. Kang, J.-Y. 2014. Augmented reality and motion capture apparel e-shopping values and usage intention. International Journal of Clothing Science and
Technology, 26 (6), 486-499. Available at:https://doi.org/10.1108/IJCST-05-2013-0055 Mensah, M. & Onyancha, O. B. 2021. Demographic factors influencing the adoption and use of social media in university libraries in Ghana: A unified theory of
acceptance and use of technology (UTAUT) approach. Journal of Electronic Resources Librarianship, 33 (3), 170-194. Available
at:https://doi.org/10.1080/1941126X.2021.1949157 Modell, S. 2015. Theoretical triangulation and pluralism in accounting research: a critical realist critique. Accounting, Auditing & Accountability Journal, 28 (7),
1138-1150. Available at:https://doi.org/10.1108/AAAJ-10-2014-1841 Mustafa, M., Nordin, M. N. & Razzaq, A. 2020. Structural Equation Modelling Using AMOS: Confirmatory Factor Analysis for Taskload of Special Education
Integration Program Teachers. Universal Journal of Educational Research, 8 (1), 127-133. Available ustafa, M., Nordin, M. N. & Razzaq, A. 2020. Structural Equation Modelling Using AMOS: Confirmatory Factor Ana
tegration Program Teachers. Universal Journal of Educational Research, 8 (1), 127-133. Available at:https://www.researchgate.net/publication/339453426_Structural_Equation_Modelling_Using_AMOS_Confirmatory_Factor_Analysis_for_Taskload_of_Specia :https://www.researchgate.net/publication/339453426_Structural_Equation_Modelling_Using_AMOS_Confirmato Nanjundeswaraswamy, T. S., Bharath, S. & Nagesh, P. 2022. Employer branding: design and development of a scale. Journal of Economic and Administrative
Sciences, 17 (1), 1-26. Available at:https://doi.org/10.1108/JEAS-01-2022-0012 Nguyen, N. P. & Adomako, S. 2021. References Environmental proactivity, competitive strategy, and market performance: The mediating role of environmental reputation. Business Strategy and the Environment, 30 (4), 2008-2020. Available at:https://doi.org/10.1002/bse.2729 Nomran, N. M. & Haron, R. 2022. Validity of zakat ratios as Islamic performance indicators in Islamic banking: a congeneric model and confirmatory factor
analysis. ISRA International Journal of Islamic Finance, 14 (1), 41-62. Available at:https://doi.org/10.1108/IJIF-08-2018-0088 Pattnaik, S. & Pattnaik, S. 2021. Exploring employee performance dimensionality in Indian public sector units. International Journal of Productivity and
Performance Management, 70 (3), 657-674. Available at:https://doi.org/10.1108/IJPPM-08-2019-0374 Peterson, R. A. & Altounian, D. 2019. Self-perceived Performance of Female-owned Firms and Male-owned Firms: Insights from Black and Mexican-American
Entrepreneurs. In: CRITTENDEN, V. L. (ed.) Go-to-Market Strategies for Women Entrepreneurs. Emerald Publishing Limited. Pim-Wusu, M., Aigbavboa, C. & Thwala, W. D. 2022. Adaptability capacity framework for sustainable practices in the Ghanaian construction industry. Built
Environment Project and Asset Management, 12 (1), 1-16. Available at:https://doi.org/10.1108/BEPAM-01-2022-0016 Potrich, A. C. G., Vieira, K. M. & Mendes-Da-Silva, W. 2016. Development of a financial literacy model for university students. Management Research Review, 39
(3), 356-376. Available at:https://doi.org/10.1108/MRR-06-2014-0143 iyashantha, A. K. H. 2022. Gifted but Not Tasted? [Online]. Daily News Available: https://www.dailynews.lk/2022/ Priyashantha, A. K. H. 2022. Gifted but Not Tasted? [Online]. Daily News Available: https://www.dailynews.lk/2022/02/10/features/272229/gifted-not-tasted Rahi, S. & Abd.Ghani, M. 2019. Investigating the role of UTAUT and e-service quality in internet banking adoption setting. The TQM Journal, 31 (3), 491-
506.Available at:https://doi.org/10.1108/TQM-02-2018-0018 Rahi, S., Othman Mansour, M. M., Alghizzawi, M. & Alnaser, F. M. 2019. Integration of UTAUT model in internet banking adoption context. Journal of Research
in Interactive Marketing, 13 (3), 411-435. Available at:https://doi.org/10.1108/JRIM-02-2018-0032 Rajeh, M., Tookey, J. E. & Rotimi, J. O. B. 2015. Estimating transaction costs in the New Zealand construction procurement. Engineering, Construction and
Architectural Management, 22 (2), 242-267. Available at:https://doi.org/10.1108/ECAM-10-2014-0130 Raji, R. A., Rashid, S. & Ishak, S. 2019. The mediating effect of brand image on the relationships between social media advertising content, sales promotion
content and behaviuoral intention. Journal of Research in Interactive Marketing, 13 (3), 302-330. Available at:https://doi.org/10.1108/JRIM-01-2018-0004 Reyes-Mercado, P. 2018. Adoption of fitness wearables. Journal of Systems and Information Technology, 20 (1), 103-127. Available
at:https://doi.org/10.1108/JSIT-04-2017-0025 Sakaya, A. J. 2023. Fear of COVID-19 and green bank service purchase intention: the mediating effect of customer empowerment and customers’ perceived
value of digital service transactions. Arab Gulf Journal of Scientific Research, 27 (1), 1-22. Available at:https://doi.org/10.1108/AGJSR-08-2022-0137 Sakaya, A. J. 2023. References Fear of COVID-19 and green bank service purchase intention: the mediating effect of customer empowerment and customers’ perceived
value of digital service transactions. Arab Gulf Journal of Scientific Research, 27 (1), 1-22. Available at:https://doi.org/10.1108/AGJSR-08-2022-0137 Samoggia, A., Bertazzoli, A. & Ruggeri, A. 2019. Food retailing marketing management: social media communication for healthy food. International Journal of
Retail & Distribution Management, 47 (9), 928-956. Available at:https://doi.org/10.1108/IJRDM-08-2018-0178 Samoggia, A., Bertazzoli, A. & Ruggeri, A. 2019. Food retailing marketing management: social media communication for healthy food. International Journal of
Retail & Distribution Management, 47 (9), 928-956. Available at:https://doi.org/10.1108/IJRDM-08-2018-0178 Page 11/14 Page 11/14 Senthilkumar, N. & Arulraj, A. 2011. SQM-HEI – determination of service quality measurement of higher education in India. Journal of Modelling in
Management, 6 (1), 60-78 Singh, N., Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and pharmacological review of Cinnamomum
verum J. Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available ngh, N., Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and ph
erum J Presl a versatile spice used in food and nutrition Food Chemistry 338 (1) 1 15 Available Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and pharmaco
Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available ngh, N., Rao, A. S., Nandal, A., Kumar, S., Yadav, S. S., Ganaie, S. A. & Narasimhan, B. 2021. Phytochemical and ph
erum J. Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available verum J. Presl-a versatile spice used in food and nutrition. Food Chemistry, 338 (1), 1-15 Available at:.https://www.sciencedirect.com/science/article/pii/S0308814620316356 /www.sciencedirect.com/science/article/pii/S0308814620316356 Singh, S. & Srivastava, S. 2019. Engaging consumers in multichannel online retail environment. Journal of Modelling in Management, 14 (1), 49-76. Available
at:https://doi.org/10.1108/JM2-09-2017-0098 Sridharan, V. G. 2021. Methodological Insights Theory development in qualitative management control: revisiting the roles of triangulation and generalization. Accounting, Auditing & Accountability Journal, 34 2, 451-479. Available at:https://doi.org/10.1108/AAAJ-09-2019-4177 Tarsakoo, P. & Charoensukmongkol, P. 2020. Dimensions of social media marketing capabilities and their contribution to business performance of firms in
Thailand. Journal of Asia Business Studies, 14 (4), 441-461. Available at:https://doi.org/10.1108/JABS-07-2018-0204 Ts, N. 2022. Nurses quality of work life: scale development and validation. Journal of Economic and Administrative Sciences, 38 (2), 371-394. References Available
at:https://doi.org/10.1108/JEAS-09-2020-0154 Venkatesh, V., Morris, M. G., Davis, G. B. & Davis, F. D. 2003. User Acceptance of Information Technology: Toward a Unified View. MIS Quarterly, 27 (3), 425-478. Available at:http://www.jstor.org/stable/30036540 Wu, C.-G. & Ho, J. C. 2022. The influences of technological characteristics and user beliefs on customers' perceptions of live chat usage in mobile banking. International Journal of Bank Marketing, 40 (1), 68-86. Available at:https://doi.org/10.1108/IJBM-09-2020-0465 Yang, K. 2010. Determinants of US consumer mobile shopping services adoption: implications for designing mobile shopping services. Journal of Consumer
Marketing, 27 (3), 262-270. Available at:https://doi.org/10.1108/07363761011038338 Zhang, H. & Gong, X. 2021a. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European
Journal of Marketing, 55 5, 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, H. & Gong, X. 2021a. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European
Journal of Marketing, 55 5, 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, H. & Gong, X. 2021b. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European
Journal of Marketing, 55 (5), 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, H. & Gong, X. 2021b. Consumer susceptibility to social influence in new product diffusion networks: how does network location matter? European
Journal of Marketing, 55 (5), 1469-1488. Available at:https://doi.org/10.1108/EJM-06-2019-0491 Zhang, J. & Du, M. 2020. Utilization and effectiveness of social media message strategy: how B2B brands differ from B2C brands. Journal of Business &
Industrial Marketing, 35 (4), 721-740. Available at:https://doi.org/10.1108/JBIM-06-2018-0190 Zhang, J. & Du, M. 2020. Utilization and effectiveness of social media message strategy: how B2B brands differ from B2C brands. Journal of Business &
Industrial Marketing, 35 (4), 721-740. Available at:https://doi.org/10.1108/JBIM-06-2018-0190 Declarations Since the cashew nut industry is related to the micro cultivation industry. Most of the business owners do not have proper awareness of social media and how
to use social media for their business. Since the cashew nut industry is related to the micro cultivation industry. Most of the business owners do not have proper awareness of social media and ho
to use social media for their business. Authors do not have any conflict of interest as far as this study is concerned. Authors do not have any conflict of interest as far as this study is concerned. Figures Page 12/14 Page 12/14 Page 12/14 Page 12/14 Figure 1
Measurement model
Figure 2
Structural model Figure 1 Measurement model Figure 2 Figure 2 Structural model Page 13/14 Page 13/14 Figure 3
SMMC Moderating effect between PE and BP
Figure 4
SMMC Moderating effect between EE and BP
Figure 5
SMMC Moderating effect between SI and BP Figure 3
SMMC Moderating effect between PE and BP
Figure 4 Figure 3 g
SMMC Moderating effect between PE and BP
Figure 4 SMMC Moderating effect between PE and BP SMMC Moderating effect between PE and BP SMMC Moderating effect between PE and BP Figure 4 SMMC Moderating effect between EE and BP
Figure 5 SMMC Moderating effect between EE and BP Figure 5 SMMC Moderating effect between SI and BP Page 14/14
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Effects of long-term sleep disruption on cognitive function and brain amyloid-β burden: a case-control study
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Thomas et al. Alzheimer's Research & Therapy (2020) 12:101
https://doi.org/10.1186/s13195-020-00668-5 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101
https://doi.org/10.1186/s13195-020-00668-5 (2020) 12:101 RESEARCH Open Access Effects of long-term sleep disruption on
cognitive function and brain amyloid-β
burden: a case-control study Jana Thomas1,2,3*
, Sharon J. Ooms2,3, Lara J. Mentink1,2,3, Jan Booij4,5, Marcel G. M. Olde Rikkert1,2,3,
Sebastiaan Overeem6,7†, Roy P. C. Kessels2,3,8† and Jurgen A. H. R. Claassen1,2,3* Jana Thomas1,2,3*
, Sharon J. Ooms2,3, Lara J. Mentink1,2,3, Jan Booij4,5, Marcel G. M. Olde Rikkert1,2,3,
Sebastiaan Overeem6,7†, Roy P. C. Kessels2,3,8† and Jurgen A. H. R. Claassen1,2,3* Abstract 1Department of Geriatric Medicine, Radboud University Medical Center, 6525,
GC, Nijmegen, The Netherlands
Full list of author information is available at the end of the article * Correspondence: Jana.Thomas@radboudumc.nl;
Jurgen.Claassen@radboudumc.nl
†Sebastiaan Overeem and Roy P. C. Kessels contributed equally to this work. 1Department of Geriatric Medicine, Radboud University Medical Center, 6525,
GC, Nijmegen, The Netherlands
Full list of author information is available at the end of the article Abstract Background: Recent evidence indicates that disrupted sleep could contribute to the development of Alzheimer’s
disease by influencing the production and/or clearance of the amyloid-β protein. We set up a case-control study to
investigate the association between long-term work-induced sleep disruption, cognitive function, and brain
amyloid-β burden. Methods: Nineteen male maritime pilots (aged 48–60 years) with chronic work-related sleep disruption and a sex-,
age-, and education-matched control sample (n = 16, aged 50–60 years) with normal sleep completed the study. Primary sleep disorders were ruled out with in-lab polysomnography. Additional sleep measurements were
obtained at home using actigraphy, sleep-wake logs, and a single-lead EEG device. Cognitive function was assessed
with a neuropsychological test battery, sensitive to early symptomatic Alzheimer’s disease. Brain amyloid-β burden
was assessed in maritime pilots using 18F-flutemetamol amyloid PET-CT. Results: Maritime pilots reported significantly worse sleep quality (Pittsburgh Sleep Quality Index (PSQI) = 8.8 ± 2.9)
during work weeks, compared to controls (PSQI = 3.2 ± 1.4; 95% CI 0.01 to 2.57; p = 0.049). This was confirmed with
actigraphy-based sleep efficiency (86% ± 3.8 vs. 89.3% ± 4.3; 95% CI 0.43 to 6.03; p = 0.03). Home-EEG recordings
showed less total sleep time (TST) and deep sleep time (DST) during work weeks compared to rest weeks (TST
318.56 (250.21–352.93) vs. TST 406.17 (340–425.98); p = 0.001; DST 36.75 (32.30–58.58) vs. DST 51.34 (48.37–69.30);
p = 0.005)). There were no differences in any of the cognitive domains between the groups. For brain amyloid-β
levels, mean global cortical standard uptake value ratios of 18F-flutemetamol were all in the normal range
(1.009 ± 0.059; 95% CI 0.980 to 1.037), confirmed by visual reads. Conclusions: Capitalizing on the particular work-rest schedule of maritime pilots, this study with a small sample
size observed that long-term intermittent sleep disruption had no effects on global brain amyloid-β levels or
cognitive function. Keywords: Sleep disruption, Shift work, Alzheimer’s disease, Amyloid-β, Cognitive function Keywords: Sleep disruption, Shift work, Alzheimer’s disease, Amyloid-β, Cognitive function * Correspondence: Jana.Thomas@radboudumc.nl;
Jurgen.Claassen@radboudumc.nl
†Sebastiaan Overeem and Roy P. C. Kessels contributed equally to this work. 1Department of Geriatric Medicine, Radboud University Medical Center, 6525,
GC, Nijmegen, The Netherlands
Full list of author information is available at the end of the article * Correspondence: Jana.Thomas@radboudumc.nl;
Jurgen.Claassen@radboudumc.nl
†Sebastiaan Overeem and Roy P. C. Kessels contributed equally to this work. Background rest week with unrestricted sleep. Their sleep disruptions
are caused by external, occupational factors, which re-
duce the bias of intrinsically caused sleep problems that
could represent an early symptom preceding the clinical
manifestation of dementia due to AD (i.e., reverse caus-
ality). In this group of maritime pilots, we sought to ex-
plore the effects of long-term, externally induced sleep
disruption on cognitive function and brain amyloid-β
burden, as biomarker of AD. g
Sleep loss has been associated with increased risk of de-
mentia in later life, specifically dementia caused by Alz-
heimer’s disease (AD). In a meta-analysis of 27 studies
with nearly 70.000 participants, sleep loss—mostly de-
fined as self-reported sleep of < 6 h per night—carried an
average relative risk of 1.68 (95% CI 1.45 to 1.86) of de-
veloping dementia caused by AD [1]. This finding is
relevant, because the etiology of late-onset AD remains
unknown and therapeutic options are limited, making
sleep a potential target for prevention or treatment of
AD [2, 3]. The association between sleep loss and AD
could be explained by reverse causality, wherein sleep
loss is an early, preclinical manifestation of Alzheimer’s
pathology [4–6]. However, the association may also be
causal, wherein sleep loss contributes to the develop-
ment of the disease. The latter hypothesis is based on a
small number of animal and human studies that have
identified mechanisms that could explain how sleep loss
may increase the risk of AD. In mouse models of genetic
and sporadic AD, for example, sleep loss increased brain
amyloid-β accumulation [7–10]. In humans, we have
previously showed that a single night of full sleep
deprivation impaired the overnight reduction in CSF
amyloid-β, causing 10% higher levels of CSF amyloid-β
the next morning [11]. Also in humans, selective disrup-
tion of slow wave sleep (SWS), without affecting other
sleep stages, led to a comparable overnight difference in
CSF amyloid-β [12]. Additionally, acute increases (5%) in
PET-amyloid-β levels in the hippocampus and thalamus
were
observed
after
a
single
night
of
full
sleep
deprivation [13]. Two mechanisms have been proposed
to explain the relationship between sleep loss and
amyloid-β accumulation. First, the clearance of soluble
toxic waste (including amyloid-β) from the central ner-
vous system, characterized by exchange of interstitial
and cerebrospinal fluids through the glymphatic path-
way, appears more effective during sleep than in wake-
fulness [14–18]. Study design The SCHIP study (Sleep Cognition Hypothesis In mari-
time Pilots) is a case-control study in healthy volunteers
under the hypothesis that repeated nights of sleep loss
may contribute to the risk of dementia due to AD by
gradually increasing amyloid-β levels. The study took
place between December 2016 and May 2019 and was
conducted and reported according to the STROBE
guidelines for case-control studies. The timing between
measurements was consistent across all participants. Sample size calculations were performed using G*power
[21] and published previously [22]. The study protocol
was peer-reviewed and published in BMJ Open [22]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 2 of 12 Page 2 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Background Second, the production of amyloid-β
may be increased during wakefulness and reduced dur-
ing sleep (especially SWS) [5, 11, 17, 19, 20]. These find-
ings have led to the hypothesis that long-term sleep loss,
through repetitive episodes of amyloid-β accumulation,
may contribute to AD. However, evidence from human
studies is lacking, and it remains unknown which quan-
tity of sleep loss, both in terms of duration and intensity,
would be required to raise the risk of developing demen-
tia due to AD. The
unique
work
of
maritime
pilots
in
the
Netherlands offers an opportunity to study the associ- Study population We included 19 middle-aged (mean age = 53; age range:
48 to 60 years) men from the national organization of
Dutch maritime pilots (Nederlandse Loodswezen). The
profession of maritime pilots in the Netherlands is
almost exclusively (99%) male. Their profession is char-
acterized
by
sleep
disruptions
caused
by
external,
occupational factors, wherein every other week is char-
acterized by sleep disruption. We recruited maritime pi-
lots with a work history of an average of 20 years
(mean = 19.8; range 10 to 30 years). Details of the study
population and their occupation have been described in
our methods paper [22] and can be found in the add-
itional information file (see Additional file 1). Maritime pilots were compared to age-, sex-, and
education-matched healthy volunteers (n = 16; mean
age = 57; age range 51 to 62 years) with occupations
comparable in intellectual demand, but with regular
working hours (no shift work). Control participants had
normal sleep, confirmed by a Pittsburg Sleep Quality
Index (PSQI) of < 5 as well as regular bed times (be-
tween 8 p.m. and midnight), and regular wake-up times
(between 5 a.m. and 9 a.m.). Participants were excluded
from taking part in the study if they were using neuroac-
tive medications, consumed > 30 alcoholic beverages per
week, had a body mass index of > 30 kg/m2, suffered
from intrinsic sleeping disorders (i.e., insomnia, REM
sleep behavioral disorder; ruled out by PSG) or, for con-
trols only, if they had self-reported cognitive complaints The
unique
work
of
maritime
pilots
in
the
Netherlands offers an opportunity to study the associ-
ation between long-term sleep disruption and AD risk. Maritime pilots have a work schedule characterized by
one week of irregular and unpredictable working hours,
leading to reduced and fragmented sleep, followed by a Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 3 of 12 Page 3 of 12 Thomas et al. Alzheimer's Research & Therapy (indicated by Cognitive Failure Questionnaire (CFQ) and
general
health
questionnaire). Vascular
health
was
assessed during study visits, using a history of health-
related events, i.e., high cholesterol, smoking, diabetes,
and hypertension in addition to blood pressure measure-
ments in maritime pilots. Baseline characteristics are
listed in Table 1; Fig. 1 provides a study flow chart. headband that records EEG signals and can differentiate
between light and deep sleep. Amyloid PET-CT imaging with 18F-flutemetamol Amyloid PET-CT imaging with
F-flutemetamol
Brain PET-CT scans were acquired in 2019 (Fig. 1) in
maritime pilots only, since outcomes can be compared
to normative data from the literature. We used the vali-
dated
tracer
18F-flutemetamol
[26],
a
tracer
that
performs comparable to 11C-PIB [27]. Previous studies
suggested that CSF and PET measurements of amyloid-β
are in high concordance [28–31], while some suggest
that PET is more powerful and more specific to AD
pathology [32, 33]. Static brain images were acquired
90–110 min post-injection (four frames of 5 min) after
bolus injection of approximately 185 MBq 18F-flutemeta-
mol on a Siemens Biograph mCT. To measure tissue up-
take ratios, PET scans of the PET-CT session were
registered to the CT scan of the PET-CT session by rigid
body
linear
registration
with
nearest
neighbor
interpolation using FSL’s FLIRT (FMRIB’s Linear Image
Registration
Tool)
[34–36]. CT
scans
were
then
registered to the MNI152 2mm skull template by affine Polysomnography (2016/2017) To ascertain that neither maritime pilots nor controls
had intrinsic sleep disorders, we performed full polysom-
nography (PSG) in a sleep lab (Kempenhaeghe, Heeze,
The Netherlands), registering total sleep time (TST),
sleep stages (N1, N2, N3, REM), wake time after sleep
onset (WASO), sleep efficiency (SEF), and sleep onset la-
tency (SOL). The PSG took place on Sunday nights dur-
ing rest weeks for maritime pilots and normal weekend
days for controls, between 2016 and 2017 (Fig. 1). PSQI PSQI was filled in twice by maritime pilots (work and
rest week) and once by controls. Cognitive assessment g
The aim of the cognitive assessment was to explore ef-
fects of long-term exposure to sleep disruption in mari-
time
pilots
on
AD-related
cognitive
impairment. Therefore, we applied a cognitive test battery that was
designed to detect cognitive dysfunction in preclinical
AD [24]. Tests focused on episodic memory (Logical
Memory Subtest from the Wechsler Memory Scale –
Fourth Edition (WMS-IV LM), Rey Auditory Verbal
Learning Test (RAVLT)), semantic memory and lan-
guage (letter and semantic fluency, Boston Naming
Test), working memory and executive function (Digit
Span subtest from the Wechsler Adult Intelligence Scale
– Fourth Edition (WAIS-IV), Trail Making Test (TMT)
parts A and B, WAIS-IV Coding), and attention (Test of
Attentional Performance 2.0, TAP). Overnight memory
consolidation was assessed using a novel paradigm based
on the Doors Test [25], assessing visual recognition
memory after short delay (10 min) and memory consoli-
dation after long delay (after sleep). An overview about
the cognitive tasks is summarized in the additional infor-
mation file (see Additional file 2) and described in detail
in our methods paper [22]. Cognitive tests were per-
formed in the morning following PSG (in 2016 and
2017). Ethical approval Ethical approval
The SCHIP study was approved by the institutional re-
view
board
(CMO
Region
Arnhem-Nijmegen,
NL55712.091.16, file number 2016-2337) and performed
in accordance with good clinical practice guidelines and
the world medical associations code of ethics (Declar-
ation of Helsinki). Written informed consent was ob-
tained from all participants after they received detailed
study information. Participants received a stipend of 50
euros for participating, travel costs were compensated. Study population Furthermore, it measures
total sleep time (TST), sleep onset latency (SOL), num-
ber of short awakenings (< 5 min awake), and number of
arousals
(> 5 min
awake). Maritime
pilots
were
instructed to wear the headband for 20 consecutive days
(10 workdays and 10 rest days). These measurements
took place in 2019, before the amyloid PET scan (Fig. 1). Sleep measurements We combined several complementary methods to assess
sleep quality. Actigraphy and sleep log Accelerometer-based sleep measurements (Actiwatch 2;
Philips Respironics, Eindhoven, The Netherlands) in
maritime pilots and controls were collected for a period
of 10 consecutive days. Data was validated with sleep-
wake diaries. For maritime pilots, these days contained a
mixture of work and rest days. Measurements were
taken during the screening phase of the study (Fig. 1),
before cognitive testing and amyloid PET scans. Home-EEG (2019) We performed objective measurements of sleep quality
during work and rest weeks in 13 of the 19 maritime pi-
lots. For this, we used a novel and innovative device with
a dry, single-lead EEG electrode (SmartSleep; Philips,
Eindhoven, The Netherlands) [23]. This device is a Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 4 of 12 Fig. 1 Flow diagram SCHIP study. Abbreviations: PSQI, Pittsburgh Sleep Quality Index; CFQ, Cognitive Failure Questionnaire; HADS, Hospital
Anxiety and Depression Scale; TAP, Test of Attentional Performance; RBD, REM sleep behavior disorder; PSG, polysomnography. Superscript letter
“a” indicates that over the course of the study, four maritime pilots went into retirement; therefore, analysis for the work days is based on the
remaining, employed maritime pilots (n = 13) Fig. 1 Flow diagram SCHIP study. Abbreviations: PSQI, Pittsburgh Sleep Quality Index; CFQ, Cognitive Failure Questionnaire; HADS, Hospital
Anxiety and Depression Scale; TAP, Test of Attentional Performance; RBD, REM sleep behavior disorder; PSG, polysomnography. Superscript letter
“a” indicates that over the course of the study, four maritime pilots went into retirement; therefore, analysis for the work days is based on the
remaining, employed maritime pilots (n = 13) cerebellum as reference region [37, 38]. Normal global
SUVR in a cognitively healthy population (aged 30–
60) was 1.3 (± 0.09) [27], comparable to mean SUVR
of 1.29 (± 0.2) reported by Thurfjell et al. [39]. As
additional step, all scans were visually rated as posi-
tive/negative for the presence of amyloid-β deposition
by an experienced and trained [40] nuclear medicine
physician (JB) using validated criteria [37]. linear registration with a mutual information cost func-
tion and nearest neighbor interpolation using FSL’s FLIR
T and by non-linear registration using FSL’s FNIRT
(FMRIB’s Non-linear Registration Tool) [34, 35]. These
transformations were combined to align the PET scan to
the MNI152 space in one single step. Tissue ratio was
used as outcome measure, which is equivalent to the
standard uptake value ratio (SUVR). The global cortical
areas as well as prefrontal and temporal cortex and the
cerebellum were selected with the MNI152 2 mm cor-
tical atlas. Home-EEG (2019) Subsequently mean uptake values of these
regions of interest (ROIs) and the tissue ratio, equiva-
lent
to
the
SUVR,
were
calculated
using
the linear registration with a mutual information cost func-
tion and nearest neighbor interpolation using FSL’s FLIR
T and by non-linear registration using FSL’s FNIRT
(FMRIB’s Non-linear Registration Tool) [34, 35]. These
transformations were combined to align the PET scan to
the MNI152 space in one single step. Tissue ratio was
used as outcome measure, which is equivalent to the
standard uptake value ratio (SUVR). The global cortical
areas as well as prefrontal and temporal cortex and the
cerebellum were selected with the MNI152 2 mm cor-
tical atlas. Subsequently mean uptake values of these
regions of interest (ROIs) and the tissue ratio, equiva-
lent
to
the
SUVR,
were
calculated
using
the In the original protocol, an additional MRI scan was
planned for PET-MRI co-registration to allow more de-
tailed regional analyses of amyloid-β uptake [22]. Be-
cause of limitations in funding, this could not be
performed for this study after all. Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 5 of 12 Page 5 of 12 Statistical analysis differences between the groups at baseline (Table 1). All
participants were Dutch, of white European descent, and
had the same level of education. y
Statistical analyses were performed using IBM SPSS Sta-
tistics for Windows, version 20.0 (IBM Corp., Armonk,
NY, USA). Alpha was set at 0.05 and tested two-sided. All
continuous variables
were assessed for normal
distribution
by
inspection
of
histograms
and
the
Shapiro-Wilk test. Normally distributed data are shown
as mean ± SD. Not normally distributed data are pre-
sented with median and interquartile ranges (IQR). For
primary outcomes of cognitive assessment, raw test
scores were transformed into z-scores for each neuro-
psychological test. Z-scores were computed in SPSS and
were based on mean and SD of the whole sample. For
the other primary outcome measure, amyloid-β burden,
we used the mean global standard value uptake ratio
(SUVR) and the dichotomous visual read of the amyloid
PET scans (positive/negative) [37]. An independent sam-
ples t-test was performed to compare normally distrib-
uted outcome measures between the groups (shown as
mean ± SD); not normally distributed outcome measures
were assessed with Mann-Whitney U tests (shown as
median (IQR)). With regard to the home-EEG data, a
Wilcoxon signed rank test was performed to compare
deep sleep time (DST) during work weeks to DST dur-
ing rest periods. Home-EEG (2019) We were able to analyze EEG-based sleep measurements
during work and rest days in 13 maritime pilots (of the
n = 19 maritime pilots, 4 had retired by 2019 when these
measurements were scheduled, and could therefore no
longer be measured during workdays; 2 could not be an-
alyzed due to technical issues) (Fig. 1). Maritime pilots
showed less TST during work weeks compared to rest
weeks (Z = −3.18; p = 0.001) as well as less DST during
work weeks compared to rest weeks (Z = −2.83; p =
0.005) (Table 3). Based on the home-EEG measure-
ments, we created hypnograms of one maritime pilot for
a work week and a rest week, illustrated in Fig. 2. Maritime pilots were on average 4 years younger than
controls (Table 1; 95% CI −6.139 to −1.716). Results
from the independent t-test did not indicate other Table 1 Baseline characteristics
Characteristics
Controls, n = 16
Maritime pilots, n = 19
Age, years
57 ± 2.9
53 ± 3.4
Educational attainment, years
17.4 ± 7.3
18 ± 0
BMI, kg/m2
25.5 ± 2.7
25.7 ± 2.7
History of diabetes
0 (0)
0 (0)
SBP, mmHg
NA
148.0 ± 16.4
DBP, mmHg
NA
90.16 ± 11.7
Medication (“yes/no”)
2 (10.5)
4 (21.1)
Smoking (“yes/no”)
3 (15.8)
3 (15.8)
History of hypertension
0 (0)
0 (0)
History of high cholesterol
0 (0)
1 (5.3)
CFQ
26.4 ± 10.8
29 ± 7.8
HADS Anxiety
4.8 ± 3
4.0 ± 1.7
HADS Depression
3.6 ± 2.5
3.7 ± 2.7
Data is shown as mean ± SD or no. (%) (for normally distributed data)
Abbreviations: BMI body mass index, SBP systolic blood pressure, DBP diastolic
blood pressure, CFQ Cognitive Failure Questionnaire, HADS Hospital Anxiety
and Depression Scale, NA not applicable Table 1 Baseline characteristics Results After exclusion and drop-out of participants (see Fig. 1),
19 maritime pilots and 16 controls completed the study
(Fig. 1, Table 1). Sleep characteristics
PSQI Maritime pilots reported worse sleep quality on the
PSQI compared to controls, during rest weeks but espe-
cially during work weeks (Table 2). When comparing
PSQI scores between work week and rest week within
maritime pilots, results of the t-test revealed that the
average PSQI score for work weeks was almost twice the
score for rest weeks, with values exceeding the validated
cutoff point (≥7) for abnormal sleep behavior (Table 2). PSG (2016/2017) Both maritime pilots and controls had normal sleep pat-
terns, including normal amount of DST (Table 2), ruling
out intrinsic sleep disorders and indicating undisturbed
sleep in maritime pilots during rest days. Actigraphy Subjective reports (PSQI) of poor sleep was confirmed
by data from 10 days of actigraphy (mix of workdays and
rest days), which indicated more awakenings and less
sleep efficiency in the maritime pilot group compared to
controls (Table 2). Cognitive assessment PSQI was administered twice for maritime pilots, including one work week and one rest week
Abbreviations: PSG polysomnography, TST total sleep time, DST deep sleep time, REM rapid eye movement sleep, WASO wake after sleep onset, SEF sleep
efficiency, SOL sleep onset latency, PSQI Pittsburgh Sleep Quality Index
*Significant at p <0 .05
**Significant at p < 0.001 participants. There
were
no
correlations
between
SUVRs and sleep quality (PSQI overall score and DST
(for rest and work weeks)). the groups. Maritime pilots performed slightly better on
the visual recognition memory after short delay com-
pared to controls. Long-term memory consolidation,
however, did not differ between the groups. All test
scores were within normal age- and education-adjusted
ranges based on available normative data (data not
shown). All results can be found in Table 4. Discussion We investigated global brain amyloid-β levels and cogni-
tive function in a unique population experiencing long-
term sleep disruption, wherein every other week was
characterized by sleep disruption due to irregular work-
ing hours. Cognitive assessment For cognitive assessment, we transformed all raw neuro-
psychological test scores into z-scores. Results from the
independent t-test did not indicate differences between
maritime pilots and controls on tests of episodic mem-
ory (WMS-IV LM recognition, RAVLT total median). Small differences were observed on semantic memory
and language, in which maritime pilots performed
slightly better on the Boston Naming Test compared to
controls. Performance on working memory and execu-
tive function (WAIS-IV, TMT, WAIS-IV Coding) and
attention (TAP 2.0) did not differ significantly between Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 6 of 12 Table 2 Comprehensive sleep characteristics of maritime pilots and controls
Measures
Controls, n = 16
Maritime pilots, n = 19
p value
PSG
TST, min
406 ± 44
403 ± 51
0.86
N1, min
46 ± 18
41 ± 14
0.40
N2, min
232 ± 36
215 ± 36
0.20
DST, min
50 ± 25
66 ± 28
0.10
REM, min
68 ± 17
79 ± 17
0.10
WASO, min
61 ± 26
53 ± 39
0.48
SEF, %
85.8 ± 7.1
86.1 ± 9.4
0.91
SOL, min
8 ± 7
11 ± 9
0.32
Actiwatch
No. awakenings
33.5 ± 11.1
37.8 ± 10.3
0.24
SEF, %
89.3 ± 4.3
86 ± 3.8
0.03*
PSQI (rest week vs. control
Overall score
3.2 ± 1.4
4.5 ± 2.2*
0.049*
PSQI (work week vs. control)
Overall score
3.2 ± 1.4
8.8 ± 2.9**
< 0.001**
Data is shown as mean ± SD (for normally distributed data)
Actiwatch data and PSQI were collected in 2016 and 2017. Actiwatch data was collected for a period of 10 consecutive days; for maritime pilots, these 10 days
were a mix of work and rest days. PSQI was administered twice for maritime pilots, including one work week and one rest week
Abbreviations: PSG polysomnography, TST total sleep time, DST deep sleep time, REM rapid eye movement sleep, WASO wake after sleep onset, SEF sleep
efficiency, SOL sleep onset latency, PSQI Pittsburgh Sleep Quality Index
*Significant at p <0 .05
**Significant at p < 0.001 Table 2 Comprehensive sleep characteristics of maritime pilots and controls (
y
)
Actiwatch data and PSQI were collected in 2016 and 2017. Actiwatch data was collected for a period of 10 consecutive days; for maritime pilots, these 10 days
were a mix of work and rest days. 18F-flutemetamol PET-CT Amyloid PET scans were administered in maritime pi-
lots only (n = 19, Fig. 1). SUVRs in healthy populations
were reported as 1.29 (± 0.2) [39] and 1.3 (± 0.09) [27]. The global cortical SUVR in maritime pilots was 1.009
(± 0.059; 95% CI 0.980 to 1.037) and therefore below
normal values for a cognitively healthy population in this
age range [27, 39]. More specifically, we detected a
SUVR of 0.860 (± 0.098; 95% CI 0.813 to 0.907) for
frontal lobes and a SUVR of 0.996 (± 0.06; 95% CI 0.967
to 1.025) for temporal lobes. In addition, all scans were
rated negative for the presence of amyloid-β depos-
ition on visual reading. Figure 3 shows examples of
amyloid
PET
images
from
two
representative Our main finding is that, in this relatively small, but
deeply phenotyped sample, this intensity and pattern of
sleep disruption was not associated with elevated brain
amyloid-β levels, nor with cognitive decline. In previous studies, a single night of full sleep
deprivation, or selective restriction of deep sleep,
and
chronic
partial
sleep
fragmentation
(rodents
only) increased brain amyloid-β levels [8–13]. These
observations have fueled the hypothesis that re-
peated nights of sleep loss may contribute to the
risk of dementia due to AD by gradually increasing
amyloid-β levels. Table 3 Results from the home-EEG measurements (maritime pilots only)
Measures
Rest week, n = 13
Work week, n = 13
p value
Home EEG
TST†, min
406.17 (340–425.98)
318.56 (250.21–352.93)
0.001*
DST†, min
51.34 (48.37–69.30)
36.75 (32.30–58.58)
0.005*
Data is shown as median (IQR) (for not normally distributed data)
Home-EEG recordings were performed in 2019 in maritime pilots only using a dry single-lead EEG device (Philips, Eindhoven, The Netherlands)
Abbreviations: TST total sleep time, DST deep sleep time
*Significant at p < 0.05
†Means calculated based on sleep periods within work week or rest week respectively Page 7 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Fig. 2 Example of a maritime pilots’ sleep schedule. a Hypnogram is based on 7 consecutive working days of sleep measurements with a dry
electrode single-lead home-EEG device. b Hypnogram is based on 7 rest days of sleep measurements with a dry electrode single-lead
home-EEG device Fig. 2 Example of a maritime pilots’ sleep schedule. a Hypnogram is based on 7 consecutive working days of sleep measurements with a dry
electrode single-lead home-EEG device. 18F-flutemetamol PET-CT b Hypnogram is based on 7 rest days of sleep measurements with a dry electrode single-lead
home-EEG device Fig. 2 Example of a maritime pilots’ sleep schedule. a Hypnogram is based on 7 consecutive working days of sleep measurements with a dry
electrode single-lead home-EEG device. b Hypnogram is based on 7 rest days of sleep measurements with a dry electrode single-lead
home-EEG device Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Page 8 of 12 Table 4 Results of cognitive assessment and memory consolidation
Measures
Controls, n = 16
Maritime pilots, n = 19
p value
WMS-IV
LM I
0.16 ± 1.07
−0.08 ± 0.99
0.49
LM II
0.29 (−0.93–1.01)
0.11 (−0.61–0.83)
0.72
LM recognition
0.25 ± 1.10
−0.12 ± 0.94
0.29
RAVLT
Total
−0.08 (−0.63–0.61)
0.77 (−1.14–1.09)
0.41
Del. recall
−0.08 ± 0.76
0.06 ± 1.25
0.70
Del. recognition
−0.10 ± 1.15
0.21 ± 0.85
0.37
Sensitivity A’
0.07 (−0.85–0.82)
0.22 (−0.30–0.82)
0.41
WAIS-IV
Coding
−0.10 ± 0.59
0.21 ± 1.26
0.37
Digit span
−0.21 ± 0.62
0.23 ± 1.24
0.21
TMT
Part A
−0.09 ± 0.75
−0.06 ± 1.15
0.94
Part B
−0.33 (−0.57–0.75)
−0.38 (−0.96–0.50)
0.24
Fluency
D-A-T
0.07 ± 0.89
−0.29 ± 1.13
0.78
Animal
0.38 (−0.88–0.73)
0.20 (−0.70–0.91)
0.84
Profession
−0.26 ± 0.85
0.26 ± 1.13
0.14
BNT
Short version
−0.11 (−0.39–0.31)
0.20 (0.10–0.62)
0.02*
TAP evening
Cued
−0.05 (−0.72–0.44)
−0.39 (−0.91–0.50)
0.37
Un-cued
−0.05 (−0.87–0.89)
−0.17 (−0.76–0.45)
0.84
TAP morning
Cued
−0.10 (−0.50–0.66)
−0.29 (−0.71–0.12)
0.27
Un-cued
−0.13 (−0.63–1.09)
−0.40 (−0.74–0.33)
0.22
Visual recognition—short delay (10 min)
Sensitivity, A’
−0.27 ± 0.90
0.46 ± 0.58
0.007*
Hits
−0.33 (−1.18–0.15)
0.64 (0.15–0.64)
0.03*
False alarms
0.19 ± 1.06
−0.27 ± 0.84
0.16
Memory consolidation—long delay (after sleep)
Sensitivity, A′
−0.08 ± 0.95
0.35 ± 0.73
0.14
Hits
0.50 (−0.21–0.70)
−0.08 (−0.69–0.54)
0.20
False alarms
0.15 ± 0.90
−0.41 ± 0.76
0.06
Data is shown as mean ± SD (for normally distributed data) or median (IQR) (for not normally distributed data)
Test results are expressed in z-scores. TAP: z-scores are based on median reaction-time. Visual recognition—short-term: assessed approximately 10 min after
targets were presented. 18F-flutemetamol PET-CT Memory consolidation after long-term took place after one night of sleep (approximately 10 h)
Abbreviations: WMS Wechsler Memory Scale, LM logical memory, RAVLT Rey Auditory Verbal Learning Test, WAIS Wechsler Adult Intelligent Scale, TMT Trail Making
test, BNT Boston Naming Test, TAP Test of Attentional Performance
*Significant at p < 0.05 Table 4 Results of cognitive assessment and memory consolidation Data is shown as mean ± SD (for normally distributed data) or median (IQR) (for not normally distributed data)
Test results are expressed in z-scores. TAP: z-scores are based on median reaction-time. Visual recognition—short-term: assessed approximately 10 min after
targets were presented. Memory consolidation after long-term took place after one night of sleep (approximately 10 h)
Abbreviations: WMS Wechsler Memory Scale, LM logical memory, RAVLT Rey Auditory Verbal Learning Test, WAIS Wechsler Adult Intelligent Scale, TMT Trail Making
test, BNT Boston Naming Test, TAP Test of Attentional Performance
*Significant at p < 0.05 The sample of maritime pilots offered a unique oppor-
tunity to explore if long-term, externally induced sleep
disruptions increase dementia risk in terms of AD-
related
impaired
cognitive
function
and
amyloid-β
burden. Their sleep behavior is characterized by work
weeks with disrupted sleep, alternating with rest weeks
of unrestricted sleep. This pattern was confirmed using
a combination of methods: self-reported disrupted sleep
during work weeks was objectified by sleep diaries, acti-
graphy, and home-EEG measurements. Relatively normal
sleep during rest weeks of maritime pilots was further-
more confirmed with PSG (compared to controls) and
home-EEG measurements. Moreover, using PSG we
were able to exclude intrinsic sleep disorders in this
group, which is important because sleep loss may be an
early manifestation of Alzheimer’s pathology and could
lead to a reverse causality association [41, 42]. To explore possible AD-related impaired cognitive perform-
ance, we applied a cognitive test battery that was chosen
for its sensitivity to cognitive changes in early, preclinical
AD [24]. On all cognitive domains, maritime pilots
showed normal cognitive performance, compared not
only to the control group, but also to normative values. This was also the case for overnight episodic memory
consolidation, which is dependent on deep sleep [5, 43]. We considered that normal cognitive function would
not rule out increased amyloid-β levels, since early
stages of amyloid-β accumulation (indicated by PET or
CSF) can have a long asymptomatic stage. Therefore, we
performed additional global brain amyloid-β imaging in
maritime pilots. 18F-flutemetamol PET-CT None of the maritime pilots had evi-
dence of elevated amyloid-β levels, with SUVR values
remaining below the values established for a healthy
population [27, 39]. In a recent meta-analysis, the Page 9 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Fig. 3 Representative transversal slides from 18F-flutemetamol PET scans of two participants. Scans were acquired 90–110 min post-injection and
show normal subcortical nonspecific uptake in the brain Fig. 3 Representative transversal slides from 18F-flutemetamol PET scans of two participants. Scans were acquired 90–110 min post-injection and
show normal subcortical nonspecific uptake in the brain levels. This argument is, however, not supported by
recent work demonstrating that a reduction in total
sleep time, but not SWS, determined the increase in
amyloid-β production [20]. estimated prevalence of PET amyloid-positivity in cogni-
tively healthy men aged 55–60 years was 13% (95% CI
10.3 to 16%) [44]. This indicates that our observation of a
prevalence of 0/19 confidently rules out elevated amyloid-
β levels, even considering the relatively small sample size. Second, it is possible that sleep disruption alone is in-
sufficient to increase AD risk, but requires the presence
of other risk factors, such as impaired glucose metabol-
ism [45], oxidative stress [46], depression [47], or general
poor vascular health [48]. Our study population was
healthy and had a low vascular risk (Table 1). What could explain the observed absence of elevated
amyloid-β levels or impaired cognitive function, despite
evidence of long-term sleep disruptions? First, assuming that the hypothesis that sleep dis-
ruption may cause AD is correct, the alternating pat-
tern of a week with unrestricted sleep following a
week of disrupted sleep may be insufficient to cause
elevated brain amyloid-β levels. Either sleep disrup-
tion during ≈50% of nights for ≈20 years is insuffi-
cient to affect amyloid-β clearance/production or the
week of normal sleep following a week of sleep dis-
ruption provides compensatory reductions in brain
amyloid-β levels. This latter option would then sug-
gest that disrupted sleep is a modifiable risk factor
and that it may not be necessary to achieve full
normalization of sleep to reduce AD risk. Whether
this can be extrapolated to the general population is
uncertain however. The maritime pilots may, due to
their profession, be better able to compensate normal
sleep in their rest weeks. 18F-flutemetamol PET-CT While most studies link re-
duced total sleep time (< 6 h) to increased AD risk [1,
20], other work suggests that the risk is specifically
linked to reduced deep sleep [12, 17]. The maritime
pilots
had
reduced
total
sleep
time
during
work
weeks, but the home-EEG recordings indicate that
they still achieved an average of 37 min of deep sleep
per sleep period. Therefore, SWS may have been in-
sufficiently impaired to result in abnormal amyloid-β Current research on this topic is still in its very early
stage, with limited evidence supporting a causal relation-
ship between sleep loss and risk of AD dementia. The
association between sleep loss and AD may be driven by
reverse causality (sleep loss as an early manifestation of
AD) or by a shared common pathway that causes sleep
loss and increases AD risk. There is also recent evidence
that found no association between sleep (subjective sleep
quality) and risk of dementia [49]. Previous evidence suggesting a link between sleep and
AD has been limited to a small number of studies in ro-
dents and humans, with variations in methodology and
study population selection. Furthermore, the human
studies focused on the relationship between poor sleep
for a short period of time (1 or 2 nights) and its effects
on amyloid-β (or tau), but have not studied actual devel-
opment of AD dementia. Longitudinal studies are available but lack rigorous
assessment of sleep and biomarker evidence of AD. Our study adds information on the long-term associ-
ation between poor sleep and AD, combining object-
ive sleep measures with established biomarkers for
AD. Page 10 of 12 Page 10 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Acknowledgements One could argue that the absence of tau measure-
ments is a limitation, as recent evidence now also sug-
gests that sleep affects tau in a similar manner as
amyloid-β [50]. We did not perform tau measurements
because the maritime pilots had no evidence of cognitive
impairment. Since tau pathology is strongly correlated
with cognitive decline [51–53], it is highly unlikely to
find evidence of tau accumulation in subjects with nor-
mal cognitive function, even more so when they are
amyloid-negative. A final limitation is that amyloid-β
status was not obtained from the controls. Instead, we
compared our outcomes to normative values from the
literature, which were acquired with additional MRI
measurements for co-registration of the amyloid PET-
CT scans. Since we used CT to identify global amyloid-β
instead of MRI, this difference in methodology has to be
kept in mind when interpreting our results. We would like to thank all participants for taking part in this study and the
secretary of the Dutch Maritime Pilot Association for helping with
recruitment of participants. Furthermore, a special thanks to the interns who
helped with data collection and to J. Doornbosch for helping with the
neuropsychological test administration. Dr. T. Tsoneva and S. Pastoor from
Philips helped us with data storage and raw data-extraction from the home-
EEG devices. J.M.F. Liebregts provided advice on and assistance with design-
ing graphics and figures. We would also like to thank Dr. K.V. Haak for his ad-
vice on the PET-CT imaging analyses. Abbreviations AD: Alzheimer’s disease; CFQ: Cognitive Failure Questionnaire; DBP: Diastolic
blood pressure; DST: Deep sleep time; HADS: Hospital Anxiety and
Depression Scale; IQR: Interquartile range; NA: Not applicable; NRA: Number
of arousals; NRI: Number of interruptions; PSG: Polysomnography;
PSQI: Pittsburgh Sleep Quality Index; RAVLT: Rey Auditory Verbal Learning
Test; REM: Rapid eye movement; ROI: Region of interest; SBP: Systolic blood
pressure; SCHIP: Sleep Cognition Hypothesis In maritime Pilots; SD: Standard
deviation; SEF: Sleep efficiency; SOL: Sleep onset latency; SUVR: Standard
uptake value ratio; SWS: Slow wave sleep; TAP: Test of Attentional
Performance; TMT: Trail Making Test; TST: Total sleep time; WAIS-IV: Wechsler
Adult Intelligence Scale (4th edition); WASO: Wake after sleep onset; WMS-IV
LM: Wechsler Memory Scale (4th edition) Logical Memory Another limitation is that controls, although matched
for sex, age, education, and general health, might not
have been matched entirely with regard to personality,
resilience, physical activity, or cognitive skills/general
intelligence. Supplementary information
S
l
i f
i
i pp
y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s13195-020-00668-5. Additional file 1. Details of the study population and their occupation. Additional file 2. Supplemental Box 1: Overview Neuropsychological
Test Battery. Additional file 1. Details of the study population and their occupation. Additional file 2. Supplemental Box 1: Overview Neuropsychological
Test Battery. The uniqueness of the population may also cause bias. Maritime pilots are healthy, have no cardiovascular risk,
and are physically active in their work, factors that may
reduce their AD risk. They may be resilient to the conse-
quences of sleep disruption, because they have success-
fully performed this work for > 10 years. One example of
such resilience could be their ability to achieve deep
sleep even under conditions of fragmented and restricted
total sleep during work weeks or their capacity to gener-
ate sufficient deep sleep during rest weeks. Conclusions A strength of the study is the comprehensive assess-
ments of all outcome measures: cognitive function was
assessed with an extensive test battery sensitive to early,
preclinical symptoms of AD; sleep was assessed with
various measurements including self-reported but also
objectively measured sleep, implementing innovative
techniques for sleep assessments (home-EEG); sleep dis-
orders were ruled out using PSG; and PET-amyloid im-
aging was used as a validated AD biomarker. A further
strength is the unique cohort of maritime pilots, with
prolonged and consistent exposure to sleep loss related
to their work, making this a highly valuable population
that allowed us to explore poor sleep as isolated variable
in relationship with the risk of AD dementia. In this study, we tested the hypothesis that prolonged
sleep loss increases the risk of dementia due to AD. We
found that a history of work-induced, long-term sleep
disruption was not associated with impairment in cogni-
tive function, nor with elevated global brain amyloid-β
levels, in a group of healthy, middle-aged men. Taking
into account the small sample size of our study, our re-
sults
do
not
necessarily
refute
the
hypothesis
we
intended to test, but neither support it. It is possible that
amyloid-β
accumulation
during
periods
with
sleep
disruption can be reduced in nights with normal sleep. Alternatively, sleep loss may only increase AD risk in
combination with other factors. Finally, the association
between sleep loss and AD in epidemiological studies
may be driven by reverse causality. These and other hy-
potheses have to be tested in future studies. Our study is limited by the small sample size. Home-EEG
measurements were available in 13 of the 19 maritime pi-
lots. However, outcomes of these sleep measurements con-
firmed observations of work-related disrupted sleep based
on PSQI, sleep-wake dairy and actigraphy data in the whole
sample, and added novel data on total and deep sleep time
during work weeks and rest weeks. Funding 9. Rothman SM, Herdener N, Frankola KA, Mughal MR, Mattson MP. Chronic
mild sleep restriction accentuates contextual memory impairments, and
accumulations of cortical Aβ and pTau in a mouse model of Alzheimer’s
disease. Brain Res. 2013;1529:200–8. This work was supported by ISAO (Internationale Stichting Alzheimer
Onderzoek (now Alzheimer Nederland), grant number: 15040) awarded to JC. Philips kindly provided the home-EEG devices that were used in this study. The funders had no role in the study design or conduct; collection, analysis,
or interpretation of data; nor in the writing of the report or in the decision
to submit the manuscript for publication. 10. Zhao HY, Wu HJ, He JL, Zhuang JH, Liu ZY, Huang LQ, et al. Chronic sleep
restriction induces cognitive deficits and cortical beta-amyloid deposition in
mice via BACE 1-antisense activation. CNS Neurosci Ther. 2017;23(3):233–40. 10. Zhao HY, Wu HJ, He JL, Zhuang JH, Liu ZY, Huang LQ, et al. Chronic sleep
restriction induces cognitive deficits and cortical beta-amyloid deposition in
mice via BACE 1-antisense activation. CNS Neurosci Ther. 2017;23(3):233–40. 11. Ooms S, Overeem S, Besse K, Rikkert MO, Verbeek M, Claassen JA. Effect of 1
night of total sleep deprivation on cerebrospinal fluid β-amyloid 42 in
healthy middle-aged men: a randomized clinical trial. JAMA neurology. 2014;71(8):971–7. Consent for publication
Not applicable. 17. Fultz NE, Bonmassar G, Setsompop K, Stickgold RA, Rosen BR, Polimeni JR,
et al. Coupled electrophysiological, hemodynamic, and cerebrospinal fluid
oscillations in human sleep. Science (80- ). 2019;366(6465):628–31. Competing interests 18. Jessen NA, Munk ASF, Lundgaard I, Nedergaard M. The glymphatic system:
a beginner’s guide. Neurochem Res. 2015;40(12):2583–99. 18. Jessen NA, Munk ASF, Lundgaard I, Nedergaard M. The glymphatic system:
a beginner’s guide. Neurochem Res. 2015;40(12):2583–99. The authors declare that they have no competing interests. Availability of data and materials
d
d
l
d d Data generated and/or analyzed during this study are included in this article
and its supplementary information files. Additionally, the datasets used/
analyzed in the current study are available from the corresponding author
on reasonable request. 12. Ju Y-ES, Ooms SJ, Sutphen C, Macauley SL, Zangrilli MA, Jerome G, et al. Slow wave sleep disruption increases cerebrospinal fluid amyloid-β levels. Brain. 2017;140(8):2104–11. 12. Ju Y-ES, Ooms SJ, Sutphen C, Macauley SL, Zangrilli MA, Jerome G, et al. Slow wave sleep disruption increases cerebrospinal fluid amyloid-β levels. Brain. 2017;140(8):2104–11. 13. Shokri-Kojori E, Wang G-J, Wiers CE, Demiral SB, Guo M, Kim SW, et al. β-
Amyloid accumulation in the human brain after one night of sleep
deprivation. Proc Natl Acad Sci. 2018;115(17):4483–8. 13. Shokri-Kojori E, Wang G-J, Wiers CE, Demiral SB, Guo M, Kim SW, et al. β-
Amyloid accumulation in the human brain after one night of sleep
deprivation. Proc Natl Acad Sci. 2018;115(17):4483–8. Received: 21 April 2020 Accepted: 13 August 2020 Received: 21 April 2020 Accepted: 13 August 2020 Received: 21 April 2020 Accepted: 13 August 2020 23. Garcia-Molina G, Tsoneva T, Jasko J, Steele B, Aquino A, Baher K, et al. Closed-loop system to enhance slow wave activity during sleep. 2018. 24. Weintraub S, Carrillo MC, Farias ST, Goldberg TE, Hendrix JA, Jaeger J, et al. Measuring cognition and function in the preclinical stage of Alzheimer’s
disease. Alzheimer’s Dementia. 2018;4:64–75. Authors’ contributions JT, SJO, SO, RPCK, and JAHRC made substantial contributions to conception
or design of the work. All authors (JT, SJO, LJM, JB, MGMO, SO, RPCK, JAHRC)
were in charge of acquisition, analysis, and interpretation of data for the
work. JT, SJO, LJM, JB, RPCK, and JAHRC were responsible for statistical data
analyses. Supervision was given by JAHRC. All authors drafted the work or
revised it critically for important intellectual content and approved the final
version of the manuscript. All authors have agreed both to be personally
accountable for their own contributions and to ensure that questions related
to the accuracy or integrity of any part of the work, even ones in which the
author was not personally involved, are appropriately investigated, resolved,
and the resolution documented in the literature. Page 11 of 12 Page 11 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Author details
1 19. Kang DW, Lee CU, Lim HK. Role of sleep disturbance in the trajectory of
Alzheimer’s disease. Clin Psychopharmacol Neurosci. 2017;15(2):89. Author details
1Department of Geriatric Medicine, Radboud University Medical Center, 6525,
GC, Nijmegen, The Netherlands. 2Donders Institute for Brain, Cognition and
Behaviour, 6525, HR, Nijmegen, The Netherlands. 3Radboud Alzheimer
Centre, 6525, GA, Nijmegen, The Netherlands. 4Department of Radiology and
Nuclear Medicine, Radboud University Medical Center, 6525, GC, Nijmegen,
The Netherlands. 5Department of Radiology and Nuclear Medicine,
Amsterdam University Medical Centers, Academic Medical Center, 1105, AZ,
Amsterdam, The Netherlands. 6Sleep Medicine Centre Kempenhaeghe, 5591,
VE, Heeze, The Netherlands. 7Eindhoven University of Technology, 5612, AZ,
Eindhoven, The Netherlands. 8Department of Medical Psychology, Radboud
University Medical Center, 6525, GA, Nijmegen, The Netherlands. 20. Lucey BP, Hicks TJ, McLeland JS, Toedebusch CD, Boyd J, Elbert DL, et al. Effect of sleep on overnight cerebrospinal fluid amyloid β kinetics. Ann
Neurol. 2018;83(1):197–204. 21. Faul F, Erdfelder E, Lang A-G, Buchner A. G* Power 3: a flexible statistical
power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175–91. 22. Thomas J, Ooms S, Verbeek M, Booij J, Rijpkema M, Kessels RPC, et al. Sleep-
Cognition Hypothesis In maritime Pilots, what is the effect of long-term
work-related poor sleep on cognition and amyloid accumulation in healthy
middle-aged maritime pilots: methodology of a case–control study. BMJ
Open. 2019;9(6):e026992. References 1. Bubu OM, Brannick M, Mortimer J, Umasabor-Bubu O, Sebastião YV, Wen Y,
et al. Sleep, cognitive impairment, and Alzheimer’s disease: a systematic
review and meta-analysis. Sleep. 2016;40(1):zsw032. 1. Bubu OM, Brannick M, Mortimer J, Umasabor-Bubu O, Sebastião YV, Wen Y,
et al. Sleep, cognitive impairment, and Alzheimer’s disease: a systematic
review and meta-analysis. Sleep. 2016;40(1):zsw032. 25. Baddeley A, Emslie H, Nimmo-Smith I. Doors and people: a test of visual
and verbal recall and recognition, Thames Valley Test Company, Bury St. Edmunds, UK. 1994. 25. Baddeley A, Emslie H, Nimmo-Smith I. Doors and people: a test of visual
and verbal recall and recognition, Thames Valley Test Company, Bury St. Edmunds, UK. 1994. 2. Mander BA, Winer JR, Jagust WJ, Walker MP. Sleep: a novel mechanistic
pathway, biomarker, and treatment target in the pathology of Alzheimer’s
disease? Trends Neurosci. 2016;39(8):552–66. 26. Curtis C, Gamez JE, Singh U, Sadowsky CH, Villena T, Sabbagh MN, et al. Phase 3 trial of flutemetamol labeled with radioactive fluorine 18 imaging
and neuritic plaque density. JAMA neurology. 2015;72(3):287–94. 3. Spira AP, Gottesman RF. Sleep disturbance: an emerging opportunity for
Alzheimer’s disease prevention? Int Psychogeriatr. 2017;29(4):529–31. 27. Lowe VJ, Lundt E, Knopman D, Senjem ML, Gunter JL, Schwarz CG, et al. Comparison of [18F] Flutemetamol and [11C] Pittsburgh Compound-B in
cognitively normal young, cognitively normal elderly, and Alzheimer’s
disease dementia individuals. NeuroImage: Clinical. 2017;16:295–302. 4. Slats D, Claassen JA, Verbeek MM, Overeem S. Reciprocal interactions
between sleep, circadian rhythms and Alzheimer’s disease: focus on the role
of hypocretin and melatonin. Ageing Res Rev. 2013;12(1):188–200. 28. Hansson O, Seibyl J, Stomrud E, Zetterberg H, Trojanowski JQ, Bittner T,
et al. CSF biomarkers of Alzheimer’s disease concord with amyloid-β PET
and predict clinical progression: a study of fully automated immunoassays
in BioFINDER and ADNI cohorts. Alzheimers Dement. 2018;14(11):1470–81. y
g
g
5. Ju Y-ES, Lucey BP, Holtzman DM. Sleep and Alzheimer disease pathology
[mdash] a bidirectional relationship. Nat Rev Neurol. 2014;10(2):115–9. 6. Roh JH, Huang Y, Bero AW, Kasten T, Stewart FR, Bateman RJ, et al. Disruption of the sleep-wake cycle and diurnal fluctuation of β-amyloid in
mice with Alzheimer’s disease pathology. Sci Transl Med. 2012;4(150):
150ra22-ra22. 29. Pannee J, Portelius E, Minthon L, Gobom J, Andreasson U, Zetterberg H,
et al. Reference measurement procedure for CSF amyloid beta (Aβ) 1–42
and the CSF Aβ1–42/Aβ1–40 ratio–a cross-validation study against amyloid
PET. J Neurochem. 2016;139(4):651–8. 7. Ethics approval and consent to participate The SCHIP study was approved by the institutional review board (CMO,
Commissie Mensgebonden Onderzoek, Region Arnhem-Nijmegen,
NL55712.091.16, file number 2016-2337) and performed in accordance with
good clinical practice guidelines and the world medical associations code of
ethics (Declaration of Helsinki). Written informed consent was obtained from
all participants after they received detailed study information. Participants re-
ceived a stipend of 50 euros for participating; travel costs were
compensated. 14. Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, et al. Sleep
drives metabolite clearance from the adult brain. Science (80- ). 2013;
342(6156):373–7. 14. Xie L, Kang H, Xu Q, Chen MJ, Liao Y, Thiyagarajan M, et al. Sleep
drives metabolite clearance from the adult brain. Science (80- ). 2013;
342(6156):373–7. 15. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A
paravascular pathway facilitates CSF flow through the brain parenchyma
and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012;4(147):147ra11-ra11. 15. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. A
paravascular pathway facilitates CSF flow through the brain parenchyma
and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012;4(147):147ra11-ra11. 16. Nedergaard M. Garbage truck of the brain. Science (80- ). 2013;
340(6140):1529–30. 16. Nedergaard M. Garbage truck of the brain. Science (80- ). 2013;
340(6140):1529–30. 7.
Kang J-E, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, et al. Amyloid-β
dynamics are regulated by orexin and the sleep-wake cycle. Science (80- ).
2009;326(5955):1005–7. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 33. Reimand J, De Wilde A, Teunissen CE, Zwan M, Windhorst AD, Boellaard
R, et al. PET and CSF amyloid-β status are differently predicted by
patient features: information from discordant cases. Alzheimers Res
Ther. 2019;11(1):100. 33. Reimand J, De Wilde A, Teunissen CE, Zwan M, Windhorst AD, Boellaard
R, et al. PET and CSF amyloid-β status are differently predicted by
patient features: information from discordant cases. Alzheimers Res
Ther. 2019;11(1):100. 34. Jenkinson M, Bannister P, Brady M, Smith S. Improved optimization for the
robust and accurate linear registration and motion correction of brain
images. Neuroimage. 2002;17(2):825–41. 34. Jenkinson M, Bannister P, Brady M, Smith S. Improved optimization for the
robust and accurate linear registration and motion correction of brain
images. Neuroimage. 2002;17(2):825–41. 35. Jenkinson M, Beckmann CF, Behrens TE, Woolrich MW, Smith SM. Fsl
Neuroimage. 2012;62(2):782–90. 36. Andersson JL, Jenkinson M, Smith S. Non-linear registration aka Spatial
normalisation FMRIB Technial Report TR07JA2. FMRIB Analysis Group of the
University of Oxford 2007. 37. Farrar G, Molinuevo JL, Zanette M. Is there a difference in regional read [18
F] flutemetamol amyloid patterns between end-of-life subjects and those
with amnestic mild cognitive impairment? Eur J Nucl Med Mol Imaging. 2019;46(6):1299–308. 38. Vandenberghe R, Van Laere K, Ivanoiu A, Salmon E, Bastin C, Triau E, et al. 18F-flutemetamol amyloid imaging in Alzheimer disease and mild cognitive
impairment: a phase 2 trial. Ann Neurol. 2010;68(3):319–29. 39. Thurfjell L, Lötjönen J, Lundqvist R, Koikkalainen J, Soininen H, Waldemar G,
et al. Combination of biomarkers: PET [18F] flutemetamol imaging and
structural MRI in dementia and mild cognitive impairment. Neurodegener
Dis. 2012;10(1–4):246–9. 40. Buckley CJ, Sherwin PF, Smith AP, Wolber J, Weick SM, Brooks DJ. Validation of an electronic image reader training programme for
interpretation of [18F] flutemetamol β-amyloid PET brain images. Nucl
Med Commun. 2017;38(3):234. 41. Winer JR, Mander BA, Helfrich RF, Maass A, Harrison TM, Baker SL, et al. Sleep as a potential biomarker of tau and β-amyloid burden in the human
brain. J Neurosci. 2019:0503–19. 42. Lucey BP, McCullough A, Landsness EC, Toedebusch CD, McLeland JS, Zaza
AM, et al. Reduced non–rapid eye movement sleep is associated with tau
pathology in early Alzheimer’s disease. Sci Transl Med. 2019;11(474):
eaau6550. 43. Mander BA, Marks SM, Vogel JW, Rao V, Lu B, Saletin JM, et al. References Kang J-E, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, et al. Amyloid-β
dynamics are regulated by orexin and the sleep-wake cycle. Science (80- ). 2009;326(5955):1005–7. 7. Kang J-E, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, et al. Amyloid-β
dynamics are regulated by orexin and the sleep-wake cycle. Science (80- ). 2009;326(5955):1005–7. 30. Palmqvist S, Zetterberg H, Blennow K, Vestberg S, Andreasson U, Brooks DJ,
et al. Accuracy of brain amyloid detection in clinical practice using
cerebrospinal fluid β-amyloid 42: a cross-validation study against amyloid
positron emission tomography. JAMA neurology. 2014;71(10):1282–9. 30. Palmqvist S, Zetterberg H, Blennow K, Vestberg S, Andreasson U, Brooks DJ,
et al. Accuracy of brain amyloid detection in clinical practice using
cerebrospinal fluid β-amyloid 42: a cross-validation study against amyloid
positron emission tomography. JAMA neurology. 2014;71(10):1282–9. 8. Minakawa EN, Miyazaki K, Maruo K, Yagihara H, Fujita H, Wada K, et al. Chronic sleep fragmentation exacerbates amyloid β deposition in
Alzheimer’s disease model mice. Neurosci Lett. 2017;653:362–9. 8. Minakawa EN, Miyazaki K, Maruo K, Yagihara H, Fujita H, Wada K, et al. Chronic sleep fragmentation exacerbates amyloid β deposition in
Alzheimer’s disease model mice. Neurosci Lett. 2017;653:362–9. Page 12 of 12 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 53. Ossenkoppele R, Smith R, Ohlsson T, Strandberg O, Mattsson N, Insel PS,
et al. Associations between tau, Aβ, and cortical thickness with cognition in
Alzheimer disease. Neurology. 2019;92(6):e601–e12. 31. Palmqvist S, Zetterberg H, Mattsson N, Johansson P, Minthon L, Blennow K,
et al. Detailed comparison of amyloid PET and CSF biomarkers for
identifying early Alzheimer disease. Neurology. 2015;85(14):1240–9. 32. Bouallègue FB, Mariano-Goulart D, Payoux P, Initiative AsDN. Comparison of
CSF markers and semi-quantitative amyloid PET in Alzheimer’s disease
diagnosis and in cognitive impairment prognosis using the ADNI-2
database. Alzheimers Res Ther. 2017;9(1):32. Thomas et al. Alzheimer's Research & Therapy (2020) 12:101 Publisher’s Note [beta]-
amyloid disrupts human NREM slow waves and related hippocampus-
dependent memory consolidation. Nat Neurosci. 2015;18(7):1051–7. 44. Jansen WJ, Ossenkoppele R, Knol DL, Tijms BM, Scheltens P, Verhey FR, et al. Prevalence of cerebral amyloid pathology in persons without dementia: a
meta-analysis. Jama. 2015;313(19):1924–38. 45. Kuehn BM. In Alzheimer Research. JAMA: Glucose metabolism moves to
center stage; 2020. 46. Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G. Oxidative stress in
Alzheimer’s disease. Biochimica et Biophysica Acta (BBA)-Molecular Basis of
Disease. 2000;1502(1):139–44. 47. Green RC, Cupples LA, Kurz A, Auerbach S, Go R, Sadovnick D, et al. Depression as a risk factor for Alzheimer disease: the MIRAGE Study. Arch
Neurol. 2003;60(5):753–9. 48. Rabin JS, Klein H, Kirn DR, Schultz AP, Yang HS, Hampton O, Pruzin J. Associations of physical activity and β-amyloid with longitudinal cognition
and neurodegeneration in clinically normal older adults. JAMA neurology. 2019;76(10):1203-10. 49. Lysen TS, Wolters FJ, Luik AI, Ikram MK, Tiemeier H, Ikram MA. Subjective
sleep quality is not associated with incident dementia: the Rotterdam study. J Alzheimers Dis. 2018;64(1):239–47. 50. Holth JK, Fritschi SK, Wang C, Pedersen NP, Cirrito JR, Mahan TE, et al. The
sleep-wake cycle regulates brain interstitial fluid tau in mice and CSF tau in
humans. Science (80- ). 2019;363(6429):880–4. 51. Bejanin A, Schonhaut DR, La Joie R, Kramer JH, Baker SL, Sosa N, et al. Tau
pathology and neurodegeneration contribute to cognitive impairment in
Alzheimer’s disease. Brain. 2017;140(12):3286–300. 52. Pontecorvo MJ, Devous MD Sr, Navitsky M, Lu M, Salloway S, Schaerf FW,
et al. Relationships between flortaucipir PET tau binding and amyloid
burden, clinical diagnosis, age and cognition. Brain. 2017;140(3):748–63.
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https://hal-cea.archives-ouvertes.fr/cea-03579858/document
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In-Can vitrification of ash
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To cite this version: Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque. In-Can vitrification of ash. IOP
Conference Series: Materials Science and Engineering, 2020, 818 (1), pp.012005. 10.1088/1757-
899X/818/1/012005. cea-03579858 In-Can vitrification of ash
Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque
To cite this version:
Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque.
In-Can vitrification of ash.
IOP
Conference Series: Materials Science and Engineering, 2020, 818 (1), pp.012005.
10.1088/1757-
899X/818/1/012005. cea-03579858 In-Can vitrification of ash Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque To cite this version:
Maxime Fournier, Nicolas Massoni, J-Francois F Hollebecque. In-Can vitrification of ash. IOP
Conference Series: Materials Science and Engineering, 2020, 818 (1), pp.012005. 10.1088/1757-
899X/818/1/012005. cea-03579858 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: cea-03579858
https://cea.hal.science/cea-03579858v1
Submitted on 18 Feb 2022 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 THERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste P Conf. Series: Materials Science and Engineering 818 (2020) 012005
doi:10.1088/1757-899X/818/1/01 M Fournier, N Massoni and J F Hollebecque
CEA, DEN, DE2D, Univ. Montpellier, Marcoule, F-30207 Bagnols-sur-Cèze M Fournier, N Massoni and J F Hollebecque
CEA, DEN, DE2D, Univ. Montpellier, Marcoule, F-30207 Bagnols-sur-Cèze maxime.fournier@cea.fr Abstract. The In-Can Melter is a metallic crucible heated in a refractory furnace using electrical
resistors allowing in-container vitrification. The In-Can Melter trial aims to demonstrate the
feasibility of the confinement in a glassy matrix of ash coming from existing incineration
processes. The ash was pelletised to allow its introduction into the can without dust emissions
and then incorporated in a 50 wt.% waste loading confinement matrix. The full-scale trial was
preceded by laboratory- and bench-scale tests. The microstructure and chemical durability of the
wasteform were characterised. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
nder licence by IOP Publishing Ltd
1 1. Introduction The Thermal treatment for radioactive waste minimisation and hazard reduction (THERAMIN) project
is a European Commission programme of work jointly funded by the Horizon 2020 Euratom research
and innovation program and various European nuclear waste management organisations (WMOs). The
THERAMIN project is being conducted between June 2017 and May 2020. Twelve European WMOs
and research and consultancy institutions from seven European countries are participating in
THERAMIN. The overall objective of THERAMIN is to provide improved, safe long-term storage and
disposal of intermediate-level waste and low-level waste suitable for thermal processing. The work
programme provides a vehicle for coordinated European Union-wide research and technology
demonstrations designed to provide improved understanding and optimisation of the application of
thermal treatment in radioactive waste management programmes across Europe, and moves
technologies higher up the Technology Readiness Level (TRL) scale. g
g
p
gy
(
)
In the framework of the THERAMIN project, the French Alternative Energies and Atomic Energy
Commission (CEA) carried out studies on the treatment and conditioning of ash resulting from the
incineration of technological surrogate waste. The trial described in this article is based on the CEA In-
Can Melter (ICM) consisting of a metallic crucible melter heated in a refractory furnace. Prior to the
full-scale trial, laboratory- and bench-scale tests were conducted to select optimised operating
conditions. Wasteforms produced were characterised after the trials. 2. Description of the CEA’s In-Can Melter p
The ICM is a metallic crucible heated in a refractory furnace using electrical resistors (Figure 1). The
can, with an approximate volume of 50 L, is renewed after each filling. The process can support either
liquid or solid waste feeds. With the current off-gas treatment system (not including a post-combustion
chamber), it can only tolerate small amounts of organics. It can also accept a small fraction of metal in
the waste. The design ensures that the process can operate remotely for high-activity waste. The design
can also be adapted for dealing with plutonium-containing material in gloveboxes. The end product can
be glass, glass ceramic, or simply a high-density waste product. 1 THERAMIN 2020 conference: thermal treatment of radioactive waste IOP Publishing IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
doi:10.1088/1757-899X/818/ IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
doi:10.1088/1757-899X/818/1/012005 Figure 1. Diagram of the CEA’s In-Can Melter. ICM has been developed for CEA needs since 2005 for military waste vitrification. The technology
is at TRL 7 and the ICM has undergone inactive commissioning in France with inactive simulants of
Can
Solid
feeding
Dust scrubber
Condenser
Liquid buffer
tank
Scrubbing column
Recycling
Vertical
sleeve
Furnace Solid
feeding Dust scrubber Vertical
sleeve Liquid buffer
tank Furnace Figure 1. Diagram of the CEA’s In-Can Melter. ICM has been developed for CEA needs since 2005 for military waste vitrification. The technology
is at TRL 7 and the ICM has undergone inactive commissioning in France with inactive simulants of
alpha effluent. The technology is currently being qualified for solid waste. ICM has been developed for CEA needs since 2005 for military waste vitrification. The technology
is at TRL 7 and the ICM has undergone inactive commissioning in France with inactive simulants of
alpha effluent. The technology is currently being qualified for solid waste. 3. Ash production The waste selected for this vitrification trial is the ash from multiple incineration tests of surrogate
technological waste (polyvinyl chloride, latex, neoprene, polyethylene, cotton, etc.) produced by the
CEA’s IRIS process (French acronym for Research facility for solid waste incineration). IRIS is a
research facility for the incineration of solids developed to treat organic waste from gloveboxes in the
nuclear industry, contaminated with alpha-bearing actinides and containing high quantities of chlorine
(Figure 2). Figure 2. Diagram of the CEA’s IRIS process for the treatment of organic waste. Organic waste
(4 to 7 kg·h
-1)
Pyrolyser
Post-combustion
(1100 °C)
Ash
Calciner
Pitch
Air
Air
Air
Cooler
High voltage
Electrostatic
filter
HEPA filter
Washing
column Figure 2. Diagram of the CEA’s IRIS process for the treatment of organic waste. The robustness and efficiency of this process is based on a decoupling of (i) the step of elimination
corrosive materials such as chlorine and (ii) the step of combustion of the organic waste. Figure 2
ows that the organic waste first goes into a pyrolysis step at a temperature of 500 °C to remove the
st corrosive gaseous compounds and then into a calciner (900 °C) fed with oxygen to complete the
mbustion while concentrating the contamination in the mineral ash. The relatively long residence time
Organic waste
(4 to 7 kg·h
-1)
Pyrolyser
Post-combustion
(1100 °C)
Ash
Calciner
Pitch
Air
Air
Air
Cooler
High voltage
Electrostatic
filter
HEPA filter
Washing
column Organic waste
(4 to 7 kg·h
-1) Air Pitch Pyrolyser Post-combustion
(1100 °C) Ash Calciner Figure 2. Diagram of the CEA’s IRIS process for the treatment of organic waste. The robustness and efficiency of this process is based on a decoupling of (i) the step of elimination
of corrosive materials such as chlorine and (ii) the step of combustion of the organic waste. Figure 2
shows that the organic waste first goes into a pyrolysis step at a temperature of 500 °C to remove the
most corrosive gaseous compounds and then into a calciner (900 °C) fed with oxygen to complete the
combustion while concentrating the contamination in the mineral ash. The relatively long residence time The robustness and efficiency of this process is based on a decoupling of (i) the step of elimination
of corrosive materials such as chlorine and (ii) the step of combustion of the organic waste. 4. Pre-treatment of ash and definition of their vitrification conditions In order to obtain a sufficient amount of ash to carry out the various tests described thereafter, 230
batches, each containing approximately 220 g of ash from various IRIS trials, were homogenised using
a Morton Mixers ribbon blender (Figure 3.a-b). This operation resulted in a decrease in the size of the
ash agglomerates, with an ash apparent density of 0.6 g·cm-3, compared to 0.2 g·cm-3 before
homogenisation, and a true density of 1.9 g·cm-3 measured by helium pycnometry. The composition of
the ash thus mixed (Table 1) was analysed by X-ray fluorescence spectroscopy (XRF). Ash is characterised by a high volatility that can lead to clogging of the feeding and off-gas treatment
system pipes and large dust carry-over. In order to limit this volatility, a temporary densification by
pelletisation was carried out using a Frogerais rotative press. The ash was pressed in the presence of a
binder to ensure the mechanical cohesion of the pellets, but without glass frit, being too abrasive for the
press. Two types of binder were tested: organic (sorbitol, C6H14O6, or fructose, C6H12O6) and mineral
(bentonite, mainly containing montmorillonite (Na,Ca)0.33(Al,Mg)2Si4O10(OH)2 (H2O)n). The addition of
10 wt.% of fructose or bentonite to the ash gave similar and satisfactory results. Bentonite was preferred
thereafter to avoid possible COx gas emissions during combustion. Approximately 60,000 pellets (9 kg
of ash), reaching a 72% densification, were produced (Figure 3.c). Figure 3. Ash in the mixer (a) before and (b) after homogenisation. (c) Ash pellets. In order to determine the vitrification conditions of the ash, laboratory-scale tests, using small
quantities of materials (≈10 g), were carried out with different amounts of ash and glass frit at 1100 °C
for 2 h. In the approach proposed in the THERAMIN project, no vitreous matrix formulation study was
carried out and FN0C77 glass frit (Table 1), simple and available, was used. At the end of the tests, the
crucibles were cut and the materials obtained were observed with a binocular magnifier and evaluated
according to two criteria: (i) obtention of a macroscopically homogeneous material and (ii) the absence
of foaming during melting. The best results were obtained for a 50:50 mixture between ash and glass
frit. Beyond this ratio, the reactivity between ash and glass frit is less efficient and the resulting material
becomes less homogeneous. (a)
(b)
5 mm
(c) (a) 5 mm
(c) Figure 3. 4. Pre-treatment of ash and definition of their vitrification conditions Ash in the mixer (a) before and (b) after homogenisation. (c) Ash pellets. (a)
(b)
5 mm
(c) (b) (a) Figure 3. Ash in the mixer (a) before and (b) after homogenisation. (c) Ash pellets. In order to determine the vitrification conditions of the ash, laboratory-scale tests, using small
quantities of materials (≈10 g), were carried out with different amounts of ash and glass frit at 1100 °C
for 2 h. In the approach proposed in the THERAMIN project, no vitreous matrix formulation study was
carried out and FN0C77 glass frit (Table 1), simple and available, was used. At the end of the tests, the
crucibles were cut and the materials obtained were observed with a binocular magnifier and evaluated
according to two criteria: (i) obtention of a macroscopically homogeneous material and (ii) the absence
of foaming during melting. The best results were obtained for a 50:50 mixture between ash and glass
frit. Beyond this ratio, the reactivity between ash and glass frit is less efficient and the resulting material
becomes less homogeneous. 3. Ash production Figure 2
shows that the organic waste first goes into a pyrolysis step at a temperature of 500 °C to remove the
most corrosive gaseous compounds and then into a calciner (900 °C) fed with oxygen to complete the
combustion while concentrating the contamination in the mineral ash. The relatively long residence time 2 HERAMIN 2020 conference: thermal treatment of radioactive waste IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
g
doi:10.1088/1757-899X/818/1/012005 in furnaces with low gas flow rates makes it possible to produce carbon-free ash, concentrating almost
all of the initial activity. The off-gas treatment system, consisting of a post-combustion chamber
followed by electrostatic filtration, ensures excellent purification. in furnaces with low gas flow rates makes it possible to produce carbon-free ash, concentrating almost
all of the initial activity. The off-gas treatment system, consisting of a post-combustion chamber
followed by electrostatic filtration, ensures excellent purification. 5. Bench-scale test and characterisation of the wasteform 5.1. Test conditions
Following the laboratory-scale tests, the quantity of treated ash was increased during a bench-scale test. This test was carried out in an Inconel™ 601 crucible having an external diameter of 100 mm and a
thickness of 5 mm, filled with 400 g of unpelletised ash and 400 g of FN0C77 glass frit (Figure 4.a). 5.1. Test conditions 3 THERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering (a)
IOP Publishing
(b) IOP Conf. Series: Materials Science and Engineering (a)
g
(b) P Conf. Series: Materials Science and Engineering (a)
(b) The tapped density of this mixture was estimated at approximately 1 g·cm-3. The mixture was heated at
300 °C·h-1 and maintained at 1100 °C for 8 h. The tapped density of this mixture was estimated at approximately 1 g·cm-3. The mixture was heated at
300 °C·h-1 and maintained at 1100 °C for 8 h. At the end of the test (Figure 4.b), the crucible was cut and the resistance of Inconel™ 601 to
corrosion was evaluated. No significant corrosion is seen at the interface between Inconel™ and the
wasteform. Above the volume occupied by the wasteform, gaseous species superficially corroded the
Inconel™ (Figure 4.c). The crystallised corrosion products are NiFe1.2Cr0.8O4 spinels and nickel oxide. However, no significant reduction in the thickness of the crucible is measured. A mass loss of 2.5 wt.%
was measured after the test and a crystallised glass of density 2.63 g·cm-3 was obtained (Figure 4.d). (d)
(c) Figure 4. (a) Ash and glass frit mixture introduced into the Inconel™ 601 crucible, (b) crucible cut
after the test, (c) inner surface of the crucible after the test and (d) wasteform produced. (a)
(b)
(d)
(c)
Concrete cast to
facilitate cutting
Wasteform (c) (b)
Concrete cast to
facilitate cutting
Wasteform (a) (c) (d) (a) (d) Figure 4. (a) Ash and glass frit mixture introduced into the Inconel™ 601 crucible, (b) crucible cut
after the test, (c) inner surface of the crucible after the test and (d) wasteform produced. 5.2. Microstructure and chemical composition of the wasteform 5.2. Microstructure and chemical composition of the wasteform
The material is mainly composed of a vitreous matrix consisting of approximately 80 wt.% of Al2O3,
B2O3, CaO, Na2O, and SiO2. This matrix includes three types of crystallisation identified by scanning
electron microscopy (SEM) and X-ray diffraction (XRD): apatites, zincochromites, and beads composed
almost exclusively of bismuth (Figure 5 and Table 1). The stoichiometries of apatites and
zincochromites evaluated by energy dispersive X-ray spectroscopy (EDS), respectively
Ca4.8P2.9Si0.3Na0.2Al0.1O12.9 and ZnCr2.6Na0.5Mg0.2Al0.1Fe0.1Ni0.1O5.8 (oxygen assumed for stoichiometry),
are consistent with the theoretical stoichiometries of these minerals, respectively Ca5(PO4)3(OH) and
ZnCr2O4. 5.1. Test conditions The crystals are homogeneously distributed in the matrix, with the exception of a layer
approximately one millimetre thick at the interface with the crucible in which they are more numerous. Figure 5. (a) SEM and (b) XRD analysis of the wasteform produced at the bench-scale. (a)
Vitreous matrix
10 µm
10
30
50
70
Counts (a.u.)
2θ (degrees)
(b)
Apatites
Zincochromites
Bismuth beads
Epoxy resin (a)
Vitreous matrix
10 µm
Epoxy resin 10
30
50
70
Counts (a.u.)
2θ (degrees)
(b)
Apatites
Zincochromites
Bismuth beads 70 2θ (degrees) (b) Figure 5. (a) SEM and (b) XRD analysis of the wasteform produced at the bench-scale. THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 HERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 P Conf. Series: Materials Science and Engineering 818 (2020) 012005
doi:10.1088/1757-899X/818/1/0 Table 1. Elemental compositions, expressed in wt.%, of ash and glass frit used for the calculation of the
theoretical composition of the wasteform. Analysed compositions of the vitreous and crystalline phases
composing the wasteform. Element
Ash
(analysed by XRF)
FN0C77 glass frit
(specified)
Wasteform
(calculated)
Vitreous matrix
(analysed by EDS)
Apatites
(analysed by EDS)
Zincochromites
(analysed by EDS)
Bismuth beads
(analysed by EDS)
Al
11.90
5.95
9.40
0.06
0.15
B
10.05
5.02
n.a.a
n.a.a
n.a.a
n.a.a
Ba
0.71
0.36
Bi
4.02
2.01
0.17
9.99
Ca
9.91
4.96
7.10
4.84
0.01
Cl
1.42
0.71
0.08
Cr
0.12
0.06
2.58
Fe
0.50
0.25
0.61
0.05
K
2.25
1.13
1.56
Mg
1.59
0.80
3.00
0.24
Na
0.55
23.74
12.14
13.71
0.16
0.48
Ni
0.69
0.35
0.05
0.17
P
1.62
0.81
0.62
2.88
S
0.31
0.16
0.32
Sb
0.15
0.08
Si
7.45
16.57
12.01
27.71
0.25
0.02
Ti
0.45
0.23
1.04
0.03
Zn
8.13
4.07
5.74
1.00
0.25
a n.a.: not analysed because boron is not detected by EDS. 5.3. Chemical durability On the first day of leaching, the alteration rate of the vitreous matrix is ≥ 2.4 g·m-2·d-1. After 3 days,
the rate is ≈1.5 g·m-2·d-1. The alteration therefore decreases over time and quickly becomes non-
stoichiometric, showing the typical behaviour of a borosilicate glass of nuclear interest. Si and Al are
retained at 30–40% in the alteration layer after one month. Such retention remains relatively low: the
amorphous layer formed on the sample surface therefore remains poor in silicon, which explains why
the hydrolysis regime remains predominant. The initial dissolution rate of the vitreous matrix leads to a
rapid increase in pH90°C to 8.5, the value at which this parameter stabilises. Figure 6. (a) Evolution of NL and pH during wasteform leaching (error bars represent the data
dispersion between the duplicates). (b) Compared evolution of NL and pH during the leaching of ISG
and In-Can-produced wasteform. The arrows are a visual guide to estimate the drop in alteration rate. 3
4
5
6
7
8
9
0
2
4
6
8
10
12
14
0
10
20
30
pH
NLi (g·m-2)
time (days)
B-ICM
B-ISG
Si-ICM
Si-ISG
pH-ICM
pH-ISG
6.0
6.5
7.0
7.5
8.0
8.5
0
2
4
6
8
10
12
0
10
20
30
pH
NLi (g·m-2)
time (days)
Al
B
Na
Si
pH
(a)
(b) 6.0
6.5
7.0
7.5
8.0
8.5
0
2
4
6
8
10
12
0
10
20
30
pH
NLi (g·m-2)
time (days) 3
4
5
6
7
8
9
0
2
4
6
8
10
12
14
0
10
20
30
pH
NLi (g·m-2)
time (days) (b) (a) Figure 6. (a) Evolution of NL and pH during wasteform leaching (error bars represent the data
dispersion between the duplicates). (b) Compared evolution of NL and pH during the leaching of ISG
and In-Can-produced wasteform. The arrows are a visual guide to estimate the drop in alteration rate. Comparison of previous results with those acquired for the reference International Simple Glass
(ISG) [4, 5] altered under the same conditions (Figure 6.b) shows similar and ‘classical’ trends. Comparison of previous results with those acquired for the reference International Simple Glass
(ISG) [4, 5] altered under the same conditions (Figure 6.b) shows similar and ‘classical’ trends. Because of its composition, the In-Can-produced vitreous matrix has a higher rate of hydrolysis. 5.3. Chemical durability y
The chemical durability of the material was studied according to a protocol adapted from the PCT-B
standardised test [1]. After crushing the material, 125–250 μm of powder were placed in contact with
pure water with a glass-surface-area-to-solution-volume ratio (S/V) of 10 m-1 at 90±2 °C in unstirred
perfluoroalkoxy (PFA) reactors. The powder specific surface area of 259 g·cm-2 was estimated by
adsorption of krypton on the sample surface (Micromeritics ASAP 2020) according to the Brunauer–
Emmett–Teller theory [2]. Solution samples, taken at regular intervals, were filtered with a cut-off of
0.45 μm, acidified with ultrapure grade HNO3 and analysed by inductively coupled plasma-atomic
emission spectroscopy (Thermo Scientific iCAP™ 6000 Series). The concentrations were used to
calculate normalised mass losses
NL
,
i
i
i
C
x
S V
where Ci is the concentration of the element i
and xi the mass percentage of i in the glass. The alteration rate,
dNL d ,
i
r
t was calculated by linear
regression. Boron is known to be an alteration tracer of the vitreous matrix, which means that it is not
retained in alteration products while released from the glass. The evolution of the normalised mass losses of Al, B, Na, and Si is shown in Figure 6.a. Only the
elements mainly integrated into the vitreous matrix are considered. The xi considered are those obtained
by EDS analysis for Na, Si, Al, and by calculation for B. Therefore, the dissolution rates of the crystalline
phases are not considered, because when a wasteform contains crystalline phases deemed to be durable, 5 THERAMIN 2020 conference: thermal treatment of radioactive waste THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 P Conf. Series: Materials Science and Engineering 818 (2020) 012005
doi:10.1088/1757-899X/818/1/01 like apatites or spinels, the wasteform dissolution is controlled by the properties of the vitreous matrix
[3]. This leaching test was duplicated, giving similar results: the average values obtained are discussed
hereafter. like apatites or spinels, the wasteform dissolution is controlled by the properties of the vitreous matrix
[3]. This leaching test was duplicated, giving similar results: the average values obtained are discussed
hereafter. 6. Full-scale trial A full-scale trial was conducted with the CEA’s ICM (Figure 1). The can is made of Inconel™ 601,
with an external diameter of 400 mm, a height of 600 mm and a wall thickness of 10 mm. The trial began with pre-loading of the container with 25 kg of FN0C77 glass frit and 17.8 kg of
unpelletised ash. This mixture was heated at 300 °C·h-1 up to 1100 °C. Then, 9.2 kg of pelletised ash
were fed into the can with a flow of 10 kg·h-1, before the introduction of 1 kg of FN0C77 frit and an
additional soaking of 2 h. Thus, the final waste loading was 50 wt.%. The test was continued with 2 h
of recycling of the dust recovered in the off-gas treatment system and 2 h of further soaking. The can
was then allowed to cool freely. A loss of mass of 2.3 wt.% was measured, consistent with that of the
test carried out at bench-scale. The extent of characterisation of the material produced during this trial is more limited than that of
the material produced at bench-scale. However, the SEM and XRD characterisations carried out on a
sample taken in the central zone of the can show a similar microstructure (Figure 7). The same bismuth
beads, apatite, and zincochromite crystals are included in a vitreous matrix. Figure 7. (a) SEM image and (b) XRD analysis of the wasteform produced using the ICM. The XRD
results are compared to those of the bench-scale test. 10
30
50
70
Counts (a.u.)
2θ (degrees)
Bench-scale test
ICM
(b)
(a)
Vitreous matrix
Apatites
Zincochromites
Bismuth beads
10 µm (a)
Vitreous matrix
10 µm 10
30
50
70
Counts (a.u.)
2θ (degrees)
Bench-scale test
ICM
(b)
Apatites
Zincochromites
Bismuth beads (a) (b) Figure 7. (a) SEM image and (b) XRD analysis of the wasteform produced using the ICM. The XRD
results are compared to those of the bench-scale test. 5.3. Chemical durability Indeed, it contains large fractions of Na2O and B2O3, which are unfavourable to the glass
durability. Therefore, in the early stages of dissolution, the evolutions of the concentrations in
solution are faster for In-Can-produced wasteform. This conclusion is also valid for the pH
evolution. The growth of the alteration layer is faster for the In-Can-produced wasteform. Thus, its
alteration rate drops faster. Between 14 and 28 days, the alteration rate of the In-Can-produced
vitreous matrix (0.07 g·m-2·d-1) is almost 1.5 time lower than that of the ISG (0.10 g·m-2·d-1),
while the hydrolysis rate of the vitreous phase of the wasteform after 1 day is higher. This result
is known for simple glasses [6]: the glasses that are hydrolysed the fastest are also those that
have the earliest rate drops. The data trends tend to show that, in the longer term, an alteration layer will form on the In-Can-
produced wasteform, causing a decrease of the alteration rate, as for the ISG. 6 THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 P Conf. Series: Materials Science and Engineering 818 (2020) 012005
doi:10.1088/1757-899X/818/1/01 Acknowledgements This project has received funding from the Euratom research and training programme 2014–2018 under
grant agreement No 755480. This paper reflects only the authors’ views, and the European Commission
is not responsible for any use that may be made of it. p
y
y
The authors are grateful to Alain Artico, Carine Castaño, and Virginie Lemaitre for carrying out the
tests, Thierry Blisson, Florian Emanuel, and Valérie Debono for the wasteform characterisation, and
PRIME Verre Company for its technical assistance. 7. Conclusions
h
l The In-Can Melter trial conducted in the framework of the THERAMIN project demonstrated a
successful process for the vitrification of ash generated from the incineration of organic waste from
glove boxes in the nuclear industry. This study presents — in a simplified version — the scale up
methodology used to demonstrate the feasibility of the thermal treatment of a given waste by a given
process. Thus, the laboratory-scale tests assess the waste loading, operating temperature, mixture
strategy and melted material quality. Next comes the bench-scale test focusing on crucible corrosion,
volatility of some species and wasteform microstructure. Finally, the study concludes with a pilot-scale
trial whose main outputs are overall corrosion and kinetic data, process performance, process and
package descriptions. p
g
p
A good waste loading of 50 wt.% was achieved in this first approach. The trial also made it possible
to begin the technical investigation required for the processing of powdery solids into the can while
avoiding dust emission: a temporary densification by pelletising was implemented. The produced wasteform consists of a crystallised glass mainly composed of SiO2, Na2O, B2O3,
Al2O3, and CaO. The term ‘crystallised glass’ refers to a vitreous matrix including crystals of apatite,
zincochromite, and bismuth alloy. The crystals are distributed homogeneously in the wasteform. The crystalline phases being durable, the durability of the wasteform is controlled by that of the
vitreous matrix. The hydrolysis rate of this glass is relatively high because of its high content of B2O3
and Na2O. Indeed, in the approach proposed in the framework of the THERAMIN project, no
optimisation study of the glass additives was conducted. However, the ‘classical’ trends observed
suggest that, in the long term, an alteration layer will form, leading to a decrease in the alteration rate. THERAMIN 2020 conference: thermal treatment of radioactive waste
IOP Conf. Series: Materials Science and Engineering 818 (2020) 012005
IOP Publishing
doi:10.1088/1757-899X/818/1/012005 THERAMIN 2020 conference: thermal treatment of radioactive waste References [1]
ASTM International 2014 Standard C1285-14. Standard Test Method for Setermining Chemical
Durability of Nuclear, Hazardeous, and Mixed Waste Glasses and Multiphase Glass
Ceramics: The Product Consistency Test (PCT) y
(
)
[2]
Brunauer S, Emmett P H and Teller E 1938 Adsorption of gases in multimolecular layers J. Am. Chem. Soc. 60 309–19 [3]
Nicoleau E, Angeli F, Schuller S, Charpentier T, Jollivet P and Moskura M 2016 Rare-earth
silicate crystallization in borosilicate glasses: Effect on structural and chemical durability
properties J. Non-Cryst. Solids 438 37–48 [4]
Gin S, Abdelouas A, Criscenti L J, Ebert W L, Ferrand K, Geisler T, Harrison M T, Inagaki Y,
Mitsui S, Mueller K T, Marra J C, Pantano C G, Pierce E M, Ryan J V, Schofield J M, Steefel
C I and Vienna J D 2013 An international initiative on long-term behavior of high-level nuclear
waste glass Mater. Today 16 243–8 [5]
Kaspar T C, Ryan J V, Pantano C G, Rice J, Trivelpiece C, Hyatt N C, Corkhill C L, Mann C,
Hand R J, Kirkham M A, Crawford C L, Jantzen C M, Du J, Lu X, Harrison M T, Cushman
C, Linford M R and Smith N J 2019 Physical and optical properties of the International Simple
Glass npj Mater. Degrad. 3 15 pj
g
[6]
Gin S, Beaudoux X, Angéli F, Jégou C and Godon N 2012 Effect of composition on the short-
term and long-term dissolution rates of ten borosilicate glasses of increasing complexity from
3 to 30 oxides J. Non-Cryst. Solids 358 2559–70 8 8
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Alleviating Class Imbalance in Actuarial Applications Using Generative Adversarial Networks
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risks
Article
Alleviating Class Imbalance in Actuarial Applications Using
Generative Adversarial Networks
Kwanda Sydwell Ngwenduna 1,2, *
1
2
3
*
Citation: Ngwenduna, Kwanda
and Rendani Mbuvha 3
School of Computer Science and Applied Mathematics, University of the Witwatersrand, West Campus,
Mathematical Sciences Building, Private Bag 3, Wits, Braamfontein 2050, South Africa
DSI-NICIS National e-Science Postgraduate Teaching and Training Platform (NEPTTP), Wits,
Braamfontein 2050, South Africa
School of Statistics and Actuarial Science, University of the Witwatersrand, West Campus, Mathematical
Sciences Building, Private Bag 3, Wits, Braamfontein 2050, South Africa; rendani.mbuvha@wits.ac.za
Correspondence: sngwenduna@gmail.com
Abstract: To build adequate predictive models, a substantial amount of data is desirable. However,
when expanding to new or unexplored territories, this required level of information is rarely always
available. To build such models, actuaries often have to: procure data from local providers, use limited
unsuitable industry and public research, or rely on extrapolations from other better-known markets.
Another common pathology when applying machine learning techniques in actuarial domains is
the prevalence of imbalanced classes where risk events of interest, such as mortality and fraud,
are under-represented in data. In this work, we show how an implicit model using the Generative
Adversarial Network (GAN) can alleviate these problems through the generation of adequate quality
data from very limited or highly imbalanced samples. We provide an introduction to GANs and how
they are used to synthesize data that accurately enhance the data resolution of very infrequent events
and improve model robustness. Overall, we show a significant superiority of GANs for boosting
predictive models when compared to competing approaches on benchmark data sets. This work
offers numerous of contributions to actuaries with applications to inter alia new sample creation,
data augmentation, boosting predictive models, anomaly detection, and missing data imputation.
Sydwell, and Rendani Mbuvha. 2021.
Alleviating Class Imbalance in
Actuarial Applications Using
Keywords: actuarial science; class imbalance; data augmentation; generative models; generative adversarial network; synthetic sampling; SMOTE
Generative Adversarial Networks.
Risks 9: 49. https://doi.org/10.3390/
risks9030049
Received: 19 October 2020
Accepted: 21 November 2020
Published: 8 March 2021
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
1.1. Background
Gaining an advantage in competitive markets through offerings of suitable tailored
products on customers relies on building and maintaining adequate predictive models.
To build these models, a substantial amount of data and a sizeable number of records
is desirable. However, when expanding to new or unexplored markets, that level of
information is rarely always available. To build such models, actuarial firms often have to
procure data from local providers, use limited unsuitable industry and public research or
rely from extrapolations from other better known markets.
In this work, we show how an implicit model using the Generative Adversarial
Network (GAN) Goodfellow et al. (2014) can alleviate this problem through the generation
of adequate quality data even from very limited small samples, from difficult domains, or
without alignment, thus handling class imbalance.
A GAN is an example of a generative model that is used to create new samples
from a latent noise space. A generative model describes how a data set is generated in
terms of a probabilistic model. This generative model pmodel mimics the training data
distribution pdata as close as possible. If this is achieved, then we can sample from pmodel to
generate realistic samples that appear to have been drawn from pdata . We are satisfied if
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our model can also generate diverse samples that are suitable different from the training
data. In some cases, the model can be estimated explicitly, and sometimes it can generate
samples implicitly. Other models are capable of doing both.
GANs were proposed in a seminal paper by Goodfellow et al. (2014). GANs are useful
for learning the structure of the data and can generate new samples without explicitly
postulating the model Goodfellow et al. (2014). They are known to be better than other
generative models due to the quality of samples they generate. GANs have been highly
successful in computer vision Brock et al. (2018); Karras et al. (2019); Zhu et al. (2017); Vondrick et al. (2016), music generation Yang et al. (2017), text generation Reed et al. (2016),
missing data imputation Li et al. (2019); Shang et al. (2017); Yoon et al. (2018), time series
generation Esteban et al. (2017); Yoon et al. (2019); Fu et al. (2019), and data augmentation Antoniou et al. (2017); Fiore et al. (2019); Mariani et al. (2018); Mottini et al. (2018);
Park et al. (2018), with remarkable results, but their application to the actuarial discipline
remains largely still unexplored.
In this work, we discuss how and where GANs can be used by actuaries. Additionally,
we provide an experiment showing how GANs can be used to boost imbalanced samples
in actuarial data sets and improve actuarial models.
1.2. Aims and Objectives
In this paper, we explain what GANs are and how they can be used to synthesize data
in order to accurately enhance very infrequent events, alleviate class imbalance and create
better prediction models. This work also provides theoretical and practical applications of
GANs.
We demonstrate a popular GAN architecture to a typical problem resembling an
actuarial use on benchmark data sets using Python Python Software Foundation (2017).
Overall, we show a significant superiority of GANs for predictive models and stochastic
simulations compared to current approaches. Specifically, this paper covers the following
aims and objectives:
•
•
•
deep overview of generative models and why GANs are of better quality than other
generative models;
an overview of GANs with practical applications in a number of areas with emphasis
for actuarial use; and
provide a practical example of a popular GAN use for alleviating class imbalance,
data augmentation, and improving predictive models.
1.3. Contribution
This work provides thorough theoretical, empirical and practical applications of
GANs, with possible leverage in actuarial science for inter alia new sample creation,
data augmentation, boosting actuarial models, anomaly detection, missing data imputation,
time series simulations and projections in life insurance, short-term insurance, health and
care, banking, investment, enterprise risk management, and other non-traditional actuarial
areas, such as telecommunications, economics, medicine, engineering, and other wider
fields.
For example, actuaries build pricing models in order to determine competitive premiums that customers should pay to be provided adequate insurance coverage O’Malley et al.
(2005). These pricing models are dependant on risk events, such as mortality, morbidity,
and lapse, which need to be estimated using an adequate and accurate model. However,
these risk events are often under-represented in data.
In this work, we show how a GAN could be used to alleviate this problem through the
generation of adequate quality data from limited or highly imbalanced samples. Essentially,
we show that synthetic data generated using GANs can augment imbalanced data sets,
leading to significantly higher predictive power of possible actuarial models fitted after.
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1.4. Structure of the Paper
The rest of the paper is organized as follows. Section 2 describes the problem of class
imbalance and its common solutions. Section 3 reviews the literature on generative models,
with particular emphasis on GANs, while Section 4 covers GAN applications, especially
for actuarial adoption. Section 5 describes the methodology followed. Section 6 outlines
the example experiments conducted. Section 7 presents the results, and Section 8 discusses
the results, while Section 9 gives conclusions, limitations, and possible future work.
2. Class Imbalance
2.1. Definition
Whilst machine learning (ML) has gained significant prevalence in the past few
decades, class imbalance, limited data sets, and missing data remain pervasive problems
Chawla et al. (2002); Fernández et al. (2018); Longadge and Dongre (2013). These issues occur due to the nature of the data space, data collection costs, data limitations, new markets,
and absolute rarity.
In binary classification problems, class imbalance occurs when one of the classes has
overwhelmingly more instances than others. ML classifiers tend to have skewed accuracy
towards the majority class when the data is imbalanced Chawla (2009); Fernández et al.
(2018). This is problematic as misclassifying a minority class can result in significant
misclassification costs than for the majority case Chawla et al. (2002). Class imbalance
arises because ML classifiers do not necessarily take into account unequal class distributions. This problem causes a significant and an unexpected performance behavior for
most classifiers.
2.2. Techniques to Alleviate Class Imbalance
Techniques as shown in Figure 1 exist to alleviate class imbalance, and these techniques
include re-sampling, algorithmic-level solutions, cost-sensitive learning, ensembles, and
generative models.
Figure 1. Taxonomy of solutions to tackle class imbalance.
2.2.1. Re-Sampling
Re-sampling techniques modify the training data such that the distribution of the
classes is evenly balanced where the majority or minority class is either under-sampled or
over-sampled. Over-sampling has been the most frequently used technique than undersampling since under-sampling eliminates important information in the majority class.
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Hybrid sampling techniques combine over-sampling with data cleaning techniques,
informed under-sampling techniques, or greedy-filtering approaches Batista et al. (2004),
thereby eliminating redundant and noisy instances, boosting the predictive accuracy of
models trained after.
2.2.2. Synthetic Sampling
A pioneering and popular method to alleviate class imbalance has been Synthetic
Minority Over-sampling TEchnique (SMOTE) Chawla et al. (2002). However, SMOTE
suffers from over-fitting, over-lapping classes, noisy examples, is less reliant on the true
probability distribution, and alters the original distribution of the minority classes, and this
may not be desirable Batista et al. (2004); Ganganwar (2012); Longadge and Dongre (2013).
There have been few empirical reviews which compare and synthesize SMOTE and
its density-based variants Gao et al. (2014). There have been few approaches which create
synthetic samples by sampling implicitly from the minority class distribution. Current
density-based approaches may be subjective as they need to pre-specify the format and
structure of the minority class distribution Das et al. (2015); Zhang and Li (2014). Generative models offer a significant alternative, yet these models have not been thoroughly
explored in imbalanced learning. In this work, we show how a popular implicit generative
model can be used to handle class imbalance and rival SMOTE.
2.2.3. Ensembles
Ensemble is where a classifier’s accuracy is increased by the use of training on different over-sampled data sets or different algorithms and combining outputs to a single outcome. These approaches tend to improve the results of re-sampling techniques
Wang and Yao (2009); Chawla et al. (2003). However, they can take a long time to compute
and still do not solve the true data distribution issue.
2.2.4. Other Methods
Algorithmic-level solutions modify the ML classifier to adjust for the presence of
class imbalance in the data. Cost-sensitive learning incorporates mis-classification costs in
the evaluation metric Ganganwar (2012). This approach is more computationally efficient
than data-level solutions He and Garcia (2008). However, mis-classification costs are often
unknown and difficult to set, making this method less popular than sampling techniques
López et al. (2013).
SMOTE and its variants remain the most studied and widely used solutions, with generative models slowly being adopted in alleviating class imbalance Fernández et al. (2018);
Fiore et al. (2019); Gao et al. (2014). Generative models are described in detail in Section 3.
3. Generative Models
This research is concerned with handling class imbalance through generative modeling.
Other approaches exist, such as synthetic sampling; however, these approaches do not take
into account the underlying structure of the data distribution and often lead to over-fitting
and over-lapping cases Gao et al. (2014). Generative models are flexible models capable of
learning the data distribution and sampling from this data distribution, thereby creating
new synthetic cases. In this section, we review generative models and explain why GANs
are of better quality than other deep generative models.
3.1. Definition
Given a data set with observations X, we assume that X has been generated from an
unknown true probability distribution pdata . A generative model pmodel mimics pdata as
close as possible. If this is achieved, then we can sample from pmodel to generate realistic
samples that appear to have been drawn from pdata .
We are satisfied if our model can also generate diverse samples that are suitable
different from X. In some cases, the model can be estimated explicitly and sometimes it
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can generate samples implicitly. Other models are capable of doing both. GANs provide
no estimate of the model but are capable of generating new data without knowing it.
Goodfellow (2016) provides a taxonomy of common deep generative models shown
in Table 1, divided into implicit and explicit models. GANs are designed to remedy most
of the disadvantages that come with explicit models and other Markov chain models.
Table 1. Taxonomy of generative models.
Approximate
Explicit Density
Tractable
Implicit Density
Variational Inference
Variational Autoencoder
Markov chain
Deep Belief Network
Restricted Boltzmann Machine
Full Visible Belief Net
NADE
MADE
PixelRNN/CNN
Change of variable models
Nonlinear ICA
Minimax GAN
Generative Adversarial Network Non-saturating GAN
GAN variants
Direct
Markov
Generative Moment Matching
Network
GMMN
Generative Stochastic Network
GSN
3.2. Explicit Models
Explicit models specify or approximate a parameterized log-likelihood representation
of the data Goodfellow et al. (2014). Parameters are then estimated and learned from the
data and this requires a maximum likelihood estimation which integrates over the entire
data space, and this may be intractable Li et al. (2015). These approximation techniques
may not always yield the best results as some of them rely on Markov chains, which are
time-consuming Goodfellow et al. (2014).
Two popular tractable models are fully visible belief networks (FVBNs)
Frey et al. (1996) and nonlinear independent component analysis (ICA). Approximate
methods improve on the design of tractable models which can be computational intensive and limited Goodfellow et al. (2014); Makhzani et al. (2015); Rezende et al. (2014).
Approximate methods use either deterministic, i.e., variational inference, or stochastic
approximations such as Markov chain Monte Carlo (MCMC) Geyer (1992). Variational
inference involves the use of Variational Autoencoders (VAEs) Kingma and Welling (2013);
Rezende et al. (2014) to approximate pmodel ( x ) using lower bounds.
3.2.1. FVBNs
FVBN estimates the probability density of the training data pmodel ( x ) into a decomposed product of one-dimensional probability distributions. This model outputs a probability for each possible value if x is discrete and outputs a network of parameters of a
simple distribution if x is continuous. Using the generated model, sampling is done one
step at a time, conditioned on all previous steps Goodfellow et al. (2014).
The problem with these models is their computational complexities as they need
to generate one point at a time. Other problems include poor learning representations,
over-emphasizing details over global data, and not closely reflecting the true generation
process Goodfellow (2016). Moreover, these models have been more useful for image
synthesis than structured data sets, such as tabular data Van den Oord et al. (2016). GANs
are known to provide new samples in parallel, thus yielding greater speed of generation
Goodfellow (2016); Li et al. (2015).
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3.2.2. Non-Linear ICA
Non-linear ICA involves defining some continuous non-linear transformations of data
between high dimensions and lower dimensional spaces. The distribution of the data pmodel
is transformed into a distribution of a latent space z defined by pz ( g), where g is some
tractable transformed version of pz . The challenge in ICA is finding tractable distributions
in the latent space, and these are limited Goodfellow et al. (2016). GANs are known to
have fewer restrictions than these models Goodfellow et al. (2014); Bengio et al. (2014);
Goodfellow (2016).
3.2.3. Variational Autoencoders
VAEs, along with FVBNs and GANs, are three of the most popular approaches for
sample generation. VAEs are an extension to AEs Bellinger et al. (2016); Larsen et al. (2015);
Rezende et al. (2014). AE learns useful representations of the data by encoding X into a
compressed latent space z using q(z| x ) and then decoding z back into X using p( x |z) by
minimizing the reconstruction error between the original data and the deconstructed data
Bellinger et al. (2016). VAE maximizes the following function:
log p( x ) ≥ Ez∼q(z| x) log p( x |z) + log p(z) − log q(z) .
(1)
Unlike auto-regressive models, VAEs are normally easy to run in parallel during
training and inference Goodfellow et al. (2016); Larsen et al. (2015); Rezende et al. (2014).
Conversely, they are normally harder to optimize than auto-regressive models Goodfellow et al. (2016); Makhzani et al. (2015). The encoder converts the input to latent space
representations through the mean and variance, and samples can be created from the
learned representation. VAEs have been criticized to be generating blurry samples and are
intractable Goodfellow et al. (2016); Salimans et al. (2016).
3.2.4. Boltzmann Machines
Boltzmann machines rely on the use of Markov chains to model pmodel ( x ) and to
sample from it Ackley et al. (1985); Hinton (2002); Salakhutdinov and Hinton (2009).
A Markov chain is a process that is used to generate samples by repeatedly drawing a
sample from a transition operator Geyer (1992). A Boltzmann machine is an energy-based
function defined as:
pmodel ( x ) = exp − E( x ) /Z,
(2)
where E( x ) is an energy function, and Z is a normalizing factor to ensure that pmodel ( x )
sums to one Ackley et al. (1985); Goodfellow et al. (2016).
These methods include restricted boltzmann machine (RBM) Ackley et al. (1985) and
deep belief networks (DBNs) Hinton et al. (2006); Hinton and Salakhutdinov (2006). DBNs
and RBMs are generative stochastic neural networks that can estimate a probability distribution Ackley et al. (1985). Samples are obtained through MCMC runs to convergence, and
this can be very expensive to run Li et al. (2015). These models were pioneers in early 2006
for deep generative models, but they have been rarely used because of poor scale-ability
for higher dimension problems and high computational costs Goodfellow et al. (2016).
3.3. Implicit Models
Implicit models learn to model the true distribution and define a stochastic procedure
to directly generate new data from a latent space. These models can be trained indirectly
without needing an explicit density function to be learned or defined. Some of these
models, such as the Generative Stochastic Network (GSN) Bengio et al. (2014), involve
MCMC methods, which impose greater computational cost and often fail to scale to
higher dimensional spaces Goodfellow et al. (2016). GANs Goodfellow et al. (2014) and
Generative Moment Matching Networks (GMMNs) Li et al. (2015) are one of the few
implicit probabilistic models capable of sampling in parallel and in a single step.
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GANs were designed to remedy most of the issues with explicit and some implicit
models. GANs are known to parallelize sample generation, have fewer restrictions,
subjectively thought to produce better samples, make no use of Markov chain or maximum
likelihood estimation, and assume no variational bounds and no distributional assumptions Arjovsky et al. (2017); Goodfellow et al. (2016). As a result, GANs have generated a
lot of interest since 2014, with wide applications in many areas.
3.3.1. GANs
GANs were originally invented in a landmark paper by Ian Goodfellow in 2014
Goodfellow et al. (2014). The setup of the framework uses an adversarial process to estimate the parameters of two artificial neural network (ANN) Rumelhart et al. (1986) models
by iteratively and concomitantly training a discriminator (D) and a generator (G), as shown
in Figure 2.
Through multiple cycles of generation and discrimination, both networks train each
other, while simultaneously trying to outwit each other Goodfellow et al. (2014); Mariani
et al. (2018); Odena et al. (2017); Zhu et al. (2017). GANs have two adversarial ANNs:
•
•
G picks z from the prior latent space Z and then generates samples from this distribution using ANN;
D receives generated samples from G and the true data examples, and it must distinguish between the two for authenticity.
Both D and G are ANNs which play a zero-sum game, where G learns to produce
realistic-looking samples and D learns to get better at discriminating between the generated
samples and the true data. Once G is trained to optimality, it can create new samples and
augment the training data set. GANs can sample in parallel better than other generative models, have fewer restrictions on the generator function, assume no use of Markov
Chains, as well as no variational bounds, unlike VAE, and produce subjectively better quality samples than other generative models Arjovsky et al. (2017); Goodfellow et al. (2016);
Goodfellow et al. (2014); Radford et al. (2015); Salimans et al. (2016).
Figure 2. Generative Adversarial Network (GAN) operation.
Whilst GANs are gaining popularity in many applications, they have notable issues.
GANs are notoriously difficult to train properly and difficult to evaluate, the likelihood
cannot be easily be computed, and they suffer from the vanishing gradient problem,
mode collapse, boundary distortion, and over-fitting Arjovsky et al. (2017); Goodfellow
et al. (2016); Salimans et al. (2016).
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Mode collapse is when many latent noise values z are mapped to the same data
point x, leading to a lack of diversity in the samples that are created, i.e., under-fitting.
The vanishing gradient problem occurs when D becomes perfect in its training without giving G the chance to improve. As a result, GANs may fail to converge, thereby
leading to poor generated samples Arjovsky et al. (2017). Figure 3 provides a nonexhaustive taxonomy of GAN variants and improved training, including common examples Creswell et al. (2018); Hitawala (2018); Hong et al. (2019); Wang et al. (2017).
Figure 3. Taxonomy of GAN variants.
For GAN reviews, (Creswell et al. (2018); Hitawala (2018); Hong et al. (2019)) provide a
comparative overview. Lucic et al. (2018) conduct an in-depth study on GANs and note no
significant performance differences on the GANs studied. There are over 300 GAN variants,
and it is impossible to review all of them. In this work, we are interested in exploring
GAN applications and showing their potential to actuaries, especially for alleviating class
imbalance, data augmentation, and improving the predictive ability of actuarial models.
3.3.2. GMMNs
GMMNs minimize the maximum mean discrepancy (MMD) between the moments of
pdata and pmodel and are known to be simpler than other generative models Li et al. (2015).
Moment matching evaluates whether the moments of the true distribution ptrue ( x ) match
those of the data pdata ( x ) through MMD. This approach is similar to GANs in terms of
training, except using a different loss function, which leads to faster sampling. However,
GMMNs have received less attention than GANs and VAEs, limiting their sample generative scheme Arjovsky et al. (2017); Goodfellow et al. (2016); Hitawala (2018).
3.4. Summary
There are a number of deep generative models for synthetic sample generation. Some
of the models are explicit with an intractable likelihood and inference. Some models are
only approximate and generate blurry samples. Other methods do not sample in parallel,
are complex, and rely on Markov chains, which are time-consuming. GANs are attractive
as they do not make any explicit density estimation, and they remedy most of these issues.
GANs have generated extremely good examples in many domains. Section 4 reviews these
GAN applications.
4. Applications of GANs
The most successful applications of GANs are in computer vision, but there have been
applications in other domains, as well. In this section, we focus on the applications where
there is some actuarial use.
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4.1. Data Augmentation
The availability of sufficient data in many domains is a necessity, especially where
predictive models are needed to make business decisions. Such models are built on
adequate training data for better generalization and meaningful accuracy Goodfellow et al.
(2016). In this work, we are interested in adopting GANs for new sample creation and data
augmentation in order to boost predictive models by supplementing training data sets
with new samples that are learned from the real data distribution in an adversarial manner.
Data augmentation is a procedure to create synthetic cases to augment the training data
and increase its size, especially for those data points that are lacking. This is where GAN
shines—the ability to create new samples and adequate data sets Goodfellow et al. (2014);
Fiore et al. (2019).
There are two main strategies to check if this augmentation really helped something:
we can train our model on fake data and check how well it performs on real samples.
We can also train our model on real data to do some classification task and only after check
how well it performs on generated data. If it works well in both cases—you can feel free to
add samples from the generative model to your real data and retrain it again—you should
expect gain of performance.
Recently, a number of papers have applied GANs to augment various data sets, with remarkable results on the performance of the predictive models applied after
Antoniou et al. (2017); Douzas and Bacao (2018); Fiore et al. (2019); Mariani et al. (2018);
Mottini et al. (2018); Park et al. (2018); Xu et al. (2019); Ding et al. (2019). Similarly, GANs
can be used to augment actuarial data sets and boost actuarial models, making them more
accurate and less biased. In this work, we demonstrate how this can be done for a number
of data sets, described in Section 6.
4.2. Anomaly Detection
Anomaly detection is the identification of rare items, events, or observations which
raise suspicions by differing significantly from the majority of the data. Anomaly detection
finds extensive use in a wide variety of applications, such as fraud detection for credit
cards, insurance, or health care. The importance of anomaly detection is due to the fact
that anomalies in data translate to significant (and often critical) actionable information
in a wide variety of application domains. There are a number of these methods, such as
clustering-based, classification-task, nearest neighbor, spectral, or statistical, but most of
them have rather strong assumptions and long training times.
Main generative models, like VAE or GAN, consist of two parts. VAE has an encoder
and the decoder, where the encoder basically models the distribution and the decoder
reconstructs from it Larsen et al. (2015). A GAN consists of the generator and the discriminator, where the generator models the distribution and the discriminator judges if it is close
to the training data Goodfellow et al. (2014). They are pretty similar in some way—there
is modeling and judging part (in VAE, we can consider reconstructing as some kind of
judgement).
The modeling part is supposed to learn the data distribution. What will happen to
the judging part if we give it some sample not from the training distribution? In case of
a well trained GAN, the discriminator will tell us 0, and reconstruction error of VAE will
be higher than average one on the training data Akcay et al. (2018). Our unsupervised
anomaly detector is then easily trained and evaluated. We can feed it with some steroids,
like statistical distances, if we want.
In medicine, Schlegl et al. (2017) propose an AnoGAN for anomaly detection of
medical images, and learn the characteristics of lesions by learning the characteristics of
health data sets. Akcay et al. (2018) present GANomaly for anomaly detection in visual
noise, noting a significant improvement on detecting anomalies on various data sets. These
methods can be leveraged for potential applications in fraud detection, lapse prediction
and claiming likelihood in insurance. A GAN useful and leveraged for anomaly detection
can rival other anomaly detection techniques.
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4.3. Time Series
Suppose we wanted to simulate the evolution of a stock price for some particular
asset using traditional simulations, such as Monte Carlo. We would need to estimate the
mean and volatility of the returns using past price evolution and then simulate new prices
under the assumption that the returns follow a Gaussian distribution with the estimated
parameters. However, this normality assumption may not be entirely true in practice where
there is a tendency for higher observed probabilities for the tail events than those predicted
by the Gaussian distribution. We could change our assumption, say, into a student-t
distribution, but neither would that assumption completely describe the reality. GANs are
capable of replicating the price evolution without making any model assumptions.
Time series and stochastic processes are widely used by financial firms for risk management, financial projections, stock prediction, extreme event monitoring, and monetary
policy making Fu et al. (2019). Traditionally, autoregressive time series models, exponential
smoothing and their variants, and, more recently, deep learning, have been introduced and
intensively studied and applied for time series data Esteban et al. (2017).
However, most of these models rely on strong dependence on model assumptions
and model parameter estimation and, thus, are less effective in the estimation of complex
distributions with time-varying features Zhou et al. (2018). GANs do not make any explicit
assumptions and are capable of learning the distributions and their dependence structures
in a non-parametric fashion. There has been a number of time series GANs proposed,
such as the recurrent conditional GAN (RCGAN) Esteban et al. (2017) and time series GAN
(TimeGAN) Yoon et al. (2019), for the generation of realistic financial time series.
Immediate actuarial uses leveraging these GANs are stochastic simulations, capital
modeling, mortality projections, reserving, asset and liability management, solvency projection, and other time series generation tasks. GANs can be used to rival Monte Carlo
or stochastic simulations without any distributional assumptions. In insurance, mortality
forecasting is an important actuarial task. Typically, mortality forecasting models, such as
Lee-Carter Lee and Carter (1992), are used, but these make strong mathematical assumptions which need to be validated by the data. Time series GANs could potentially be used
to simulate and project mortality rates into the future, potentially competing with existing
models.
4.4. Privacy Preservation
Data of a lot of companies can be secretive, confidential, or sensitive. Sometimes,
we need to share it with third parties like consultants or researchers. If we want to share a
general idea about our data that includes the most important patterns, details, and shapes
of the objects, we can use GANs directly to sample examples of our data to share with
other people without sharing identifiable features. This way we will not share any exact
confidential data, just something that looks exactly like it.
Privacy-preservation GANs are capable of accomplishing this task Beaulieu-Jones et al.
(2019). In actuarial valuation models where model points are used to determine the amount
of money to hold for an individual/groups, such GANs may be useful for the creation of
synthetic samples to be fed into the valuation model, without needing the details of any
policy. In particular, GANs can be used to share synthesized data and make them publicly
available, increasing the scope for actuarial research, collaboration, and comparisons.
4.5. Missing Data Imputation
Missing data causes an issue in analysis as most standard data analytic methods are
not designed for missing data. Techniques, such as single imputation (SI) and multiple
imputation (MI) Rubin (2004), exist, but there is no consensus on which of the MI method
is superior, even though MI is known to be better than SI Schafer and Olsen (1998).
Generative Adversarial Imputation Net (GAIN) Yoon et al. (2018) provides an alternative generative modeling approach to create new cases that can be used to impute missing
information. View Imputation via GANs (VIGAN) Shang et al. (2017) deals with data that
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are collected from heterogeneous sources, resulting in multi-view or multi-modal data sets
where missing data occurs in a number of these sources. These methods were shown to be
better than SI/MI methods, thereby improving the effectiveness of ML algorithms trained
after. These GANs can be used to impute missing data points in experience investigations
and assumption setting in both short-term and life insurance when conducting valuation
or pricing, increasing the number of data points available for boosting the predictive power
of models built after.
4.6. Semi-Supervised Learning
The purpose of a Semi-Supervised GAN (SGAN) is to train the discriminator into a
classifier which can achieve superior classification accuracy from as few labeled examples
as possible Sricharan et al. (2017), thereby reducing the dependency of classification tasks
on enormous labeled data sets. It has been shown that an SGAN generalizes from a small
number of training examples much better than a comparable, fully-supervised classifier
Chongxuan et al. (2017); Liu et al. (2019); Miyato et al. (2018). This has been lauded as the
most useful GAN application with good performance with a small number of labels on
data sets Odena (2016); Salimans et al. (2016).
For imbalanced data sets, such as mortality, morbidity, fraud, lapses, extreme events,
large claims, and sub-standard risks, SGAN may offer a superior alternative predictive
model compared to ML models which require significant training data for improved
accuracy. Typically, one has to deal with imbalanced classes either through synthetic
sample generation using some heuristic method, such as SMOTE, cost sensitive adjustment
to the evaluation metric, or adding uncertainty margins, which can be subjective. Through
the training of an SGAN, it is possible to have a sample generative scheme whilst having a
classifier, as well. This has tremendous advantages over many generative and ML models.
4.7. Domain Adaptation
It is quite possible that the training data used to learn a classifier has a different
distribution from the data which is used for testing. This results in degradation of the
classifier performance and highlights the problem known as domain adaptation Hong et al.
(2018). In domain adaptation, the training and test data are from similar but different
distributions. This area has become interesting for GANs in the past few years.
These methods include CycleGAN Zhu et al. (2017), Discover GAN (DiscoGAN)
Kim et al. (2017), DualGAN Yi et al. (2017), and StarGAN Choi et al. (2018), which can be
used for multiple domains. With these methods, one can transfer an algorithm learned
from a different data set to a new one and achieve similar performance. Such approaches
are also able to learn representation adaptation, which is learning feature representations
that a discriminator cannot differentiate which domain they belong to Tzeng et al. (2017).
By using synthetic data and domain adaptation, the number of real-world examples that
are needed to achieve a given level of performance is reduced significantly, utilizing only
randomly generated simulated points Hoffman et al. (2017). Domain adaptation can learn
transfers between different domains, by synthesizing different data sets. This can be useful
in combing public data sets or other market data with internal company data in actuarial
firms.
4.8. Summary
Given the above taxonomy of GAN applications, Table 2 depicts specific actuarial
areas where GANs can be useful.
To our knowledge, there has been limited applications of GANs in actuarial areas,
such as insurance, health care, banking, investment, and enterprise risk management.
This is compounded by the fact that GANs have been highly successful on computer vision,
with less emphasis on tabular data sets. However, there have been recent applications
of GANs on other tabular data sets, such as airline passengers Mottini et al. (2018) and
medical records Armanious et al. (2018).
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Table 2. Potential GAN applications in actuarial disciplines.
Actuarial Discipline
Description
Product Design,
Propensity and Customer
Behavior
Create wider and more model points; Boost propensity models with more data per cell,
leading to better and accurate models.
Actuarial Models
Experience monitoring and experience rates derived using a large credible data set.
Boost models using data augmentation, semi-supervised learning, missing data
imputation and domain adaptation for pricing, assumption setting, anomaly detection,
risk estimation, time series and attention prediction in insurance, reinsurance, banking,
investment, healthcare, and enterprise risk management.
Projections
Network modeling by looking at driving dependencies rather than correlation
assumptions, i.e., use generative models. Strategic flexible and more decision-based
models based on the environment. More GAN-based time series models driven by the
environment. Enhanced solvency projection models and stress tests which are based on
rich data sets.
Reserving
Make projections more predictive through a large enough credible data at all model
points, i.e., accurate assumptions per risk cell with less margins.
Surplus Distribution
More granular individual information from alternative data sources through leveraging
generative models.
Investment Strategy
Granular data for asset/liability modeling, i.e., use GANs to simulate scenarios that
depend entirely on the adopted investment strategy and boosting the model. Enhanced
market risk monitoring. Improvements to portfolio optimization.
Data Cleaning
Reduce errors; fill in gaps using imputation; increase the sample size; query other data
sets and verify patterns using Cycle GANs.
Research
Make actuarial data sets more publicly available through synthesized data generated by
GANs, boosting industry data. This is helpful for creating accurate and more up-to-date
standard tables and encouraging actuarial research.
External Data Sources
Leverage other data sets through combining multiple data sets. For example, DualGAN
or CycleGAN can be leveraged to learn a representation that encompasses different data
sets.
GANs can equally be adopted or leveraged for similar tasks to boost limited actuarial
data sets and improve actuarial models, especially in areas where models are needed to
make business decisions. Examples of actuarial applications with limited data and the
class imbalance problem include claim frequency modeling, claim amount estimation,
lapse prediction, fraud detection, mortality/morbidity rate estimation, catastrophe modeling, extreme event models, and risk estimation. Leveraging GANs to increase the data size
on these data sets could lead to better actuarial models. In particular, GANs could allow
less reliance on using stochastic simulations that are based on subjective distributions and
err less on margins used.
5. Methodology
This section describes in detail the theoretical operation of GANs, their challenges,
and tricks to improve their training. Throughout this paper, it is assumed that both GAN
networks are implemented with ANNs. For comparative purposes, we also implement
a popular synthetic data generative mechanism using Synthetic Minority Over-sampling
Technique (SMOTE) Chawla et al. (2002).
5.1. SMOTE
This section describes the theoretical operation of SMOTE for comparative purposes
with the GAN applied in this work. SMOTE creates new synthetic cases by linearly
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interpolating between two nearest neighbor (NN) instances of the minority class. Chawla
et al. (2002) show that SMOTE improves the effectiveness of ML classifiers compared to
random over-sampling and under-sampling approaches. Over time, SMOTE has become
the default method for synthetic sample generation and has proven to be popular among
researchers, becoming a pioneer in imbalanced learning Fernández et al. (2018).
Considering a random minority instance x, a new instance s is generated by considering its k-NNs. These k-NNs are found by using the Euclidean distance metric. Initially,
an instance y is generated at random from the k-NNs. Then, a new synthetic minority
instance s is generated, as follows:
s = x+α y−x ,
(3)
where α is randomly generated from the Uniform distribution [0, 1].
SMOTE parameters are the value of k and the number of minority cases to generate.
These parameters can be tuned to ensure an optimal metric is achieved. SMOTE is the
benchmark method for addressing class imbalance in binary classification problems.
5.2. Vanilla GAN
This section describes the original GAN formulation, called MiniMax GAN (MMGAN). This is the baseline model over which all other GAN variants are based.
5.2.1. The Discriminator
The discriminator (D) receives generated samples from a generator G and the true data
examples from pdata ( x ), and must distinguish between the two for authenticity through a
deep ANN Goodfellow et al. (2014). The resulting output Dθd ( x ) for an input x is the probability of x being sampled from pdata ( x ) instead of p g , where p g is the implicit distribution
defined by G. The vector θd represents learned parameters from D. The discriminator’s
goal is to yield D ( x ) near 1 for x ∼ pdata and D ( G (z)) closer to 0 for p ∼ pz (z) using the
sigmoid function in the output layer. This is achieved by maximizing D’s loss over θd :
h
i
MM− GAN
JD
= EX ∼ pdata (x) log Dθd ( x ) + EZ∼ pz (z) log(1 − Dθd ( Gθg (z)) .
(4)
5.2.2. The Generator
The generator (G) randomly picks a sample z from the prior latent space defined by
p(z) and then generates samples from this distribution using an ANN. This deep ANN
must learn the parameters Θ g given an input z ∼ pz (z), that will give the output Gθg (z). G
is trained to fool D, i.e., to make D’s output for fake/generated sample D ( G (z)) closer to 1.
The parameters of G are learned by minimizing G’ loss over Θ g :
h
i
JGMM−GAN = EZ∼ pz (z) log(1 − Dθd ( Gθg (z)) .
(5)
5.2.3. GAN Loss
Combining the losses for D and G, GANs solve the following minimax game in
alternate steps through Gradient Descent (GD) Ruder (2016):
h
i
min max EX ∼ pdata ( x) log Dθd ( x ) + EZ∼ pz (z) log(1 − Dθd ( Gθg (z)) .
(6)
θg
θd
The above losses for D and G are the original formulation proposed by Goodfellow in
2014, called minimax GAN (MM-GAN). Since we are minimizing over θ g and maximizing
over θd , training of GANs alternate between GD on G and gradient ascent on D Goodfellow
et al. (2016). Typically, for every training of G, D is trained k times although an optimal
choice is debatable among researchers. This is shown in Algorithm 1.
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Remark 1. Gradient based updates on the networks can be accomplished using one of the GD
optimizers. Typically, Stochastic GD (SGD) with Momentum Qian (1999) for D, Root Mean
Square propagation (RMSprop) Hinton and Tieleman (2012), or Adaptive Moment estimation
(Adam) Kingma and Ba (2014) for G tend to work well in practice Goodfellow et al. (2014);
Radford et al. (2015).
Algorithm 1: Mini-batch SG ascent of GANs with the original objective for MM-GAN. The number of steps to
apply to D, k, is a hyper-parameter. For every training of G, we train D k times. Goodfellow et al. (2014) used
k = 1.
1: for number of epochs do
2:
update the discriminator
3:
4:
for k steps do
•
Sample mini-batch of m noise samples {z(1) , . . . , z(m) } from the noise prior p g (z).
•
Sample mini-batch of m true examples { x (1) , . . . , x (m) } from the training data distribution pdata ( x ).
•
Update the discriminator D by ascending its stochastic gradient on these
mini-batches:
"
#
1 m
∆θd ∑ log D xi + log 1 − D G (zi )
.
m i =1
5:
end for
6:
update the generator
7:
•
•
8:
Sample mini-batch of m noise samples {z(1) , . . . , z(m) } from the noise prior p g (z).
Update the generator by descending its stochastic gradient computed on this
mini-batch:
1 m
∆θ g ∑ log 1 − D G (zi ) .
m i =1
end for
5.2.4. Non-Saturating GAN
While the above loss function is useful for theoretical results, unfortunately, it does not
work well in practice, and there are challenges getting the GAN to convergence, stabilize
its training, and getting diverse samples Arjovsky et al. (2017); Mirza and Osindero (2014);
Radford et al. (2015); Salimans et al. (2016). In practice, rather than training the above loss
function for G, to provide better gradients in earlier training, Goodfellow et al. (2014)
suggest to maximize the following objective function for G instead:
JGNS−GAN = EZ∼ pz (z) log Dθd ( Gθg (z) .
(7)
This version of GAN is called non-saturating GAN (NS-GAN) and is typically used as
the benchmark in most studies and in practice. This leads to the following NS-GAN loss
function:
max max EX ∼ pdata ( x) log Dθd ( x ) + EZ∼ pz (z) log Dθd ( Gθg (z) .
(8)
θg
θd
With this new loss function, we alternate between gradient ascent on D and gradient
ascent on G. Algorithm 1 is based on the original MM-GAN formulation; however, it can
easily be tweaked to represent NS-GAN.
5.2.5. Optimal Solution
Theoretically, it can be shown that for p g = pdata , the GAN zero-sum game in Equation
(6) has a global optima. Given enough capacity for both networks and D is trained to
optimality for a fixed G, convergence of the GAN algorithm is guaranteed Goodfellow
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et al. (2014); Manisha and Gujar (2018); Mirza and Osindero (2014); Nowozin et al. (2016);
∗ ( x ) for a fixed G is:
Radford et al. (2015). The optimal discriminator DG
∗
DG
(x) =
pdata ( x )
.
pdata ( x ) + p g ( x )
(9)
∗ ( x ) into Equation (6) for
Assuming that D is perfectly trained and if we substitute DG
G’s loss, this gives rise to the Jensen-Shannon (JS) divergence Lin (1991). The JS divergence
can be written as a function of the Kullback-Leibler (KL) divergence Kullback (1997);
Kullback and Leibler (1951).
Definition 1. The KL divergence between two probability distributions pdata and p g is defined as
KL( pdata , p g ) = DKL pdata || p g =
Z
pdata ( x ) log
!
pdata ( x )
dx.
pg (x)
Definition 2. The JS divergence between two probability distributions pdata and p g is defined as
JS( pdata , p g ) = D JS pdata || p g
pdata + p g
pdata + p g
1
1
+ KL p g ,
.
= KL pdata ,
2
2
2
2
∗ ( x ) into Equation (6), the minimum loss for G is reached if and
If we substitute DG
only if p g = pdata ; thus, one can show that:
JG = − log 4 + 2JS pdata , p g .
(10)
This equation tells us that, when D has no capacity limitation and is optimal, the GAN
loss function measures the similarity between pdata and p g using JS divergence. However,
although the above results provide a nice theoretical result, in practice, D is rarely ever fully
optimal when optimizing G Goodfellow et al. (2014). Thus, alternative GAN architectures
have been proposed to fix this issue and to get closer to optimality. Below, we describe
what causes this failure to convergence and how to fix it.
5.3. Challenges with GANs
GANs are notoriously difficult to train properly and to evaluate, the likelihood cannot
be easily be computed, and they suffer from the vanishing gradient problem, mode collapse,
boundary distortion, and over-fitting Arjovsky et al. (2017); Creswell et al. (2018); Goodfellow et al. (2016); Hitawala (2018); Hong et al. (2019); Salimans et al. (2016). This section
describes key challenges on GAN training.
5.3.1. Mode Collapse
Mode collapse is when many latent noise values z are mapped to the same data point
x, leading to a lack of diversity in the samples that are created, i.e., under-fitting. This is
regarded as the most significant problem with GANs Manisha and Gujar (2018). Many
studies have spent lots of time in varied contexts to fix this.
5.3.2. Vanishing Gradient
This occurs when D becomes perfect in its training without giving G the chance to
improve. As a result, GANs may fail to converge, thereby leading to poor generated
samples Arjovsky et al. (2017).
5.4. Improved GAN Training
There are many GAN architectures which avoid the problems that come with the
vanilla GAN. We briefly describe some of the most common and popular GAN solutions.
Salimans et al. (2016) look at ways to improve GANs (called hacks), while other authors
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propose variants to the vanilla GAN by changing the cost function, adding gradient
penalties (GPs), adding labels, avoiding over-fitting, and finding better ways of optimizing
GANs. Given the vast number of taxonomies, we are not able to cover all of them but only
discuss the most popular and those subsequently used in this work.
5.4.1. Conditional GANs
The first extension of GAN was the conditional GAN (cGAN) which gave the generator
the label Y in the latent space, making them class conditional Mirza and Osindero (2014).
Most of the GAN variants can be modified to include cGAN. cGAN allows to create
diversified samples and forcing G to create specific samples, thereby fixing mode collapse
problem.
5.4.2. Deep Convolutional GAN
Until the introduction of deep convolutional GAN (DCGAN) Radford et al. (2015),
training GANs was still unstable. DCGANs provide some further tricks using convolutional and deconvolutional layers. Given that DCGANs use convolutional NNs which are
typically used for images, we do not review this architecture in detail as our main focus
in on tabular data. Despite this, the structure of the DCGAN is very useful in providing
stable training for most GANs Lucic et al. (2018).
5.4.3. Loss Variants
There are a number of GAN architectures which change the loss function to improve
GAN training and stability. The loss function for GAN measures the similarity between
pdata and p g using JS. Unfortunately, JS tends not to be smooth enough to ensure a stable
training Hong et al. (2019); Manisha and Gujar (2018). There are a number of GAN loss variants which have been proposed over the years. Broadly, there are two loss function groups
with better properties, i.e., f-divergence Nowozin et al. (2016) and Integral Probability
Metrics (IPMs) Hong et al. (2019); Müller (1997).
Among these loss groups, Wasserstein GAN (WGAN) Arjovsky et al. (2017) is arguably the most popular and well-studied Hitawala (2018); Wang et al. (2017). WGAN is
considered a general unified framework under the recently proposed Relativistic GAN
(RGAN) Jolicoeur-Martineau (2018). Thus, we adopt to describe WGAN as it has become
the most widely used GAN architecture since DCGANs.
5.5. WGAN
This section describes WGAN and its improved training using WGAN-GP.
5.5.1. Wasserstein Distance
IPM generalizes a critic function f belonging to an arbitrary function class, where IPM
measures the maximal distance between two distributions under some functional frame
f Hitawala (2018). Among the IPMs, the Wasserstein distance is the most common and
widely used metric Manisha and Gujar (2018).
Informally, the Earth mover (EM) Rubner et al. (2000) distance W ( pdata , p g ) measures
the minimal changes needed to transform p g into pdata . More formally, EM between two
probability distributions pdata and p g is:
W pdata , p g =
inf
γ∼Π( pdata ,p g )
E(x,y)∼γ k x − y k ,
(11)
where Π( pdata ,p g ) represents a set of all joint probability distributions in which marginal
distributions are, respectively, pdata ( x ) and p g ( x ). Precisely, γ( x, y) is a transport plan,
i.e., percentage of mass that should be moved from x to y to transform p g into pdata .
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The infimum in Equation (11) is intractable as it is tricky to exhaust all the elements of
Π( pdata ,p g ) Arjovsky et al. (2017). This is solved using the following functional format:
W pdata , p g = sup Ex∼ pdata f ( x ) − Ex∼ pg f ( x ) ,
(12)
k f k L ≤1
where the supremum is taken over a 1-Lipschitz function f . A function f is 1-Lipschitz if
for all x1 , x2 : | f ( x1 ) − f ( x2 )| ≤ | x1 − x2 |.
5.5.2. The Critic
In WGAN, D’s output is not a probability anymore but can instead be any number,
and, for this reason, D is typically called the critic. The WGAN critic tries to maximize
the difference between its predictions for real samples and generated samples, with real
samples scoring higher. Arjovsky et al. (2017) force the critic to be 1-Lipschitz continuous for
the loss function to work well:
JWGAN = max EX ∼ pdata ( x) D ( x ) + EZ∼ pz (z) 1 − D ( G (z)) ,
(13)
w ∈W
where W is the set of 1-Lipschitz continuous functions. Typically, to enforce the Lipschitz
constraint, the critic weights w are clipped to lie within a small range, usually [−0.01, 0.01]
after each training batch Arjovsky et al. (2017); Gulrajani et al. (2017).
The critic is trained to convergence so that the gradients of G are accurate, thus
removing the need to balance the training of G and D by simply training D several times
between G’s updates, to ensure it is close to convergence. Typically, 5 critic updates to
1 generator update is used Arjovsky et al. (2017). WGAN used the RMSProp version of
gradient GD with a small learning rate and no momentum Arjovsky et al. (2017). However,
Adam may also be used as it is a combination of RMSProp with Momentum.
5.6. Improved WGAN Training
Even though WGAN has been shown to stabilize GAN training, it is not generalized
for deeper training due to weight clipping which tends to localize most parameters at
−0.01 and 0.01 Gulrajani et al. (2017); Manisha and Gujar (2018). This effect dramatically
reduces the modeling capacity for D. Gulrajani et al. (2017) amend WGAN through an
addition of a gradient-penalty (GP) to the loss function, coming with WGAN-GP. In total,
three changes are made to WGAN critic to convert it to WGAN-GP: include a GP to the
loss function; do not clip critic weights; and do not use batch normalization layers in the
critic. WGAN-GP is defined using the following loss function:
h
2 i
EX ∼ pdata (x) D ( x ) + EZ∼ pz (z) 1 − D ( G (z)) + λEx̃∼ pdata k ∆D ( x̃ ) k2 −1 ,
(14)
where x̃ samples uniformly along the straight line between points sampled from pdata
and p g , and λ is the GP term. Gulrajani et al. (2017) show a better distribution of learned
parameters compared to WGAN, and this method has been the default method in most
GAN loss variants.
We adopt the conditional version of WGAN-GP, called WCGAN-GP, as an alternative
to current actuarial/statistical approaches for synthetic sample generation. Once WGANGP is trained to convergence, G can be used to create new samples by feeding it the latent
space Z.
6. Experiments
This section outlines the experiments conducted, showing a popular GAN application
for data augmentation and boosting predictive models. We compare WGAN with SMOTE.
This exercise can be similarly adopted for any actuarial modeling problem, such as mortality,
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morbidity, medical segmentation, credit risk, extreme events, regression, Value-at-Risk,
and anomaly detection in insurance, investment, banking, and healthcare.
6.1. Data Sets
We considered 5 publicly available imbalanced data sets from the Center for Machine
Learning and Intelligent Systems database Dua and Graff (2017). The data sets are described
below and shown in Table 3.
Table 3. Imbalanced data sets used in the experiments.
Imbalanced Data Set
Majority Cases
Minority Cases
Number of Features
Numeric Features
Ordinal Features
Credit Card Fraud
Pima Indians Diabetes
Glass Identification
German Credit Scoring
Breast Cancer Wisconsin
284,807
500
144
700
357
492
268
70
300
212
31
8
9
20
28
31
8
9
14
28
0
0
0
6
0
6.1.1. Credit Card Fraud
European public credit card fraud transactions made in 2013 are utilized Pozzolo (2015).
This data is highly imbalanced, with 492 fraudulent transactions out of a total of 284,807
transactions, representing a mere 0.172% of fraud cases. This data set contains 31 anonymized
features (Time, Amount, V0,V1, . . . V28) and the Class indicator showing 1 for frauds and 0
for non-fraudulent cases. All the variables are numeric.
6.1.2. Pima Indians Diabetes
This data set contains the prediction of the onset of diabetes within 5 years in Pima
Indians given some medical details, representing 34.90% of diabetic cases out of a total of
768 samples Smith et al. (1988). There are 8 independent variables.
6.1.3. German Credit Scoring
This data comes from the German credit scoring from the Machine Learning Repository Dua and Graff (2017). There are 1000 observations with 20 independent variables.
The dependent variable is the evaluation of customer’s current credit status, which indicates whether a borrower’s credit risk is good or bad.
6.1.4. Breast Cancer Wisconsin
This data represents the characteristics of a cell nuclei that is present in the digitized
image of a breast mass Street et al. (1993). The data is used to predict the presence of benign
or malignant cancer, with 37.25% being malignant samples from a total of 569 cases.
6.1.5. Glass Identification
This data set determines whether the glass type is float or not in term of their oxide
content Evett and Spiehler (1987). There are 32.71% of float glass types out of a total of
214 cases.
6.2. Scaling the Data
Many ML methods expect data to be of the same scale to avoid the dominance of certain
variables and this can affect the accuracy of specific models Ioffe and Szegedy (2015); Mitchell
(2006). Normalization re-scales the data to the range between 0 and 1. Standardization
centers the data distribution to N (0, 1). We adopt normalization as it does not assume any
specific distribution. This will potentially speed up convergence Goodfellow et al. (2016);
Mitchell (2006).
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6.3. Train-Test Split
ML models are usually trained and tested on unseen data. Two approaches to split
the data are cross-validation (CV) and train-test split Friedman et al. (2001). CV divides the
data into K subsets that can lack sufficient credibility and can result in higher variability of
predictions, if the data size is too small Friedman et al. (2001). Train-test split, however, can
allow a larger subset of the data to be used for estimating model coefficients and results in
more reasonable results Mitchell (2006).
Existing literature typically uses a 70–90% train-test split, especially if the data is
large. This technique is simple, easy to understand and widely used, despite giving noisy
estimates sometimes Friedman et al. (2001); Goodfellow et al. (2016); Mitchell (2006). CV
is typically used to optimize parameters of a classifier. This work adopts 75% training data
and 25% testing data. Other train-test splits are possible; however, we leave this for future
work.
6.4. SMOTE Implementation
Over-sampling is performed on the 75% training data using the R imbalance library.
The R imbalance library contains functions for performing SMOTE and other variants.1
The two parameters to tune are the number of neighbors and the over-sampling rate.
We kept the over-sampling rate the same to ensure balanced class distributions within each
data set. Using SMOTE, we create additional synthetic cases to augment the above training
data sets.
We varied the number of k-NNs for each data set to ensure optimal parameters are
chosen through a 10-fold CV. This was done through a grid search scheme, with values of
k-NN ranging from 1 to 15, optimized using the Area under the Precision-Recall Curve
(AUPRC). The best parameter values for each data set are shown in Table 4 below.
Table 4. Optimal parameter values for k-NN for each data set.
Data Set
Value of k-NN
Credit Card Fraud
Pima Indians Diabetes
Glass Identification
German Credit Scoring
Breast Cancer Wisconsin
6
9
10
12
10
6.5. GAN Implementation
Given its popularity and wide use, WGAN is adopted for an alternative synthetic sample generation. Specifically, we adopt the conditional version of WGAN with GP; thus, we
use WCGAN-GP Gulrajani et al. (2017); Mirza and Osindero (2014). Below, we describe
how parameters are chosen and results generated.
6.5.1. Software
Due to its simplicity and faster computations, the high-level Keras François (2015)
library with Tensorflow back-end is chosen to implement WCGAN-GP.2 This is trained
using all minority cases of each data set.
6.5.2. The Generator
This section describes how the parameters for G are chosen. The random noise for z is
generated from N (0, 1) with 100 dimensions. This is based on GAN hacks which suggest
to sample from a spherical distribution Salimans et al. (2016).
1
The imblearn package in Python can also do SMOTE and its notable variants. Other packages exist in R, such as ROSE, unbalanced, smotefamily,
DMwR, ebmc, and IRIC.
2
Pytorch and Tensorflow are also popular packages available in Python for implementing GANs.
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Rectified Linear Unit (ReLU) Glorot et al. (2011) is adopted in the hidden layers
Salimans et al. (2016). For G’s output later, hyperbolic tangent (tanh) is adopted. No drop
out or batch normalization is applied in the hidden layers, following advice by Gulrajani
et al. (2017) for WGAN-GP.
The layers are chosen such that they are ordered in an ascending manner for G. For
simplicity, after a number of iterations, 3 layers were chosen for each data set. In the first
layer, there were 128 units; in the second layer, 256 units; and, in the third layer, 512 units.
These layers worked well in the experiments conducted. The output layer had the data
dimension of the data as the number of units.
Weights are initialized using the He initialization method He et al. (2015). Adam is
used to optimize the weights of G Radford et al. (2015); Salimans et al. (2016). For Adam,
we used default values with β 1 = 0.5 and β 2 = 0.9 for G Kingma and Ba (2014).
We used a batch size of 128 when optimizing the gradients for faster training Ioffe and
Szegedy (2015). Initial learning rate η for G was fixed at 0.00004. The number of epochs
were found to be 5000 where the GAN training was found to be stable.
6.5.3. The Critic
ReLu is adopted with a negative slope of 0.2 Glorot et al. (2011); Radford et al. (2015).
Similarly to the generator, 3 layers were used in the hidden layers. The layers were arranged
in a descending manner, with 512 units in the first layer, 256 units in the second layer, and
128 units for the last layer. The critic gives the output a single value using a linear function
Arjovsky et al. (2017). Adam was used with default parameters in Keras François (2015),
as per Table 5.
Table 5. Adam parameters for the critic François (2015); Kingma and Ba (2014).
Parameter
Value
η
β1
β2
ε
0.00001
0.5
0.90
10−8
Critic weights were also initialized using the He method, and a similar batch size as in
the generator was used. We pre-trained the critic 100 times at each adversarial training step
Arjovsky et al. (2017). This ensures faster convergence at each step before G is updated.
We used WGAN with a GP with the default values as per the original paper Gulrajani et al.
(2017). The GP value was left unchanged at 10. We call this model WGAN-GP. We found
that, after 5000 epochs, the losses plateaued and did not change much.
6.5.4. Labels
Typically, to boost faster training and fix mode collapse, additional information can
be incorporated in both G and D using cGAN Mirza and Osindero (2014). We used the
conditional version of WGAN-GP where class labels were added to the minority cases.
To accomplish this, clustering was done on the minority cases in order to induce class labels
on the training data.
We explored a number of common mechanisms considering k-means clustering Hartigan
and Wong (1979), Agglomerative Hierarchical Clustering (AHC) Voorhees (1986), Hierarchical DBSCAN Ester et al. (1996), and t-distributed Stochastic Neighborhood Embedding
(t-SNE) Maaten and Hinton (2008). The details of these algorithms are beyond the scope of
this work.
Due to its wide use and simplicity, we adopted k-means clustering with 2 clusters
for each data set. This yielded labels that could be fed into G and D to induce generated
samples. We call the final model WCGAN-GP after incorporating these class labels into the
training.
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6.5.5. Training WGAN-GP
Figure 4 presents the experiments of training WCGAN with GP. For comparative purposes, using similar parameters, we show the quality of samples generated for WCGAN3
with GP, WGAN, cGAN and non-saturating GAN on the credit card fraud data.
Figure 4. Comparison of GAN experiments ran on fraud data cases.
We consider this for two combinations of the features for illustrative purposes up to
5000 epochs. The results show the superiority of samples generated by WCGAN with
GP. There is a clear mode collapse problem on the vanilla GAN and cGAN. WGAN and
WCGAN with GP show better samples. There are clear damped oscillations and unstable
losses for GAN and cGAN where Wasserstein GANs exhibit stable training and losses,
especially after 1000 iterations, where it seems to settle and stabilize.
Figure 5 shows the critic loss for each epoch, where, after 1000 epochs, the loss starts
to plateau. Thus, we decided to stop the training after 5000 epochs. We repeated this
experiment for each data set and adopted WCGAN with GP after 5000 epochs as the model
to use for synthetic sample generation.
6.5.6. Generating Synthetic Samples
Once the WCGAN with GP is trained to 5000 epochs, the learned generator distribution is used to create more synthetic samples by feeding it the number of samples to
output.
3
The version of the WCGAN was incorporated with an improved WGAN training using the GP term as per the paper by Gulrajani et al. (2017).
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Figure 5. Difference between generated and real data critic loss.
6.6. Logistic Regression
For simplicity, and given the wide use with actuaries, a Logistic Regression (LR)
McCullagh (1984) model is trained using Python 3.7 Python Software Foundation (2017)
on both the imbalanced training data and over-sampled data sets to predict the likelihood
of each minority case using this equation:
!
d
hθ ( x (d) )
=
θ
+
θi Xi , 0 < hθ ( x (d) ) < 1,
(15)
log
0
∑
1 − hθ ( x (d) )
i =1
where hθ ( x (d) ) is the probability of the given minority case, θi ’s are the estimated coefficients
using SGD, Xi is the feature vector for sample i, and d is the number of features to include
in the LR model. The coefficients are estimated by minimizing a loss function through a
SGD. Typically, classification is such that, when hθ ( x (d) ) ≥ 50% for each instance, assign
the minority case and, otherwise, the majority case.
6.7. Evaluation
The confusion matrix returns a report showing how predicted classes on unseen test
data using the LR model compare to actual observed classes, as depicted in Table 6.
Table 6. The confusion matrix.
Confusion Matrix
Predicted: Minority
Predicted: Majority
Actual: Minority
Actual: Majority
True Positive (TP)
False Positive (FP)
False Negative (FN)
True Negative (TN)
True Negative (TN) is the number of majority cases that were correctly classified as
such. False Positive (FP) is the number of majority cases that were incorrectly classified as
minority. True Positive (TP) is the number of minority cases that were correctly classified
as minority. False Negative (FN) is the number of minority cases that were incorrectly
classified as majority. Using these definitions, Table 7 presents the most well known
evaluation metrics for binary problems.
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Table 7. Evaluation metrics for binary problems.
Metric
Accuracy
Precision
Recall
F1-Score
Formula
TP + TN
TP + TN+ FP + FN
TP
TP
+ FP
TP
TP
+ FN
Precision × Recall
2×
Precision + Recall
Precision is the ability of the LR model not to label a minority case that is actually
majority. Recall is the ability of the LR model to find all minority cases. F1-Score is a
harmonic mean between Precision and Recall He and Garcia (2008). F1-Score puts equal
weight to both Precision and Recall. Accuracy can be misleading and inappropriate when
there are imbalanced classes and, thus, may be biased towards majority cases Chawla et al.
(2002); Ganganwar (2012); He and Garcia (2008). Thus, we do not use rely on it in this
work. Accuracy, Precision, Recall, and F1-Score should be close to 100% for a LR model
to do well on the testing data. However, these scores are influenced by what threshold is
used to decide between the two binary classes.
The Receiver Operating Characteristic (ROC) curve Bradley (1997); Hanley and McNeil
(1982) measures a classifier’s performance on a test set over different decision thresholds
by varying the Precision and the FP rate. The Area under the Curve (AUC) measures the
performance of the LR model trained on both imbalanced and over-sampled data sets and
tested on unseen data with values close to 100% considered excellent performance Bekkar
et al. (2013); Hanley and McNeil (1982). We also compute the Precision-Recall curve and
compute the Area Under the Precision-Recall Curve (AUPRC) to get a weighted score.
A method that gives the highest score is better.
6.8. Statistical Hypothesis Testing
Friedman test Friedman (1937), followed by a post-hoc Nemenyi test Nemenyi (1962),
are performed to verify the statistical significant differences between WCGAN-GP and
SMOTE.
6.8.1. Friedman Test
The Friedman test is a non-parametric ranking test to determine whether SMOTE
and WCGAN-GP methods perform similarly in mean performance rankings based on the
measures above, when normality does not hold Friedman (1937).
6.8.2. Post-Hoc Nemenyi Test
If the null hypothesis is rejected, a post-hoc test can be applied where WCGANGP is considered as the control method. The post-hoc Nemenyi test evaluates pairwise
comparisons between the over-sampling methods if the Friedman test suggests that there
is a difference in performance Nemenyi (1962); Pohlert (2014). We adopt WCGAN-GP as
the control method.
6.8.3. Implementation
Both tests are conducted using the Pairwise Multiple Comparison Ranks Package
(PMCMR) Pohlert (2014) available in R. We assume statistical significance of the alternative
hypothesis at p-values < 0.05. In other words, we fail to reject the null hypothesis when
the resulting p-value is higher than 0.05, suggesting that there is no difference between
SMOTE and WCGAN-GP.
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7. Results
This section presents the results of all the LR models applied on the baseline and
over-sampled data sets, with metrics on Precision, Recall, F1-Score, AUC, and AUPRC
computed on the same unseen test data.
7.1. Comparisons
Table 8 presents the evaluation metrics (based on the testing set) of the LR model
applied on the baseline and over-sampled data sets for a default threshold of 50%. Bold
shows an algorithm that performs the best for that data set, i.e., a higher score for that
metric. Figure 6 shows the average performance across all data sets from each evaluation
metric.
Table 8. Evaluation metrics based on a default threshold of 50%.
Method
Precision
Recall
F1-Score
AUPRC
AUC
85.71%
5.11%
86.24%
63.41%
93.33%
76.42%
72.90%
9.69%
81.03%
74.60%
72.28%
81.35%
81.70%
98.36%
88.20%
74.47%
53.54%
75.51%
56.45%
80.30%
59.68%
64.22%
64.24%
66.67%
72.49%
68.18%
74.10%
73.61%
75.48%
75.22%
60.31%
47.83%
46.51%
51.34%
70.51%
81.08%
55.47%
56.99%
59.11%
63.02%
58.84%
66.60%
68.57%
69.61%
70.94%
50.00%
73.91%
55.00%
42.86%
70.83%
78.57%
46.15%
72.34%
64.71%
53.83%
87.29%
69.56%
63.93%
72.86%
78.03%
94.34%
92.59%
96.23%
94.34%
100.00%
96.23%
94.34%
96.15%
96.23%
95.39%
98.56%
96.93%
95.50%
96.45%
97.00%
Credit Card Fraud
Baseline
SMOTE
WCGAN-GP
Pima Indians Diabetes
Baseline
SMOTE
WCGAN-GP
German Credit Scoring
Baseline
SMOTE
WCGAN-GP
Glass Identification
Baseline
SMOTE
WCGAN-GP
Breast Cancer Wisconsin
Baseline
SMOTE
WCGAN-GP
Figure 6. Average performance across all data sets.
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In general, SMOTE improves Recall at the expense of a lower Precision. This results in
a lower F1-Score than Baseline results. As a result of a much lower Precision for SMOTE,
AUPRC is penalized and lower than both Baseline and WCGAN-GP. SMOTE compromises
the Precision significantly, whereas WCGAN-GP improves Recall, while not significantly
penalizing Precision.
Overall, WCGAN-GP shows a higher F1-Score. Thus, using a default threshold, WCGANGP performs the best on F1-Score, followed by Baseline and SMOTE being last (on the average).
The lower Precision on SMOTE may be due to the strict assumed probability distributions and
possible creation of over-lapping and noisy samples Bellinger et al. (2015); Das et al. (2015);
Gao et al. (2014); Mathew et al. (2015); Zhang and Li (2014). While the univariate results on
Precision, Recall, and F1-Score are useful, they do not give the entire picture over different
thresholds Bekkar et al. (2013).
Since AUC and AUPRC are based on varied thresholds, these metrics are typically
preferred over one dimension measurements, such as Precision, Recall, and F1-Score
Bekkar et al. (2013); Ganganwar (2012); López et al. (2013). Since we are also comparing
the above results with the Baseline model, these metrics are impacted by class imbalance
He and Garcia (2008). Thus, we rely on the AUC and AUPRC.
7.1.1. AUC
The ROC curve represents the trade-off between Precision and the FP rate, while the
AUC is the area under the ROC curve Bekkar et al. (2013). SMOTE reports higher AUC
values than the Baseline. In general, WCGAN-GP is better on 3 of the 5 data sets except
on credit card fraud and diabetes data sets. Overall, the average AUC value is not too
different between WCGAN-GP and SMOTE. This result conflicts the AUPRC scores where
WCGAN-GP shows a clear dominant superiority over SMOTE.
Whilst AUC may be useful, it does not consider Recall, which may be the most
important metric for minority cases. AUC may be affected by skewed data sets and the
data distribution He and Garcia (2008). ROC curves are appropriate when the data is
balanced, whereas Precision-Recall curves are appropriate for imbalanced data sets Bekkar
et al. (2013); He and Garcia (2008). AUC may tend to provide an overly optimistic view
than AUPRC He and Garcia (2008).
In general, an algorithm that dominates in AUC may not necessarily dominate
the AUPRC space He and Garcia (2008). Saito and Rehmsmeier (2015) suggest that the
Precision-Recall curve and AUPRC are more informative than the ROC curve and AUC.
Since we are also comparing with the Baseline which is imbalanced, ROC and AUC may
be inappropriate; thus, AUPRC provides a sensible measure for all methods.
7.1.2. AUPRC
AUPRC has all the characteristics of the AUC; thus, for the purposes of this work,
we rely more on AUPRC than AUC He and Garcia (2008); Saito and Rehmsmeier (2015).
Overall, WCGAN-GP shows better improvements over SMOTE. WCGAN-GP is highest
on AUPRC, suggesting this algorithm performs the best across many thresholds and all the
data sets used. On the average, SMOTE does not provide a superior predictive performance
than the Baseline on all the metrics. Below, we further provide conclusive evidence on the
statistical significance of the above results on the AUPRC.
7.2. Statistical Hypothesis Testing
Table 9 shows the results of the Friedman test applied on AUPRC to verify the
statistical significance of WCGAN-GP compared to SMOTE.
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Table 9. Results for Friedman’s test.
Data Set
p-Value
Significance
Credit Card Fraud
Pima Indians Diabetes
German Credit Scoring
Glass Identification
Breast Cancer Wisconsin
2.9560 × 10−23
0.188386
1.0683 × 10−11
0.465622
4.0085 × 10−12
Yes
No
Yes
No
Yes
There is enough evidence at 5% significance level to reject the null hypothesis on 3
of the data sets, except German credit scoring and glass identification, suggesting that
over-sampling methods are not performing similarly and are different.
Since the null hypothesis was rejected for 3 of the data sets, a post-hoc test was applied
to further determine pairwise comparisons using the Nemenyi test where WCGAN-GP is
the control method. Table 10 shows the results of the post-hoc test.
Table 10. Results for the post-hoc test.
Test
Credit Card Fraud
German Credit
Breast Cancer
WCGAN-GP vs.
SMOTE
0.001
0.003
0.001
The above results confirm the significant superiority of WCGAN-GP over SMOTE
as all the p-values are less than 0.05 for the 3 data sets where Friedman’s test suggested
a difference. These results confirm the findings shown in Figure 6 and Table 8 where
the average performance seen on both the AUC and AUPRC was lower for SMOTE
compared to WCGAN-GP. In general, WCGAN-GP provides statistically significant better
performance on 3 of the 5 data sets.
8. Discussion
8.1. Results
Overall, SMOTE improves the AUC/AUPRC when applied on the imbalanced data
set but significantly penalizes Precision, leading to a lower AUPRC on 2 of the data sets
used. SMOTE samples synthetic points along line segments joining minority instances
using the Euclidean distance. This approach may end up using majority instances, thus
creating noisy examples and over-lapping cases Han et al. (2005). SMOTE is not based on
the true distribution of the minority class data Gao et al. (2014). The poor performance of
SMOTE (especially on Precision on the credit card fraud data set) may be attributed to these
effects. Overall, SMOTE alters the data distribution as was observed by the significant
compromise on Precision and generally lower F1-Score, AUPRC, and AUC values.
Other SMOTE variants, such as density-based approaches, are meant to improve the
above SMOTE weaknesses Bellinger et al. (2015); Gao et al. (2014). However, they make
strict assumptions about the structure and distribution of the minority class data. SMOTE
was the quickest to over-sample. WCGAN-GP requires a significant pre-training of both
the critic and the generator.
GANs are well-known for their training and computing powers Creswell et al. (2018);
Lucic et al. (2018). Thus, they have expensive run-times. However, current GANs, such as
WGAN and WGAN-GP, remedy this impact with stable training. The quality of generated
samples may be worth it compared to the training times. In this study, the GANs reached
stable training even for small samples, such as credit card fraud cases. This means that
GANs may still be used even for smaller data sets with enough training capacity.
Using WCGAN-GP to over-sample minority cases provided the best performance on
the AUPRC and on 3 of the data used on AUC. GANs do not make explicit assumptions
about the probability distribution of the minority class data. This idea has been used to
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create new samples in a number of data sets. Recent works on this Douzas and Bacao (2018);
Fiore et al. (2019); Mariani et al. (2018); Park et al. (2018); Ding et al. (2019) report GAN
superior performances over SMOTE for image data sets. There is a significant potential to
create new samples using GANs, leveraging them to augment limited actuarial data sets.
Given the current surge in interest for GANs, optimizing and training GANs is becoming straightforward as there are many implementations in Keras, Pytorch, and Tensorflow.
Thus, running times for GANs might not necessarily be an issue, enabling GANs to provide
a superior over-sampling approach to supplement imbalanced data sets. Because GANs
have become so popular, their limitations have been improved tremendously.
However, there are still open challenges for GANs. GANs rely on the generated
examples being completely differentiable with respect to the generative parameters. As a
result, GANs cannot product discrete data directly. Another key challenge is the evaluation
of GANs after training, even though there are measures to compute the quality of results
generated.
Research for GANs grows each year. Practitioners may need to add GANs to their
toolkit as this will significantly improve their models and aid on decision-making as GANs
will be characterized by advancements in deep learning, training process maturity, and
open acceptance and their wide use in commercial applications.
8.2. Implications for Actuaries
Given the superiority of GANs over other generative models and their wide applications, there is scope for actuarial use. The most obvious use is data augmentation and
boosting predictive models used for assumption setting, propensity modeling, pricing,
reserving, capital, and solvency projections, as demonstrated in the experiments conducted.
Using synthetic data sets created through GANs could allow actuaries to share salient
features of their data without sharing the full data set, enabling actuarial data sets to be
more widely available for public use and research purposes.
Given the surge in marketing and social promotions, info-graphics are the main ingredient of social media marketing. GANs can help marketers and designers in the creative
process. Other applications include anomaly detection, joint distribution learning, discriminative modeling, semi-supervised learning, domain adaptation, attention prediction, data
manipulation, missing data imputation, time series generation, privacy preservation, and
computer vision.
These GAN applications have potential actuarial adoption in insurance, banking,
health and care, and other non-traditional areas, as GANs have been shown to provide
better alternatives to current approaches. Research for GANs grows each year, and actuaries
may need to add GANs to their toolkit, as this will significantly improve their models and
aid on decision-making.
9. Conclusions, Limitations and Future Research
9.1. Conclusions
Gaining an advantage in competitive markets through offerings of suitable tailored
products on customers relies on building and maintaining adequate predictive models.
To build these models, a substantial amount of data and a sizeable number of records
is desirable. However, when expanding to new or unexplored markets, that level of
information is rarely always available. As a result, actuarial firms have to procure data
from a local provider, through purchasing reinsurance from a re-insurer, through limited
unsuitable industry and public research or rely from extrapolations from other betterknown markets. In this work, we show how an implicit model using GANs can alleviate
this problem through the generation of adequate quality data even from very limited small
samples, from difficult domains, or without alignment.
This example is a classic data augmentation application of GANs where we showed
their superiority of SMOTE and improving the original results. SMOTE improved the
classification performance. However, SMOTE is not based on the true underlying minority
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class distribution. SMOTE density estimation approaches remedy this issue; however, they
are not based on the true data distribution as they make strong data assumptions.
Using WCGAN-GP, it is possible to create synthetic cases implicitly, and this turned
out to offer a significantly better improvement over SMOTE. This work comprehensively
reviews GAN theory and applications in a number of domains, with possible adoption for
actuarial use. These applications have scope for actuarial science, and actuaries can add
them to their toolkit to aid predictive models.
9.2. Summary of Applications
In our opinion, the future of GANs will be characterized by open acceptance of
GANs and their applications by the research community and being used in commercial
applications. Given their impressive results and advancement in deep learning techniques,
we expect a wider use of GANs. The training instability of GANs will soon be done without
any problems as the maturity of the training improves with new techniques being invented
at a rapid speed.
There are potential applications of GANs for actuarial use in insurance, health and
care, banking, investment, and enterprise risk management to inter alia new sample creation, data augmentation, boosting predictive models, anomaly detection, semi-supervised
learning, attention prediction, time series generation, and missing data imputation. In conclusion, GANs have the potential to boost actuarial models and make better business
decisions.
9.3. Limitations and Future Research
We repeated training and testing of each over-sampling method 30 times to minimize
stochastic effects—this sample size can be increased for more robustness. Alternatively,
a bootstrapping approach can be applied to better understand the distributional attributes
of the model errors.
Given that we considered train-test split to split the data, changing this could potentially change the outcome of the results. Given that we have provided the significance of the
results using 30 multiple samples, this adds further comfort to the outcome. However, the
potential use of other data splitting methods, such as bootstrap, CV, and different train-test
splits, can enhance this work further. Different data splitting methods for different data
sets may provide further research work.
Our future work includes comparing current traditional actuarial approaches, such
as stochastic simulations and pricing models versus each GAN approach in each domain,
extensively. Time series GANs have been gaining interest in the past few years. An
interesting research area is using recurrent conditional GANs to simulate and project
mortality, compared with the traditional Lee-Carter model and its variants. Below are
possible future research to improve this work:
•
•
•
•
Consideration on other data sets to apply the same techniques, especially complex
data sets that include small disjuncts, over-lapping, mixed data types, and multiple
classes, particularly actuarial data sets.
Alternative consideration for other ML algorithms would show which ML technique
is best and for which data set and domain.
Empirical comparison of these results with other tabular data sets where GANs were
applied.
Implementation and leveraging of the GANs in R or Python for actuarial use.
Author Contributions: Conceptualization, K.S.N. and R.M.; methodology, K.S.N.; software, K.S.N.;
validation, K.S.N.; formal analysis, K.S.N.; investigation, K.S.N. and R.M.; resources, K.S.N.; writing—
original draft preparation, K.S.N.; writing—review and editing, R.M.; visualization, K.S.N.; supervision, R.M.; project administration, K.S.N. and R.M.; and funding acquisition, K.S.N. All authors have
read and agreed to the published version of the manuscript.
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Funding: This research was funded by the DSI-NICIS National e-Science Postgraduate Teaching and
Training Platform (NEPTTP) (http://www.escience.ac.za/).
Acknowledgments: The authors would like to thank the reviewers for their helpful comments.
The support of the DSI-NICIS National e-Science Postgraduate Teaching and Training Platform
(NEPTTP) towards this research is hereby acknowledged. Opinions expressed and conclusions
arrived at, are those of the author and are not necessarily to be attributed to the NEPTTP.
Conflicts of Interest: The authors declare that there are no conflict of interest regarding the publication of this paper. The funders had no role in the design of the study; in the collection, analyses, or
interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
References
Goodfellow, Ian, Jean Pouget-Abadie, Mehdi Mirza, Bing Xu, David Warde-Farley, Sherzil Ozair, Aaron Courville, and Yoshua Bengio.
2014. Generative adversarial nets. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 2672–80.
Ackley, David H., Geoffrey Everest Hinton, and Terrence Joseph Sejnowski. 1985. A learning algorithm for boltzmann machines.
Cognitive Science 9: 147–69. [CrossRef]
Akcay, Samet, Amir Atapour-Abarghouei, and Toby P. Breckon. 2018. Ganomaly: Semi-supervised anomaly detection via adversarial
training. In Asian Conference on Computer Vision. Cham: Springer, pp. 622–37.
Antoniou, Antreas, Amos Storkey, and Harrison Edwards. 2017. Data augmentation generative adversarial networks. arXiv,
arXiv:1711.04340.
Arjovsky, Martin, Soumith Chintala, and Leon Bottou. 2017. Wasserstein gan. arXiv, arXiv:1701.07875.
Armanious, Karim, Chenming Jiang, Marc Fischer, Thomas Küstner, Tobias Hepp, Konstantin Nikolaou, Sergio Gatidis, and Bin Yang.
2018. Medgan: Medical image translation using gans. arXiv, arXiv:1806.06397.
Batista, Gustavo Enrique, Ronaldo Cristiano Prati, and Maria-Carolina Monard. 2004. A study of the behavior of several methods for
balancing machine learning training data. ACM SIGKDD Explorations Newsletter 6: 20–29. [CrossRef]
Beaulieu-Jones, Brett K., Zhiwei Steven Wu, Chris Williams, Ran Lee, Sanjeev P. Bhavnani, James Brian Byrd, and Casey S. Greene.
2019. Privacy-preserving generative deep neural networks support clinical data sharing. Circulation: Cardiovascular Quality and
Outcomes 12: e005122. [CrossRef]
Bekkar, Mohamed, Hasiba Kheliouane Djemaa, and Akrouf Alitouche Taklit. 2013. Evaluation measures for models assessment over
imbalanced data sets. Journal of Information Engineering and Applications 3: 27–38.
Bellinger, Collin, Chris Drummond, and Nathalie Japkowicz. 2016. Beyond the boundaries of smote. In Joint European Conference on
Machine Learning and Knowledge Discovery in Databases. Berlin: Springer, pp. 248–63.
Bellinger, Collin, Nathalie Japkowicz, and Chris Drummond. 2015. Synthetic oversampling for advanced radioactive threat detection.
Paper presented at the IEEE 14th International Conference on Machine Learning and Applications (ICMLA), Miami, FL, USA,
December 9–11, pp. 948–53.
Bengio, Yoshua, Eric Thibodeau-Laufer, Guillaume Alain, and Jason Yosinski. 2014. Deep generative stochastic networks trainable by
backprop. Paper presented at the International Conference on Machine Learning, Bejing, China, June 22–24, pp. 226–34.
Bradley, Andrew P. 1997. The use of the area under the roc curve in the evaluation of machine learning algorithms. Pattern
Recognition 30: 1145–59. [CrossRef]
Brock, Andrew, Jeff Donahue, and Karen Simonyan. 2018. Large scale gan training for high fidelity natural image synthesis. arXiv,
arXiv:1809.11096.
Chawla, Nitesh Vijay. 2009. Data mining for imbalanced datasets: An overview. In Data Mining and Knowledge Discovery Handbook.
Berlin: Springer, pp. 875–86.
Chawla, Nitesh Vijay, Kevin Bowyer, Lawrence Hall, and W. Phillip Kegelmeyer. 2002. Smote: synthetic minority over-sampling
technique. Journal of Artificial Intelligence Research 16: 321–57. [CrossRef]
Chawla, Nitesh Vijay, Alecsander Lazarevic, Lawrence Hall, and Kevin Bowyer. 2003. Smoteboost: Improving prediction of the
minority class in boosting. In European Conference on Principles of Data Mining and Knowledge Discovery. Berlin: Springer, pp.
107–19.
Choi, Yunjei, Minje Choi, Munyoung Kim, Jung-Woo Ha, Sunghun Kim, and Jaegul Choo. 2018. Stargan: Unified generative adversarial
networks for multi-domain image-to-image translation. Paper presented at the IEEE Conference on Computer Vision and Pattern
Recognition, Salt Lake City, UT, USA, June 18–23, pp. 8789–97.
Chongxuan, Lee, Kun Xu, Jun Zhu, and Bo Zhang. 2017. Triple generative adversarial nets. In Advances in Neural Information Processing
Systems. Cambridge: The MIT Press, pp. 4088–98.
Creswell, Antonia, Tom White, Vincent Dumoulin, Kai Arulkumaran, Biswa Sengupta, and Anil Bharath. 2018. Generative adversarial
networks: An overview. IEEE Signal Processing Magazine 35: 53–65. [CrossRef]
Das, Barnan, Narayana Chatapuram Krishnan, and Diane J. Cook. 2015. Racog and wracog: Two probabilistic oversampling techniques.
IEEE Transactions on Knowledge and Data Engineering 27: 222–34. [CrossRef] [PubMed]
Ding, Zihan, Xiao-Yang Liu, Miao Yin, Wei Liu, and Linghe Kong. 2019. Tgan: Deep tensor generative adversarial nets for large image
generation. arXiv, arXiv:1901.09953.
Risks 2021, 9, 49
30 of 33
Douzas, George, and Fernando Bação. 2018. Effective data generation for imbalanced learning using conditional generative adversarial
networks. Expert Systems With Applications 91: 464–71. [CrossRef]
Dua, Dheeru, and Graff Casey. 2017. UCI Machine Learning Repository. University of California, Irvine, School of Information and
Computer Sciences. Available online: https://archive.ics.uci.edu/ml (accessed on 18 May 2019).
Esteban, Cristóbal, Stephanie L. Hyland, and Gunnar Rätsch. 2017. Real-valued (medical) time series generation with recurrent
conditional gans. arXiv, arXiv:1706.02633.
Ester, Martin, Hans-Peter Kriegel, Joerg Sander, and Xiaowei Xu. 1996. A density-based algorithm for discovering clusters in large
spatial databases with noise. Kdd 96: 226–31.
Evett, I. W., and E. J. Spiehler. 1987. Rule induction in forensic science. In KBS in Goverment. Burlington: Jones & Bartlett Publishers,
pp. 107–18.
Fernández, Alberto, Salvador Garcia, Francisco Herrera, and Nitesh Vijay Chawla. 2018. Smote for learning from imbalanced data:
progress and challenges, marking the 15-year anniversary. Journal of Artificial Intelligence Research 61: 863–905. [CrossRef]
Fiore, Ugo, Alfredo De Santis, Francesca Perla, Paolo Zanetti, and Francesco Palmieri. 2019. Using generative adversarial networks for
improving classification effectiveness in credit card fraud detection. Information Sciences 479: 448–55. [CrossRef]
François, Chollet. 2015. keras. Available online: https://github.com/fchollet/keras (accessed on 20 August 2019).
Frey, Brendan J., Geoffrey Everest Hinton, and Peter Dayan. 1996. Does the wake-sleep algorithm produce good density estimators?
In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 661–667.
Friedman, Jerome, Trevor Hastie, and Robert Tibshirani. 2001. The Elements Of Statistical Learning. Springer Series in Statistics New
York. New York: Springer, vol. 1.
Friedman, Milton. 1937. The use of ranks to avoid the assumption of normality implicit in the analysis of variance. Journal of the
American Statistical Association 32: 675–701. [CrossRef]
Fu, Rao, Jie Chen, Shutian Zeng, Yiping Zhuang, and Agus Sudjianto. 2019. Time series simulation by conditional generative
adversarial net. arXiv, arXiv:1904.11419.
Ganganwar, Vaishali. 2012. An overview of classification algorithms for imbalanced datasets. International Journal of Emerging
Technology and Advanced Engineering 2: 42–47.
Gao, Ming, Xia Hong, Sheng Chen, C. J. Harris, and Emad Khalaf. 2014. Pdfos: Pdf estimation based over-sampling for imbalanced
two-class problems. Neurocomputing 138: 248–59. [CrossRef]
Geyer, Charles J. 1992. Practical markov chain monte carlo. Statistical Science 7: 473–83. [CrossRef]
Glorot, Xavier, Antoine Bordes, and Yoshua Bengio. 2011. Deep sparse rectifier neural networks. Paper presented at the Fourteenth
International Conference on Artificial Intelligence and Statistics, Fort Lauderdale, FL, USA, April 11–13, pp. 315–23.
Goodfellow, Ian. 2016. Nips 2016 tutorial: Generative adversarial networks. arXiv, arXiv:1701.00160.
Goodfellow, Ian, Yoshua Bengio, and Aaron Courville. 2016. Deep Learning. Cambridge: MIT Press, vol. 1.
Gulrajani, Ishaan, Faruk Ahmed, Martin Arjovsky, Vincent Dumoulin, and Aaron Courville. 2017. Improved training of wasserstein
gans. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 5767–77.
Han, Hui, Wenyuan Wang, and Binghuan Mao. 2005. Borderline-smote: A new over-sampling method in imbalanced data sets
learning. In International Conference on Intelligent Computing. Berlin: Springer, pp. 878–87.
Hanley, James A, and Barbara J. McNeil. 1982. The meaning and use of the area under a receiver operating characteristic (roc) curve.
Radiology 143: 29–36. [CrossRef] [PubMed]
Hartigan, J. A., and M. A. Wong. 1979. Algorithm as 136: A k-means clustering algorithm. Journal of the Royal Statistical Society. Series c
(Applied Statistics) 28: 100–8. [CrossRef]
He, Haibo, and Edwardo A. Garcia. 2008. Learning from imbalanced data. IEEE Transactions on Knowledge & Data Engineering 21:
1263–84.
He, Kaiming, Xiangyu Zhang, Shaoqing Ren, and Jain Sun. 2015. Delving deep into rectifiers: Surpassing human-level performance on
imagenet classification. Paper presented at the IEEE International Conference on Computer Vision, Santiago, Chile, December
7–13, pp. 1026–34.
Hinton, Geoffrey Everest. 2002. Training products of experts by minimizing contrastive divergence. Neural Computation 14: 1771–800.
[CrossRef]
Hinton, Geoffrey Everest, Simon Osindero, and Yee-Why Teh. 2006. A fast learning algorithm for deep belief nets. Neural
Computation 18: 1527–54. [CrossRef]
Hinton, Geoffrey Everest, and Ruslan Russ Salakhutdinov. 2006. Reducing the dimensionality of data with neural networks. Science 313:
504–7. [CrossRef]
Hinton, Geoffrey Everest, and Tiejman. Tieleman. 2012. Lecture 6.5—Rmsprop, Coursera: Neural Networks for Machine Learning.
Available online: https://www.cs.toronto.edu/~tijmen/csc321/slides/lecture_slides_lec6.pdf (accessed on 28 August 2019).
Hitawala, Saifuddin. 2018. Comparative study on generative adversarial networks. arXiv, arXiv:1801.04271.
Hoffman, Judy, Eric Tzeng, Taesung Park, Jun-Yan Zhu, Phillip Isola, Kate Saenko, Alexei Efros, and Trevor Darrell. 2017. Cycada:
Cycle-consistent adversarial domain adaptation. arXiv, arXiv:1711.03213.
Hong, Weixiang, Zhenzhen Wang, Ming Yang, and Junsong Yuan. 2018. Conditional generative adversarial network for structured
domain adaptation. Paper presented at the IEEE Conference on Computer Vision and Pattern Recognition, Salt Lake City, UT,
USA, June 18–23, pp. 1335–44.
Risks 2021, 9, 49
31 of 33
Hong, Yongjun, Uiwon Hwang, Jaeyoon Yoo, and Sungroh Yoon. 2019. How generative adversarial networks and their variants work:
An overview of GAN. ACM Computing Surveys (CSUR) 52: 10. [CrossRef]
Ioffe, Sergey, and Christian Szegedy. 2015. Batch normalization: Accelerating deep network training by reducing internal covariate
shift. arXiv, arXiv:1502.03167.
Jolicoeur-Martineau, Alexia. 2018. The relativistic discriminator: A key element missing from standard gan. arXiv, arXiv:1807.00734.
Karras, Terro, Samuli Laine, and Timo Aila. 2019. A style-based generator architecture for generative adversarial networks. Paper
presented at the IEEE Conference on Computer Vision and Pattern Recognition, Long Beach, CA, USA, June 15–20, pp. 4401–10.
Kim, Taeskoo, Moonsu Cha, Hyunsoo Kim, Jungkwon Lee, and Jiwon Kim. 2017. Learning to discover cross-domain relations with
generative adversarial networks. Paper presented at 34th International Conference on Machine Learning, Sydney, Australia,
August 6–11, vol. 70, pp. 1857–65.
Kingma, Diederik Pieter, and Jimmy Ba. 2014. Adam: A method for stochastic optimization. arXiv, arXiv:1412.6980.
Kingma, Diederik Pieter, and Max Welling. 2013. Auto-encoding variational bayes. arXiv, arXiv:1312.6114.
Kullback, Solomon. 1997. Information Theory and Statistics. North Chelmsford: Courier Corporation.
Kullback, Solomon, and Richard Arthur Leibler. 1951. On information and sufficiency. The Annals of Mathematical Statistics 22: 79–86.
[CrossRef]
Larsen, Anders Boesen Lindbo, Søren Kaae Sønderby, Hugo Larochelle, and Ole Winther. 2015. Autoencoding beyond pixels using a
learned similarity metric. arXiv, arXiv:1512.09300.
Lee, Ronald D., and Lawrence R. Carter. 1992. Modeling and forecasting us mortality. Journal of the American Statistical Association 87:
659–71.
Li, Stevan Cheng-Xian, Bo Jiang, and Benjamin Marlin. 2019. Misgan: Learning from incomplete data with generative adversarial
networks. arXiv, arXiv:1902.09599.
Li, Yujia, Kevin Swersky, and Richard Zemel. 2015. Generative moment matching networks. Paper presented at the 32nd International
Conference on Machine Learning, Lille, France, July 6–11, pp. 1718–27.
Lin, Jianhua. 1991. Divergence measures based on the shannon entropy. IEEE Transactions on Information Theory 37: 145–51. [CrossRef]
Liu, Zhiyue, Jiahai Wang, and Zhiwei Liang. 2019. Catgan: Category-aware generative adversarial networks with hierarchical
evolutionary learning for category text generation. arXiv, arXiv:1911.06641.
Longadge, Rushi, and Snehalata Dongre. 2013. Class imbalance problem in data mining review. arXiv, arXiv:1305.1707.
López, Victoria, Alberto Fernández, Salvado García, Vasile Palade, and Francisco Herrera. 2013. An insight into classification with
imbalanced data: Empirical results and current trends on using data intrinsic characteristics. Information Sciences 250: 113–41.
[CrossRef]
Lucic, Mario, Karol Kurach, Marcin Michalski, Sylvain Gelly, and Olivier Bousquet. 2018. Are gans created equal? A large-scale study.
In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 700–9.
Van der Maaten, Laurens, and Geoffrey Everest Hinton. 2008. Visualizing data using t-sne. Journal of Machine Learning Research 9:
2579–605.
Makhzani, Alireza, Jonathon Shlens, Navdeep Jaitly, Ian Goodfellow, and Brendan Frey. 2015. Adversarial autoencoders. arXiv,
arXiv:1511.05644.
Manisha, Padala, and Sujit Gujar. 2018. Generative adversarial networks (gans): What it can generate and what it cannot? arXiv,
arXiv:1804.00140.
Mariani, Giovanni, Florian Scheidegger, Roxana Istrate, Costas Bekas, and Cristiano Malossi. 2018. Bagan: Data augmentation with
balancing gan. arXiv, arXiv:1803.09655.
Mathew, Josey, Ming Luo, Chee Khiang Pang, and Hian Leng Chan. 2015. Kernel-based smote for svm classification of imbalanced
datasets. Paper presented at the IECON 2015-41st Annual Conference of the IEEE Industrial Electronics Society, Yokohama,
Japan, November 9–12, pp. 1127–32.
McCullagh, Peter. 1984. Generalized linear models. European Journal of Operational Research 16: 285–92. [CrossRef]
Mirza, Mehdi, and Simon Osindero. 2014. Conditional generative adversarial nets. arXiv, arXiv:1411.1784.
Mitchell, Tom M. 2006. The Discipline of Machine Learning. Pittsburgh: Machine Learning Department, School of Computer Science,
Carnegie Mellon University, vol. 9.
Miyato, Takeru, Shin-ichi Maeda, Masanori Koyama, and Shin Ishii. 2018. Virtual adversarial training: a regularization method for
supervised and semi-supervised learning. IEEE Transactions on Pattern Analysis and Machine Intelligence 41: 1979–93. [CrossRef]
[PubMed]
Mottini, Alejandro, Alix Lheritier, and Rodrigo Acuna-Agost. 2018. Airline passenger name record generation using generative
adversarial networks. arXiv, arXiv:1807.06657.
Müller, Alfred. 1997. Integral probability metrics and their generating classes of functions. Advances in Applied Probability 29: 429–43.
Nemenyi, Peter. 1962. Distribution-free multiple comparisons. In Biometrics. Princeton: Princeton University, vol. 18, p. 263.
Nowozin, Sebastian, Botond Cseke, and Ryota Tomioka. 2016. f-gan: Training generative neural samplers using variational divergence
minimization. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 271–279.
Odena, Augustus 2016. Semi-supervised learning with generative adversarial networks. arXiv, arXiv:1606.01583.
Odena, Augustus, Christopher Olah, and Jonathon Shlens. 2017. Conditional image synthesis with auxiliary classifier gans. Paper
presented at the 34th International Conference on Machine Learning, Sydney, Australia, August 6–11, vol. 70, pp. 2642–51.
Risks 2021, 9, 49
32 of 33
O’Malley, B. R., Robert E. Dorrington, Stephen C. Jurisich, J. A. Valentini, T. M. Cohen, and B. J. Ross. 2005. An investigation of the
mortality of south african assured lives, 1995–1998. South African Actuarial Journal 5: 27–59. [CrossRef]
Park, Noseong, Mahmoud Mohammadi, Kshitij Gorde, Sushil Jajodia, Hongkyu Park, and Youngmin Kim. 2018. Data synthesis based
on generative adversarial networks. Proceedings of the Very Large Data Bases Endowment 11: 1071–83. [CrossRef]
Pohlert, Thorsten 2014. The pairwise multiple comparison of mean ranks package (pmcmr). R Package 27: 10.
Pozzolo, Andrea Dal. 2015. Adaptive Machine Learning for Credit Card Fraud Detection. Edited by Machine Learning Group. Brussels:
Université Libre de Bruxelles.
Python Software Foundation. 2017. Python Language Reference (Version 3.6. 3.). Amsterdam: Python Software Foundation.
Qian, Ning. 1999. On the momentum term in gradient descent learning algorithms. Neural Networks 12: 145–51. [CrossRef]
Radford, Alec, Luke Metz, and Soumith Chintala. 2015. Unsupervised representation learning with deep convolutional generative
adversarial networks. arXiv, arXiv:1511.06434.
Reed, Scott, Zeynep Akata, Xinchen Yan, Lajanugen Logeswaran, Bernt Schiele, and Honglak Lee. 2016. Generative adversarial text to
image synthesis. arXiv, arXiv:1605.05396.
Rezende, Danilo Jimenez, Shakir Mohamed, and Daan Wierstra. 2014. Stochastic backpropagation and approximate inference in deep
generative models. arXiv, arXiv:1401.4082.
Rubin, Donald Bruce. 2004. Multiple Imputation for Nonresponse in Surveys. Hoboken: John Wiley & Sons, vol. 81.
Rubner, Yossi, Carlo Tomasi, and Leonidas Guibas. 2000. The earth mover’s distance as a metric for image retrieval. International
Journal of Computer Vision 40: 99–121. [CrossRef]
Ruder, Sebastian. 2016. An overview of gradient descent optimization algorithms. arXiv, arXiv:1609.04747.
Rumelhart, David Everett, Geoffrey Everest Hinton, and Ronald J. Williams. 1986. Learning representations by back-propagating
errors. Nature 323: 533. [CrossRef]
Saito, Takaya, and Mark Rehmsmeier. 2015. The precision-recall plot is more informative than the roc plot when evaluating binary
classifiers on imbalanced datasets. PLoS ONE 10: e0118432. [CrossRef]
Salakhutdinov, Ruslan Russ, and Geoffrey Everest Hinton. 2009. Deep boltzmann machines. In Artificial Intelligence and Statistics.
Berlin: Springer, pp. 448–55.
Salimans, Tim, Ian Goodfellow, Wojciech Zaremba, Vicki Cheung, Alec Radford, and Xi Chen. 2016. Improved techniques for training
gans. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 2234–42.
Schafer, Joseph L., and Maren K. Olsen. 1998. Multiple imputation for multivariate missing-data problems: A data analyst’s perspective.
Multivariate Behavioral Research 33: 545–71. [CrossRef]
Schlegl, Thomas, Philipp Seeböck, Sebastian M. Waldstein, Ursula Schmidt-Erfurth, and Georg Langs. 2017. Unsupervised anomaly
detection with generative adversarial networks to guide marker discovery. In International Conference on Information Processing in
Medical Imaging. Cham: Springer, pp. 146–157.
Shang, Chao, Aaron Palmer, Jiangwen Sun, Ko-Shin Chen, Jin Lu, and Jinbo Bi. 2017. Vigan: Missing view imputation with generative
adversarial networks. Paper presented at 2017 IEEE International Conference on Big Data (Big Data), Boston, MA, USA, December
11–14, pp. 766–75.
Smith, Jack W., James E. Everhart, W. C. Dickson, William C. Knowler, and Robert Scott Johannes. 1988. Using the adap learning
algorithm to forecast the onset of diabetes mellitus. In Proceedings of the Annual Symposium on Computer Application in Medical Care.
Bethesda: American Medical Informatics Association, pp. 261–65.
Sricharan, Kumar, Raja Bala, Matthew Shreve, Hui Ding, Kumar Saketh, and Jin Sun. 2017. Semi-supervised conditional gans. arXiv,
arXiv:1708.05789.
Street, W. Nick, William H. Wolberg, and O. L. Mangasarian. 1993. Nuclear feature extraction for breast tumor diagnosis. In Biomedical
Image Processing and Biomedical Visualization. Washington, DC: International Society for Optics and Photonics, vol. 1905, pp.
861–70.
Tzeng, Eric, Judy Hoffman, Kate Saenko, and Trevor Darrell. 2017. Adversarial discriminative domain adaptation. Paper presented at
the IEEE Conference on Computer Vision and Pattern Recognition, Honolulu, HI, USA, July 21–26, pp. 7167–76.
Van den Oord, Aaron, Nal Kalchbrenner, Lasse Espeholt, Oriol Vinyals, and Alex Graves. 2016. Conditional image generation with
pixelcnn decoders. In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 4790–4798.
Vondrick, Carl, Hamed Pirsiavash, and Antonio Torralba. 2016. Generating videos with scene dynamics. In Advances in Neural
Information Processing Systems. Cambridge: The MIT Press, pp. 613–621.
Voorhees, ELLEN M. 1986. Implementing agglomerative hierarchic clustering algorithms for use in document retrieval. Information
Processing & Management 22: 465–76.
Wang, Kunfeng, Chao Gou, Yanjie Duan, Yilun Lin, Xinhu Zheng, and Fei-Yue Wang. 2017. Generative adversarial networks:
Introduction and outlook. IEEE/CAA Journal of Automatica Sinica 4: 588–98. [CrossRef]
Wang, Shuo, and Xin Yao. 2009. Diversity analysis on imbalanced data sets by using ensemble models. Paper presented at 2009 IEEE
Symposium on Computational Intelligence and Data Mining, Nashville, TN, USA, March 30–April 2, pp. 324–31.
Xu, Lei, Maria Skoularidou, Alfredo Cuesta-Infante, and Kalyan Veeramachaneni. 2019. Modeling tabular data using conditional gan.
In Advances in Neural Information Processing Systems. Cambridge: The MIT Press, pp. 7335–45.
Yang, Li-Chia, Szu-Yu Chou, and Yi-Hsuan Yang. 2017. Midinet: A convolutional generative adversarial network for symbolic-domain
music generation. arXiv, arXiv:1703.10847.
Risks 2021, 9, 49
33 of 33
Yi, Zili, Hao Zhang, Ping Tan, and Minglun Gong. 2017. Dualgan: Unsupervised dual learning for image-to-image translation. Paper
presented at the IEEE International Conference on Computer Vision, Venice, Italy, October 22–29, pp. 2849–57.
Yoon, Jinsung, Daniel Jarrett, and Mihaela Van der Schaar. 2019. Time-series generative adversarial networks. In Advances in Neural
Information Processing Systems. Cambridge: The MIT Press, pp. 5508–18.
Yoon, Jinsung, James Jordon, and Mihaela Van Der Schaar. 2018. Gain: Missing data imputation using generative adversarial nets.
arXiv, arXiv:1806.02920.
Zhang, Huaxiang, and Mingfang Li. 2014. Rwo-sampling: A random walk over-sampling approach to imbalanced data classification.
Information Fusion 20: 99–116. [CrossRef]
Zhou, Xingyu, Zhisong Pan, Guyu Hu, Siqi Tang, and Cheng Zhao. 2018. Stock market prediction on high-frequency data using
generative adversarial nets. Mathematical Problems in Engineering 2018: 4907423. [CrossRef]
Zhu, Jun-Yan, Taesung Park, Phillip Isola, and Alexei A. Efros. 2017. Unpaired image-to-image translation using cycle-consistent
adversarial networks. Paper presented the IEEE International Conference on Computer Vision, Venice, Italy, October 22–29,
pp. 2223–32.
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Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer
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Ambrose, Sarah; Gordon, Naheema; Goldsmith, James; Wei, Wenbin; Zeegers, Maurice; Document Version
Publisher's PDF, also known as Version of record Citation for published version (Harvard):
Ambrose, S, Gordon, N, Goldsmith, J, Wei, W, Zeegers, M, James, N, Knowles, M, Bryan, R & Ward, D 2015,
'Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients
with Bladder Cancer', Proteomes, vol. 3, no. 3, pp. 266-282. https://doi.org/10.3390/proteomes3030266 Link to publication on Research at Birmingham portal Link to publication on Research at Birmingham portal Publisher Rights Statement: Citation for published version (Harvard):
Ambrose, S, Gordon, N, Goldsmith, J, Wei, W, Zeegers, M, James, N, Knowles, M, Bryan, R & Ward, D 2015,
'Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients
with Bladder Cancer', Proteomes, vol. 3, no. 3, pp. 266-282. https://doi.org/10.3390/proteomes3030266 Proteomes 2015, 3, 266-282; doi:10.3390/proteomes3030266
Article
OPEN ACCESS
proteomes
ISSN 2227-7382
www.mdpi.com/journal/proteomes
Use of Aleuria alantia Lectin Affinity Chromatography to
Enrich Candidate Biomarkers from the Urine of Patients
with Bladder Cancer
Sarah R. Ambrose 1, Naheema S. Gordon 1, James C. Goldsmith 1, Wenbin Wei 1,
Maurice P. Zeegers 1,2, Nicholas D. James 3, Margaret A. Knowles 4, Richard T. Bryan 1
and Douglas G. Ward 1,*
1 School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK;
E-Mails: SRA295@student.bham.ac.uk (S.R.A.); n.gordon@bham.ac.uk (N.S.G.);
jgoldsmith38@googlemail.com (J.C.G.); w.wei@bham.ac.uk (W.W.);
m.zeegers@maastrichtuniversity.nl (M.P.Z.); r.t.bryan@bham.ac.uk (R.T.B.)
2 Department of Complex Genetics, NUTRIM School of Nutrition and Translational Research in
Metabolism, Maastricht University Medical Centre, Maastricht 6200 MD, The Netherlands
3 Clinical Trials Unit, University of Warwick, Coventry CV4 7AL, UK;
E-Mail: N.D.James@warwick.ac.uk
4 Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James’s’ University
Hospital, Beckett Street, Leeds LS9 7TF, UK; E-Mail: M.A.Knowles@leeds.ac.uk
* Author to whom correspondence should be addressed; E-Mail: d.g.ward@bham.ac.uk;
Tel.: +44-121-414-9528.
Academic Editor: Michael A. Tainsky
Received: 30 June 2015 / Accepted: 27 August 2015 / Published: 3 September 2015
Abstract: Developing a urine test to detect bladder tumours with high sensitivity and
specificity is a key goal in bladder cancer research. We hypothesised that bladder
cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the
binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer
patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins
from the urine of bladder cancer patients and control subjects for analysis by shotgun
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ll li Proteomes 2015, 3, 266-282; doi:10.3390/proteomes3030266 Take down policy Take down policy
While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been
uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to
the work immediately and investigate. Download date: 24. Oct. 2024 1. Introduction Urothelial bladder cancer is the fourth most common cancer in men and ninth most common cancer
in women in western societies [1]. This highly heterogeneous disease presents as a spectrum from
low-grade non-invasive tumours with a good prognosis but high recurrence rate through to high-grade
muscle invasive tumours with a very poor prognosis. Low grade tumours tend to have a near-normal
karyotype with few genomic rearrangements and often have activating mutations in FGFR3 and the
MAPK pathway, whereas high-grade tumours typically have inactivating mutations in TP53 and/or
other tumour suppressor genes and multiple chromosomal aberrations [2]. Algorithms based on
clinicopathological factors can be used to guide treatment, which ranges from transurethral resection
and surveillance for recurrence for low-risk disease through to cystectomy and systemic chemotherapy
for muscle-invasive and metastatic disease [3]. Bladder tumours are typically detected by flexible cystoscopy, a burdensome and resource-intensive
procedure. Patients undergoing surveillance for bladder cancer will require this procedure at regular
intervals for many years [4]. There is thus a need for a urine or blood-based test to reduce reliance on
cystoscopy. Despite extensive research, most candidate urinary biomarkers for bladder cancer do not
show sufficient sensitivity and specificity to replace cystoscopy [5]. Most of the proposed biomarkers
are particularly poor at detecting low-grade NMIBC. Indeed, no urinary biomarkers have been
validated as having sufficient sensitivity and specificity to be widely adopted in clinical practice [5]. A number of urinary biomarkers have been proposed for the detection of bladder cancer including
tests based on miRNA [6], RNA [7], DNA [8], metabolites [9] and proteins [10]. The latter may be
measured in exfoliated cells (e.g., ImmunoCyt®) or as soluble proteins in the urine e.g., NMP22 and
BTA. Unfortunately, none of these tests are both highly specific and sensitive for early stage and low
grade disease. Nucleic acid tests based on DNA methylation and mutations have the advantage over
other biomarkers in identifying the presence of a disease-specific variant of the molecule that is being
detected, rather than the total level of a molecule which may be released from both cancer and normal
cells. Because bladder cancer is highly heterogeneous at the molecular level it is likely that a panel of
biomarkers will be required to detect all tumours. Theoretically, highly specific markers can be
combined to generate an effective test. Proteomes 2015, 3 267 Proteomes 2015, 3 glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity
chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1
as proteins warranting further investigation as urinary biomarkers for low-grade bladder
cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring
lectin binding to unfractionated proteomes, but it is possible that more specific reagents
and/or a focus on individual proteins may produce clinically useful biomarkers. Keywords: bladder cancer; urine; biomarker; lectin; glycoproteome Keywords: bladder cancer; urine; biomarker; lectin; glycoproteome Keywords: bladder cancer; urine; biomarker; lectin; glycoproteome Use of Aleuria alantia Lectin Affinity Chromatography to
Enrich Candidate Biomarkers from the Urine of Patients
with Bladder Cancer Sarah R. Ambrose 1, Naheema S. Gordon 1, James C. Goldsmith 1, Wenbin Wei 1,
Maurice P. Zeegers 1,2, Nicholas D. James 3, Margaret A. Knowles 4, Richard T. Bryan 1
and Douglas G. Ward 1,* Sarah R. Ambrose 1, Naheema S. Gordon 1, James C. Goldsmith 1, Wenbin Wei 1,
Maurice P. Zeegers 1,2, Nicholas D. James 3, Margaret A. Knowles 4, Richard T. Bryan 1
and Douglas G. Ward 1,* 1 School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK;
E-Mails: SRA295@student.bham.ac.uk (S.R.A.); n.gordon@bham.ac.uk (N.S.G.);
jgoldsmith38@googlemail.com (J.C.G.); w.wei@bham.ac.uk (W.W.);
m.zeegers@maastrichtuniversity.nl (M.P.Z.); r.t.bryan@bham.ac.uk (R.T.B.) 1 School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK;
E-Mails: SRA295@student.bham.ac.uk (S.R.A.); n.gordon@bham.ac.uk (N.S.G.);
jgoldsmith38@googlemail.com (J.C.G.); w.wei@bham.ac.uk (W.W.);
m.zeegers@maastrichtuniversity.nl (M.P.Z.); r.t.bryan@bham.ac.uk (R.T.B.) 2 Department of Complex Genetics, NUTRIM School of Nutrition and Translational Research in
Metabolism, Maastricht University Medical Centre, Maastricht 6200 MD, The Netherlands 3 Clinical Trials Unit, University of Warwick, Coventry CV4 7AL, UK;
E-Mail: N.D.James@warwick.ac.uk 4 Section of Experimental Oncology, Leeds Institute of Cancer and Pathology, St James’s’ University
Hospital, Beckett Street, Leeds LS9 7TF, UK; E-Mail: M.A.Knowles@leeds.ac.uk * Author to whom correspondence should be addressed; E-Mail: d.g.ward@bham.ac.uk;
Tel.: +44-121-414-9528. Academic Editor: Michael A. Tainsky Academic Editor: Michael A. Tainsky Received: 30 June 2015 / Accepted: 27 August 2015 / Published: 3 September 2015 Received: 30 June 2015 / Accepted: 27 August 2015 / Published: 3 September 2015 Abstract: Developing a urine test to detect bladder tumours with high sensitivity and
specificity is a key goal in bladder cancer research. We hypothesised that bladder
cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the
binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer
patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins
from the urine of bladder cancer patients and control subjects for analysis by shotgun
proteomics. None of the lectins showed a strong preference for bladder cancer cell lines
over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over
those from non-cancer controls. However, several lectins showed a strong preference for
bladder cell line glycans over serum glycans and are potentially useful for enriching Proteomes 2015, 3 Numerous proteomic approaches have been applied to analyse urine in the search for bladder cancer
biomarkers (reviewed in [10]). The urinary proteome is challenging to mine in depth, in part due to an
abundance of plasma proteins, and many proteomic studies have suggested plasma proteins as biomarkers
despite the fact that they are unlikely to be specific for bladder cancer. For example, Chen et al. carried
out quantitative shotgun proteomics on pooled bladder cancer urine samples and non-cancer controls
using iTRAQ for relative quantitation, followed by MRM quantitation of candidate biomarkers
producing a multimarker panel with a ROC AUC of 0.814 [11,12]. The “biomarkers”, however, are
moderately abundant plasma proteins rather than cancer-specific proteins. Top-down approaches have
the ability to detect proteoforms not readily distinguishable in bottom-up approaches. However, to
date, CE-MS and MALDI based profiling have failed to generate a highly sensitive and specific test
for bladder cancer [13–15]. A small number of studies have used lectin affinity chromatography in
studies of the urinary glycoproteome, however these studies have used broad specificity lectins
(expected to capture most glycoproteins) rather than focussing on alterations in glycosylation [16,17]. Kreunin et al. [16] compared the glycoproteome of urine from bladder cancer patients and non-cancer
patients using Concanavalin A (ConA) affinity chromatography combined with nano-liquid
chromatography-tandem mass spectrometry (LC-MS/MS). Alpha-1β-glycoprotein was identified as
the most discriminatory protein, but again, this is a plasma protein. It has been reported that protein glycosylation is significantly altered in many cancers including
bladder cancer [18]. Alterations in glycosylation patterns in cancer reflect changes in expression of
glycosyltransferases and glycosidases [19]. The changes to glycan structure that can occur in cancer
include O-glycan truncation, increased branching of N-glycans, and increased sialylation, sulfation and
fucosylation [18]. There have been several studies on the glycosylation state of specific proteins found
in the sera of cancer patients. For example, increased fucosylation and sialylation of PSA have been
reported in prostate cancer [20]. Wu and colleagues used a fucose specific lectin, Aleuria aurantia
lectin (AAL) to characterise the fucosylation of haptoglobin in ovarian cancer and found increased
levels of fucosylated haptoglobin in patient sera [21]. Another study observed glycan-specific changes
in periostin and thrombospondin in ovarian cancer [22]. All of these studies found that the specific
protein glycoforms were able to better differentiate cancer sera from control serum samples than the
total concentration of the protein. 1. Introduction However, total levels of protein markers may be influenced by
non-malignant conditions and haematuria, limiting specificity and therefore suitability for inclusion in
multimarker tests. A test based on bladder cancer-specific variants of proteins would be expected to
outperform a test based on the total levels of these proteins. 268 2.2. Patient Samples Patient urine and serum samples were collected as part of the Bladder Cancer Prognosis Programme
(BCPP) [25]. Recruitment to BCPP was undertaken between 2005 and 2011 and consists of samples
from patients with suspected primary bladder cancer. Midstream urine was collected, centrifuged at
2000 rpm for 10 min and the supernatant stored in aliquots at −80°C. After sample collection, each
patient underwent TURBT and definitive diagnosis by histopathological examination of the resected
tissue. Urine samples obtained from patients with non-malignant conditions were retained in the BCPP
collection and have been used as non-cancer controls. For the lectin ELISA experiments, urine samples
were pooled into four control pools (two normal and two from patients with cystitis or inflammation)
and six cancer pools (two from each of stages pTa, pT1 and pT2+). For the proteomics experiments,
two pools of pTa patient urine were used (n = 75; n = 36) and a non-cancer control urine pool (n = 28). All samples used were negative for haematuria by dipstick test. Further patient information is provided
in Supplemental Materials. 2.1. Materials and Cell Lines All lectins were purchased from Vector Laboratories Ltd. (Peterborough, UK). All other chemicals
and materials were purchased from Sigma-Aldrich (St. Louis, MI, USA) unless otherwise stated. All lectins were purchased from Vector Laboratories Ltd. (Peterborough, UK). All other chemicals
and materials were purchased from Sigma-Aldrich (St. Louis, MI, USA) unless otherwise stated. The bladder cancer cell lines 5637 and HB-CLS-2 were purchased from CLS Cell Lines Service
GmbH (Eppelheim, Germany). NHU-TERT, VM-CUB-1, MGH-U3, RT4, RT112, SW780 and T24
were provided by Professor Margaret Knowles (University of Leeds, Leeds, UK). The UROtsa cell
line was a gift from Alexander Dowell (University of Birmingham, Birmingham, UK). Cell lines were
cultured as previously described [24]. The bladder cancer cell lines were derived from tumours of
different grades as detailed in Supplemental Table S1. Cell lysates were prepared by sonication in PBS
containing cOmplete EDTA-free protease inhibitor cocktail followed by a 10 min centrifugation at
13,000 rpm. The protein concentration of the supernatant was determined by Bradford assay. Proteomes 2015, 3 Perhaps the best example of a cancer specific glycoform is AFP-L3. An increase in total AFP concentration in the serum was originally used as an indicator for
hepatocellular carcinoma (HCC); however, measuring the total AFP concentration cannot always
discriminate between small HCCs and chronic liver disease. Further study of the protein identified a
core-fucosylated form of AFP known as AFP-L3 which is specific to HCC and which can be measured
in the serum to distinguish between HCC and chronic liver disease, making it a clinically useful
biomarker [23]. Identifying a similarly cancer-specific glycoprotein biomarker for bladder cancer
could be the answer to finding an accurate non-invasive test for disease detection and long-term
surveillance of patients. In the experiments reported here we test the hypothesis that incorporating selective lectin
chromatography into urinary proteomics workflows has the potential to uncover urinary biomarkers for
bladder cancer. We focus on low grade non-invasive bladder cancer as this is the form of the disease
which is most challenging to detect using currently available non-invasive tests. 269 Proteomes 2015, 3 Proteomes 2015, 3 2.6. Filter-Aided Sample Preparation and Tryptic Digestion Up to 200 µg of protein was dissolved in 9 M Urea, 1% CHAPS (Melford Laboratories, Ipswich, UK)
in 100 mM triethylammonium bicarbonate (TEAB), and incubated with 20 mM dithiothreitol (DTT)
for 30 min at room temp. Following addition of 50 mM iodoacetamide the proteins were captured in
0.5 mL 30 kDa MWCO centrifugal filters, centrifuged at 13,000 rpm for five minutes and washed four
times with 100 mM TEAB. Proteins were digested by incubating overnight at 37 °C with 5 μg of
sequencing grade trypsin (Promega). Peptides were collected by centrifugation. 2.4. Lectin Dot Blots Samples were diluted (cell lysate or serum diluted to 3 ug protein/mL in PBS and urine diluted ×50
in PBS and 2 μL spotted onto nitrocellulose membrane. Once dry, the membrane was blocked with 1%
BSA in PBS for 40 min and then washed three times with PBST. The membrane was incubated in a
lectin solution at 10 ug/mL in PBS for 30 min, washed with PBST and incubated with streptavidin-HRP
(as above). The membrane was washed with PBST and imaged using ECL and photographic film. 2.5. Lectin Affinity Chromatography Lectin conjugated agarose beads were washed 10 times with PBS to remove sugars in their storage
solution and 500 µL of 50% slurry mixed with 5 mL of pooled urine and 500 μL of 12 × PBS. Binding
was allowed to occur during a 2 h incubation on a rotating mixer at 4°C. The beads were captured on
filters and the flow through was collected and stored. The beads were then washed thoroughly with PBS
and bound glycoproteins eluted with 2 × 400 μL of 100 mM L-fucose or 200 mM N-aceytl-D-galactosamine
(GalNAc). In experiments using UEA1 and DBA (which require divalent cations) PBS was substituted
with 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 100 uM MnCl2, 100 uM ZnSO4 and 20 mM MOPS,
pH 7.4. All lectin affinity-chromatography–shotgun-proteomics experiments were performed as 2
independent replicates. Proteomes 2015, 3 Proteomes 2015, 3 270 2.7. Stable Isotope Labelling After tryptic digestion, formaldehyde was added to the control urine peptides to a final
concentration of 0.2% w/v and deuterated formaldehyde was added to the pTa patient urine peptides. Sodium cyanoborohydride was added to both samples to a final concentration of 25 mM. After 30 min,
0.5 M ammonium bicarbonate was added to quench the reaction. The control and cancer urine samples
were then combined and acidified with 10% trifluoroacetic acid (TFA), the peptides were captured on
a C18 cartridge, washed with 0.1% TFA and peptides eluted with 600 μL of 60% acetonitrile
(ACN)/0.1% TFA. 2.3. Lectin ELISAs Urine samples were diluted ×50 in PBS and cell lysates and serum samples were diluted to 3 ug
protein/mL in PBS and 100 µL added to 96 well Maxisorb Immuno-plates followed by a 1 hour
incubation at 37°C to adsorb proteins. The wells were washed three times with 200 μL PBS containing
0.05% w/v Tween® 20 (PBST, Sigma-Aldrich). The plates were blocked with 1% BSA in PBS for one
hour. After washing with PBST, 100 μL of biotinylated lectin at 10 μg/mL in PBS was added to the
wells and incubated for 30 min. After washing with PBST, 100 μL of a 1 in 200 dilution of
streptavidin conjugated to horseradish peroxidase (R&D Systems, Abingdon, UK) in 1% BSA in PBS
was added and incubated for 30 min. The plates were then washed five times with PBST and 100 μL of
substrate solution (3,3′,5,5′-tetramethlbenzidine) was added. The reaction was stopped with 40 μL of
2M H2SO4 and the absorbance measured at 450 nm. All ELISAs were performed in triplicate and
means compared across experimental groups using t-tests. 3.1. Lectin Binding to Urothelial Cell Line Lysates Lectin ELISAs were used to evaluate the ability of 25 different lectins to bind to cell lysates of 2
normal urothelial human cell lines and 8 human bladder cancer cell lines derived from tumours of
different grades (potentially allowing us to determine how glycosylation differs between low- and
high-grade disease). Although different cell lines show quite different lectin binding profiles, none of
the lectins showed consistent, substantially different binding between the normal and cancer cell lines
or between cancer cell lines derived from low or high grade tumours (p > 0.05 in all cases). The data
are summarised in Figure 1. Figure 2 demonstrates the specificity of AAL for fucose containing
substrates: including 100 mM L-Fucose during the incubation of AAL on urothelial cell line lysate
coated plates effectively prevents any binding from taking place. Proteomes 2015, 3 used was 0%–50% of buffer B (2 mM ammonium formate, pH 3.0, 80% ACN) for 45 min followed by
50%–100% buffer B over five minutes and then 100%–0% buffer B for the last 10 min. Fractions were
dried and reconstituted in 0.1% formic acid (FA) in water. The peptides in each fraction were analysed
by LC-MS/MS using a 60 min gradient of 0%–36% ACN in 0.1% FA at a flow rate of 350 nL/min
with an Acclaim® Pepmap C18, 3 μm, 100 Å column (25 cm × 75 μm) (Thermo Scientific, Loughborough,
UK) attached to an Ultimate 3000 RS HPLC system coupled to an Impact Quadrupole-TOF mass
spectrometer (Bruker Daltonics, Coventry, UK) working in data-dependent mode at 5 MS/MS per cycle. 2.9. Peptide Identification and Analysis All MS/MS spectra were searched against a database containing Swissprot human sequences and
randomized versions thereof using MASCOT (version 2.3 Matrix Science Ltd, London, UK). Search
parameters were as follows: (i) species: Homo sapiens; (ii) enzyme: trypsin; (iii) ≤2 missed cleavages;
(iv) 10 ppm precursor ion tolerance; (v) 0.02 Da fragment ion tolerance; (vi) fixed modifications:
cysteine carbamidomethylation; (vii) variable modifications: methionine oxidation; (viii) a peptide
score of >25. For quantitative experiments light and heavy dimethylation of N-termini and lysine
residues were also included as variable modifications. Proteinscape 3 software (Bruker Daltonics) was
used to combine multiple search results and filter the data using a protein false discovery rate of 1%. WARP-LC (Bruker Daltonics) was used for relative quantitation based on extracted ion chromatograms
and limma used for statistical analysis of differential expression [26]. 2.8. Peptide Fractionation and LC-MS/MS Peptides were dried and dissolved in mixed mode buffer A (110 μL 20 mM ammonium formate,
pH 6.5, 3% ACN) and separated into 16 fractions using an Acclaim® Mixed-Mode WAX-1, 3 um, 120 Å
(2.1 × 150 mm) column (Dionex, Camberley, UK) at a flow rate of 100 μL/min. The elution gradient 271 3.2. Lectin Binding Properties of Urinary Proteins The binding of the same panel of 25 lectins to the proteins in pooled urine from patients without
bladder cancer or with pTa, pT1 or pT2+ bladder cancer was tested by ELISA. None of the lectins
demonstrated a statistically significant higher or lower binding (p > 0.05) to the proteins in the urine of
cancer patients relative to urine of non-cancer controls (Figure 3). 272 Proteomes 2015, 3 272 teomes 2015, 3
2
Figure 1. Lectin binding to urothelial cell lysates. Cell lines are shown from left to right,
non-cancer (NHU-TERT & UROtsa), grade 1 (MGH-U3, RT4, SW780), grade 2 (RT112,
VM-CUB-1, 5637) and grade 3 bladder cancer (T24, HB-CLS-2). The relative binding
level of each lectin to the cell lines is shown on a sliding scale from high (red) to low (blue). ,
Figure 1. Lectin binding to urothelial cell lysates. Cell lines are shown from left to right,
non-cancer (NHU-TERT & UROtsa), grade 1 (MGH-U3, RT4, SW780), grade 2 (RT112,
VM-CUB-1, 5637) and grade 3 bladder cancer (T24, HB-CLS-2). The relative binding
level of each lectin to the cell lines is shown on a sliding scale from high (red) to low (blue). Figure 1. Lectin binding to urothelial cell lysates. Cell lines are shown from left to right,
non-cancer (NHU-TERT & UROtsa), grade 1 (MGH-U3, RT4, SW780), grade 2 (RT112,
VM-CUB-1, 5637) and grade 3 bladder cancer (T24, HB-CLS-2). The relative binding
level of each lectin to the cell lines is shown on a sliding scale from high (red) to low (blue). Absorbance 450 nm
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
- Fucose
+ Fucose Absorbance 450 nm
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
- Fucose
+ Fucose
Figure 2. Fucose inhibition of AAL binding to cell lysates. Lectin ELISA results are
shown for AAL binding (absorbance 450 nm) to cell lysates ±100 mM L-fucose. Figure 2. Fucose inhibition of AAL binding to cell lysates. Lectin ELISA results are
shown for AAL binding (absorbance 450 nm) to cell lysates ±100 mM L-fucose. 273 Proteomes 2015, 3 273 Figure 3. Lectin binding to pooled urine samples. Each pooled urine samples consists of
urine from >8 patients with: C1&C2 = no abnormality detected, C3&C4 = no malignant
disease (cystitis/inflammation), Ta1 & Ta2 = pTa UBC, T1-1 &T1-2 = pT1 UBC, T2-1 &
T2-2 = MIBC. 3.3. Lectins with Selectivity for Urothelial Glycans Relative to Plasma Glycans A lectin that could enrich urothelial glycoproteins relative to plasma glycoproteins in urine would
be a useful tool in urine proteomics. We therefore compared the binding of the 25 lectins to bladder
cell line lysates with their binding to serum from non-cancer control subjects by lectin ELISA and
confirmed selected results with lectin dot blots (Figure 4). Whilst RCA1 and PHA-E bound more
strongly to serum than to cell lysate, the majority of lectins preferred the lysate with 10 lectins showing
very low binding to serum. These lectins, in particular UEA1 and DBA, showed very high cell:serum
binding ratios. Lectins that bind mannose, glucose or sialic acid tended to show low binding to
urothelial glycans whereas fucose, galactose and N-acetylgalactosamine binding lectins exhibited high
binding to urothelial glycans relative to serum. 3.2. Lectin Binding Properties of Urinary Proteins The relative binding level of each lectin to the 8 pooled urines is shown on
a sliding scale from high (red) to low (blue). Figure 3. Lectin binding to pooled urine samples. Each pooled urine samples consists of
urine from >8 patients with: C1&C2 = no abnormality detected, C3&C4 = no malignant
disease (cystitis/inflammation), Ta1 & Ta2 = pTa UBC, T1-1 &T1-2 = pT1 UBC, T2-1 &
T2-2 = MIBC. The relative binding level of each lectin to the 8 pooled urines is shown on
a sliding scale from high (red) to low (blue). 3.4. Glycoproteome Analysis of Pooled pTa UBC Patient Urine Shotgun proteomics was used to assess the ability of AAL, UEA and DBA affinity chromatography
to extract subproteomes from a pooled bladder cancer urine sample (75 patients with G1-G3 pTa
disease). UEA1 and DBA were chosen for glycoprotein enrichment due to their striking preference for
urothelial proteins over serum proteins and AAL was chosen because it not only displayed a
preference for urothelial proteins over serum proteins, but has previously been shown to have an
affinity for cancer-related glycoproteins. LC-MS/MS analysis of the proteins bound to UEA1 or DBA
and eluted with 100 mM L-fucose or 200 mM N-aceytl-D-galactosamine respectively identified
surprisingly few proteins: DBA captured 140 proteins of which 75 were found in two experimental
replicates and UEA1 captured 122 proteins of which 69 were common to both replicates. Furthermore,
these proteins included keratins and abundant proteins such as uromodulin and albumin indicative of
non-specific binding. In contrast, in both experimental replicates, more than 500 proteins were eluted
from AAL with 100 mM L-Fucose. There was a high degree of overlap between the proteins identified 274 Proteomes 2015, 3 Proteomes 2015, 3 in the AAL eluates in both replicates with 436 protein identifications common to both (Figure 5). Of
these 436 proteins, 285 contain glycosylation sites (65%). Of these 285 glycoproteins, 274 possess N-
linked glycans and 24 possess O-linked glycans. Figure 4. Lectin binding to urothelial cell line lysates and serum. The histogram shows
data from lectin ELISAs run in triplicate (pooled cell lysate and pooled serum). The inserted
panel shows confirmatory dot blots With the exception of PHA E SNA and LCA all Figure 4. Lectin binding to urothelial cell line lysates and serum. The histogram shows
data from lectin ELISAs run in triplicate (pooled cell lysate and pooled serum). The inserted
panel shows confirmatory dot blots. With the exception of PHA-E, SNA and LCA all
lectins showed significantly different binding to lysates and serum (p < 0.05). Figure 4. Lectin binding to urothelial cell line lysates and serum. The histogram shows
data from lectin ELISAs run in triplicate (pooled cell lysate and pooled serum). The inserted
panel shows confirmatory dot blots. With the exception of PHA-E, SNA and LCA all
lectins showed significantly different binding to lysates and serum (p < 0.05). AAL eluate duplicates
Flow-through duplicates
114
436
119 130
449
94
161
275
174
Figure 5. The number of proteins identified by LC-MS/MS in the AAL flow-through and
eluted fractions. The upper Venn diagrams show the number of proteins identified in the
flow-through and eluted fractions of two independent AAL chromatographies of a pooled
urine sample from patients with pTa bladder cancer. The lower Venn diagram shows the
overlap between the proteins identified in both eluates and both flow-throughs. AAL eluate duplicates
Flow-through duplicates
114
436
119 130
449
94
161
275
174 174 161 Figure 5. The number of proteins identified by LC-MS/MS in the AAL flow-through and
eluted fractions. The upper Venn diagrams show the number of proteins identified in the
flow-through and eluted fractions of two independent AAL chromatographies of a pooled
urine sample from patients with pTa bladder cancer. The lower Venn diagram shows the
overlap between the proteins identified in both eluates and both flow-throughs. Proteomes 2015, 3 275 The AAL flow throughs (i.e., proteins not captured by AAL) were also analysed by LC-MS/MS and
the protein identifications compared with the proteins identified in the AAL eluate to determine which
proteins were enriched by the AAL affinity chromatography. We defined proteins as enriched by AAL
if they were present in both eluates and were identified by at least twice as many peptides in the
eluates as in the flow throughs. Using these criteria, AAL enriched 186 proteins. Of these 186 enriched
proteins, 115 (62%) contain glycosylation sites and 84 of these proteins were not identified in the
flow-throughs. Of the 186 enriched proteins, 70 are associated with the extracellular space and 71 with
the plasma membrane. This suggests that AAL lectin affinity chromatography may be able to identify
proteins originating from the urothelial cell surface or proteins released from the urothelium. The
levels of abundant plasma proteins were decreased in the AAL eluates relative to the flow-thoughs:
albumin decreased from 8307 peptide spectrum matches in the flow-throughs to 585 in the eluates and
serotransferrin decreased from 950 to 76 peptide spectrum matches. Information on the protein
identifications are provided in Supplemental Materials. 3.5. Quantitative Comparison of AAL Binding Proteins in the Urine of Control Subject and Patients
with G1 pTa Bladder Cancer 3.5. Quantitative Comparison of AAL Binding Proteins in the Urine of Control Subject and Patients
with G1 pTa Bladder Cancer AAL affinity chromatography was performed on pooled urine samples from patients with G1 pTa
bladder cancer (n = 36) and non-cancer controls (n = 28). The eluted proteins were digested and the
peptides stable isotope labelled as described in the method section, the samples combined and analysed
by shotgun proteomics. Duplicate experiments again proved reproducible with 394 protein identified
in both AAL eluates. We also analysed the 2 pooled urine samples without AAL enrichment in
duplicate with 501 protein identifications common to replicates. The heavy/light peptide intensity
ratios were used to estimate the relative levels of proteins in the two pooled urine samples in the AAL
eluate and whole urine datasets. In the AAL eluates the concentrations of 21 proteins were
significantly (p << 0.01) and substantially (≥2-fold increase in both experimental replicates) higher in
the pooled pTa urine than the control urine. Of these, 12 proteins were also increased in the cancer
sample in the whole urine experiments whereas 9 of the proteins were increased in cancer only in the
AAL eluates. The 12 proteins elevated in cancer in both the whole urine and AAL eluates are likely to
be present at a higher total concentration in the cancer sample whereas the 9 proteins increased in the
AAL eluates but not whole urine could be cancer specific glycoforms. Of the 9 proteins with apparent
altered glycosylation (rather than simply an increase in total concentration), 6 were previously
identified as released by bladder cancer cell lines in vitro [24] (mucin-1 (MUC1), golgi apparatus
protein 1 (GLG1), endoplasmin (HSP90B1), prostatic acid phosphatase (ACPP), Ig gamma-2 chain C
region (IGHG2), and deoxyribonuclease-2-alpha (DNASE2A)), and 3 were not (voltage-dependent
anion-selective channel 1, carbonic anhydrase 1 and bile salt-activated lipase 11) . The cancer:normal
intensity ratio in the AAL eluate and the whole urine data for the proteins previously identified as
released by bladder cancer cell lines is shown in Figure 6. 276 Proteomes 2015, 3 Cancer: normal ratio
4
3.5
3
2.5
2
1.5
1
0.5
0
Cancer: normal ratios in whole urine and AAL enriched urine
MUC1
HSP90B1
GLG1
ACPP
IGHG2
DNASE2A
Proteins
whole urine
AAL
Figure 6. AAL binding proteins and their cancer: normal peptide intensity ratios for the
whole urine experiment and the AAL experiment. 3.5. Quantitative Comparison of AAL Binding Proteins in the Urine of Control Subject and Patients
with G1 pTa Bladder Cancer Mucin-1 (MUC1), endoplasmin
(HSP90B1), golgi apparatus protein 1 (GLG1), prostastic acid phosphatase (ACPP),
Ig gamma-2 chain C region (IGHG2), and deoxyribonuclease-2-alpha (DNASE2A) all
have a greater cancer: normal ratio after AAL enrichment. Cancer: normal ratios in whole urine and AAL enriched urine Cancer: normal ratios in whole urine and AAL enriched urine Cancer: normal ratios in whole urine and AAL enriched urine Cancer: normal ratio
4
3.5
3
2.5
2
1.5
1
0.5
0
Cancer: normal ratios in whole urine and AAL enriched urine
MUC1
HSP90B1
GLG1
ACPP
IGHG2
DNASE2A
Proteins
whole urine
AAL Figure 6. AAL binding proteins and their cancer: normal peptide intensity ratios for the
whole urine experiment and the AAL experiment. Mucin-1 (MUC1), endoplasmin
(HSP90B1), golgi apparatus protein 1 (GLG1), prostastic acid phosphatase (ACPP),
Ig gamma-2 chain C region (IGHG2), and deoxyribonuclease-2-alpha (DNASE2A) all
have a greater cancer: normal ratio after AAL enrichment. Proteomes 2015, 3 277 cancer [28]. The literature on lectin binding to bladder cancer tissues is complex with no clear
consensus as to which lectins bind preferentially to tumours over normal tissue or relationships of
lectin binding to stage, grade and outcome [29,30]. These data indicate that specific glycoprotein
markers will be required rather than global changes in glycosylation. Perhaps our most important finding is that several lectins are selective for urothelial proteins over
plasma proteins. This is a useful characteristic for bladder cancer urine proteomics because low
abundance urothelial proteins can be masked from detection by mass spectrometry by the presence of
highly abundant serum proteins in the urine. The broad specificity lectins WGA and ConA that have
been used in previous urinary glycoprotoemic studies [16,17] however, are not selective for urothelial
proteins over plasma proteins. When we tested the ability of the 2 lectins with the greatest selectivity
for urothelial proteins, UEA1 and DBA, to enrich urothelial glycoproteins from urine we identified
only a small number of proteins. Perhaps these unexpected results are because the lectins recognise
cellular proteins that are not released into the urine or because the eluting sugars were unable to
effectively compete with the glycoprotein-lectin interaction. Nonetheless, it would seem that the other
lectins with a high lysate/serum ratio (PNA, SJA, GSL I, SBA, PHA-L, STL, VVA) should be
considered in urinary glycoproteomic workflows. AAL was selected in this study partly because it
showed a small preference for urothelial proteins but more so because it has previously been used to
enrich aberrantly glycosylated proteins in HCC [31]. AAL is specific for core and terminal fucose
structures including fucose (α-1,6) linked to N-acetylglucosamine and fucose (α-1,3) linked to
structures related to N-acetyllactosamine (85). The fucose (α-1,6) residue is a core fucose structure that
is present in many mammalian tissues and has been reported to be altered in pathological settings. Of the 580 unique proteins captured by AAL, 6 behave as if they are aberrantly glycosylated
in bladder cancer: mucin-1, golgi apparatus protein 1, prostatic acid phosphatase, endoplasmin,
deoxyribonuclease-2-alpha and Ig gamma-2 chain C region. The selection was based on the fact that
they are all released by bladder cell lines [24] and it appears that it is their glycosylation status rather
than the total quantity of these proteins which change in bladder cancer. 4. Discussion Current urinary protein biomarkers for bladder cancer lack the sensitivity and/or specificity required
to have utility in the clinic. Aberrant glycosylation has been widely reported in cancer and some cancer
biomarkers utilise changes in protein glycosylation to improve biomarker effectiveness. In this study
we investigated whether cancer specific glycoforms are a feature of bladder cancer and whether lectin
affinity chromatography is a useful tool in urine proteomics. In summary, the binding of 25 different
lectins to bladder cancer cell lines or urine from control subjects and patients with bladder cancer did
not detect any major global changes in glycosylation. We did, however, find that many lectins have a
preference for bladder cancer cell line proteins over plasma proteins and this may be useful for
discriminating between proteins that are released into the urine from the urothelium and those that are
filtered through the kidney or leak in due to haematuria. A recent paper by Yang et al. reported that the binding of many lectins differed significantly between
a normal urothelial cell line (HCV29) and several bladder cancer cell lines, and went on to show
increased binding of LCA and SNA and decreased binding of ConA to bladder cancer tissue relative to
adjacent normal tissue [27]. The use of different reference cell lines (NHU-TERT and UROtsa) in our
study may be the reason that we did not see clear differences between the non-cancer and cancer cell
lines, although differences between individual cell lines were observed. The lack of evidence for
cancer-specific changes in glycosylation in our experiments does not exclude the possibility that they
exist since we looked globally at the whole glycoproteome rather than determining the glycosylation
status of individual proteins. The fact that no lectins demonstrated preferential binding to bladder
cancer patient urine samples over control urine samples may reflect that the vast majority of the
proteins in the urine samples are not tumour derived. Consistent with this finding, a recent study of
urinary glycans found only small changes in the N- and O-linked glycomes of patients with bladder 5. Conclusions Our data demonstrate a role for certain lectins in urinary biomarker discovery. The urinary
glycoproteome has not been fully explored to date and using the lectins with a strong preference for
urothelial proteins over serum proteins (Figure 4) in conjunction with shotgun proteomics may identify
much needed biomarkers for bladder cancer. To assess the biomarker potential of the candidate
glycoprotein biomarkers suggested by the current work, initial validation could be carried out by lectin
affinity chromatography combined with an ELISA using specific antibodies against the target
glycoprotein. If the glycoprotein concentration in urine samples from bladder cancer patients is
confirmed as significantly greater than the concentration in control samples then a more streamlined
assay such as a sandwich ELISA combining lectin and antibody binding would be required for full
validation and ultimately clinical use. Proteomes 2015, 3 278 bladder cancer but sensitivity for early disease is poor [38]. An investigation of urinary levels of
MUC1 31 patients with TCC and 30 control patients found no significant difference between patient
groups [40]. However, total MUC1 was measured whereas our data suggest that it is an alternatively
glycosylated form of MUC1 that is increased in the urine of bladder cancer patients. The golgi apparatus protein 1 (GLG1), also known as CFR, ESL-1 and MG-160, is a 135 kDa
glycosylated single pass type I transmembrane protein that contains 16 cysteine-rich GLG1 repeats [41]. It is found in the golgi apparatus and on the cell surface membrane. GLG1 is able to bind with many
different proteins making it an important regulatory protein and signal transducer. The localisation
of GLG1 is a crucial determinant of GLG1 function and is influenced by two mechanisms: the
transmembrane domain and cytoplasmic tail retain GLG1 in the golgi apparatus, whereas the cys-rich
repeats destabilise the protein and GLG1 is recruited to the cell surface via processes of stability
control [41]. It has also been reported that GLG1 can be released from the cell by proteolytic cleavage
at the juxtamembrane region [42]. These mechanisms of localisation and proteolytic cleavage may be
altered in cancer and may play a role in the increased presence of GLG1 in the urine. GLG1 has been
shown by immunohistochemistry to be highly or intermediately expressed in both high and low grade
urothelial cancer tissue samples [43]; furthermore, GLG1 was detected on the cell surface of bladder
cancer cell lines (Ward, unpublished data). Therefore, it seems plausible that the GLG1 detected in
pTa patient urine is tumour derived. Proteomes 2015, 3 For two of these proteins,
Deoxyribonuclease-2-alpha (DNASE2) and Ig gamma-2 chain C region (IGHG2) there is a lack of
further evidence for a role in bladder. Endoplasmin (HSP90B1) is a ubiquitously expressed molecular
chaperone of plasma membrane associated and secreted proteins [32]. Heat shock proteins are widely
reported as overexpressed in cancer and one study reported that HSP90B1 is overexpressed in canine
bladder cancer [33]. Prostatic acid phosphatase (ACPP) is a 100 kDa tyrosine phosphatase that
dephosphorylates a diverse array of substrates under acidic conditions [34]. ACPP exists as intracellular
and secreted forms that possess different glycosylation patterns and different hydrophobicities [35]. Although serum ACPP can be used to monitor prostate cancer it is reportedly not expressed in bladder
cancer [36] and is not likely be a good biomarker for bladder cancer (because urinary ACPP may be
primarily derived from the prostate) unless a genuinely bladder cancer specific glycoform exists. MUC1 and GLG1 appear to be the most interesting of the 6 candidates as discussed below. MUC1 is a transmembrane protein present in normal urothelium on the apical surfaces of umbrella
cells and acts to protect the cells from adhesion of bacteria [37]. Overexpression and changes in
glycosylation of MUC1 have been reported in lung, breast, ovary, colon and bladder cancer [38]. In an
immunohistochemistry study of 539 bladder tumours MUC1 was expressed in 62% of the tumours and
increased with tumour grade [39]. Serum levels of MUC1 are elevated in patients with late stage Author Contributions Experimental work: SRA, NSG, JCG and DGW. Data analysis: WW. Provision and
characterization of cell lines: MAK. BCPP management team (biospecimen collection): MPZ, NDJ,
RTB. Study design and writing: DGW, SJA and RTB. Conflicts of Interest The authors declare no conflict of interest. References 1. Ferlay, J.; Soerjomataram, I.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman,
D.; Bray, F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in
GLOBOCAN 2012. Int. J. Cancer 2015, 136, E359–E386. 2. Knowles, M.; Hurst, C. Molecular biology of bladder cancer: New insights into pathogenesis and
clinical diversity. Nat. Rev. Cancer 2015, 15, 25–41. 2. Knowles, M.; Hurst, C. Molecular biology of bladder cancer: New insights into pathogenesis and
clinical diversity. Nat. Rev. Cancer 2015, 15, 25–41. 3. Sylvester, R.; van der Meijden, A.; Oosterlinck, W.; Witjes, J.; Bouffioux, C.; Denis, L.;
Newling, D.W.; Kurth, K. Predicting recurrence and progression in individual patients with stage
Ta T1 bladder cancer using EORTC risk tables: A combined analysis of 2596 patients from seven
EORTC trials. Eur. Urol. 2006, 49, 466–477. 4. Babjuk, M.; Burger, M.; Zigeuner, R.; Shariat, S.; van Rhijn, B.; Compérat, E.; Sylvester, R.J.;
Kaasinen, E.; Böhle, A.; Palou Redorta, J.; et al. EAU guidelines on non-muscle-invasive
urothelial carcinoma of the bladder: Update 2013. Eur. Urol. 2013, 64, 639–653. 4. Babjuk, M.; Burger, M.; Zigeuner, R.; Shariat, S.; van Rhijn, B.; Compérat, E.; Sylvester, R.J.;
Kaasinen, E.; Böhle, A.; Palou Redorta, J.; et al. EAU guidelines on non-muscle-invasive
urothelial carcinoma of the bladder: Update 2013. Eur. Urol. 2013, 64, 639–653. 5. Schmitz-Dräger, B.; Droller, M.; Lokeshwar, V.; Lotan, Y.; Hudson, M.; van Rhijn, B.; Marberger,
M.J.; Fradet, Y.; Hemstreet, G.P.; Malmstrom, P.U.; et al. Molecular markers for bladder cancer
screening, early diagnosis, and surveillance: The WHO/ICUD consensus. Urol. Int. 2015, 94, 1–24. 6. Miah, S.; Dudziec, E.; Drayton, R.; Zlotta, A.; Morgan, S.; Rosario, D.; Hamdy, F.C.; Catto, J.W. An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer. Br. J. Cancer
2012, 107, 123–128. 7. O’Sullivan, P.; Sharples, K.; Dalphin, M.; Davidson, P.; Gilling, P.; Cambridge, L.; Harvey, J.;
Toro, T.; Giles, N.; Luxmanan, C.; et al. A multigene urine test for the detection and stratification
of bladder cancer in patients presenting with hematuria. J. Urol. 2012, 188, 741–747. 8. Hurst, C.; Platt, F.; Knowles, M. Comprehensive mutation analysis of the TERT promoter in
bladder cancer and detection of mutations in voided urine. Eur. Urol. 2014, 65, 367–369. 9. Pasikanti, K.; Esuvaranathan, K.; Ho, P.; Mahendran, R.; Kamaraj, R.; Wu, Q.; Chiong, E.;
Chan, E.C. Noninvasive urinary metabonomic diagnosis of human bladder cancer. J. Proteome Res. 2010, 9, 2988–2995. 10. Acknowledgements We thank all the West Midlands Consultant Urologists and their units involved with BCPP, as well
as the BCPP research nurses and Margaret Grant, Deborah Bird, Jennifer Barnwell, Duncan Nekeman
and Eline van Roekel. BCPP is funded by Cancer Research UK, the University of Birmingham and the
Birmingham and The Black Country and West Midlands North and South Comprehensive Local
Research Networks, and sponsored by the University of Birmingham. The BCPP biospecimen
collection is supported by funding from the Birmingham ECMC. Douglas Ward is funded by a
philanthropic donation to the University of Birmingham in support of bladder cancer research. 279 Proteomes 2015, 3 Proteomes 2015, 3 Proteomes 2015, 3 280 11. Chen, Y.; Chen, H.; Domanski, D.; Smith, D.; Liang, K.; Wu, C.; Chen, C.L.; Chung, T.; Chen, M.C.;
Chang, Y.S.; et al. Multiplexed quantification of 63 proteins in human urine by multiple reaction
monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers. J. Proteomics 2012, 75, 3529–3545. 12. Chen, C.; Lin, T.; Tsai, C.; Wu, C.; Chung, T.; Chien, K.; Wu, M.; Chang, Y.S.; Yu, J.S.; Chen, Y.T. Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled
with quantitative proteomics. J. Proteomics 2013, 85, 28–43. 13. Bryan, R.; Wei, W.; Shimwell, N.; Collins, S.; Hussain, S.; Billingham, L.; Murray, P.G.;
Deshmukh, N.; James, N.D.; Wallace, D.M.; et al. Assessment of high-throughput high-resolution
MALDI-TOF-MS of urinary peptides for the detection of muscle-invasive bladder cancer. Proteomics Clin. Appl. 2011, 5, 493–503. 14. Schiffer, E.; Vlahou, A.; Petrolekas, A.; Stravodimos, K.; Tauber, R.; Geschwend, J.; Neuhaus, J.;
Stolzenburg, J.U.; Conaway, M.R.; Mischak, H.; et al. Prediction of muscle-invasive bladder
cancer using urinary proteomics. Clin. Cancer Res. 2009, 15, 4935–4943. 15. Vlahou, A.; Schellhammer, P.F.; Mendrinos, S.; Patel, K.; Kondylis, F.; Gong, L.; Nasim, S.;
Wright, G.L., Jr. Development of a novel proteomic approach for the detection of transitional cell
carcinoma of the bladder in urine. Am. J. Pathol. 2001, 158, 1491–1502. 16. Kreunin, P.; Zhao, J.; Rosser, C.; Urquidi, V.; Lubman, D.; Goodison, S. Bladder cancer
associated glycoprotein signatures revealed by urinary proteomic profiling. J. Proteome Res. 2007, 6,
2631–2639. 17. Yang, N.; Feng, S.; Shedden, K.; Xie, X.; Liu, Y.; Rosser, C.; Lubman, D.M.; Goodison, S. Urinary glycoprotein biomarker discovery for bladder cancer detection using LC/MS-MS and
label-free quantification. Clin. Cancer Res. 2011, 17, 3349–3359. 18. Ohyama, C. Glycosylation in bladder cancer. Int. J. Clin. Oncol. 2008, 13, 308–313. 19. Meany, D.; Chan, D. Aberrant glycosylation associated with enzymes as cancer biomarkers. Clin. Proteomics 2011, 8, 7, doi:10.1186/1559-0275-8-7. 20. Dwek, M.; Jenks, A.; Leathem, A. A sensitive assay to measure biomarker glycosylation
demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate
cancer compared with benign prostatic hyperplasia. Clin. Chim. Acta 2010, 411, 1935–1939. 21. Wu, J.; Zhu, J.; Yin, H.; Buckanovich, R.; Lubman, D. Analysis of glycan variation on
glycoproteins from serum by the reverse lectin-based ELISA assay. J. Proteome Res. 2014, 13,
2197–2204. 22. Abbott, K.; Lim, J.; Wells, L.; Benigno, B.; McDonald, J.; Pierce, M. References Wood, S.; Knowles, M.; Thompson, D.; Selby, P.; Banks, R. Proteomic studies of urinary
biomarkers for prostate, bladder and kidney cancers. Nat. Rev. Urol. 2013, 10, 206–218. Proteomes 2015, 3 Proteomes 2015, 3 281 25. Zeegers, M.; Bryan, R.; Langford, C.; Billingham, L.; Murray, P.; Deshmukh, N.; Hussain, S.;
James, N.; Wallace, D.M.; Cheng, K.K. The West Midlands Bladder Cancer Prognosis Programme:
Rationale and design. BJU Int. 2010, 105, 784–788. 25. Zeegers, M.; Bryan, R.; Langford, C.; Billingham, L.; Murray, P.; Deshmukh, N.; Hussain, S.;
James, N.; Wallace, D.M.; Cheng, K.K. The West Midlands Bladder Cancer Prognosis Programme:
Rationale and design. BJU Int. 2010, 105, 784–788. 26. Smyth, G. Linear models and empirical Bayes methods for assessing differential expression in
microarray experiments. Stat. Appl. Genet. Mol. Biol. 2004, 3, 3, doi:10.2202/1544-6115.1027. 27. Yang, G.; Tan, Z.; Lu, W.; Guo, J.; Yu, H.; Yu, J.; Sun, C.; Qi, X.; Li, Z.; Guan, F. Quantitative
glycome analysis of N-glycan patterns in bladder cancer vs normal bladder cells using an
integrated strategy. J. Proteome Res. 2015, 14, 639–653. 28. Takeuchi, M.; Amano, M.; Kitamura, H.; Tsukamoto, T.; Masumori, N.; Hirose, K.;
Ohashi, T.; Nishimura, S.I. N- and O-glycome analysis of serum and urine from bladder cancer
patients using a high-throughput glycoblotting method. J. Glycomics Lipidomics 2013, 3,
doi:10.4172/2153-0637.1000108. 29. Langkilde, N.; Wolf, H.; Orntoft, T. Binding of wheat and peanut lectins to human transitional
cell carcinomas. Correlation with histopathologic grade, invasion, and DNA ploidy. Cancer 1989,
15, 849–853. 30. Neal, D.; Charlton, R.; Bennett, M. Histochemical study of lectin binding in neoplastic and
non-neoplastic urothelium. Br. J. Urol. 1987, 60, 399–401. 31. Zhang, S.; Jiang, K.; Zhang, Q.; Guo, K.; Liu, Y. Serum fucosylated paraoxonase 1 as a potential
glycobiomarker for clinical diagnosis of early hepatocellular carcinoma using ELISA Index. Glycoconj. J. 2015, 32, 119–125. 32. Melnick, J.; Dul, J.; Argon, Y. Sequential interaction of the chaperones BiP and GRP94 with
immunoglobulin chains in the endoplasmic reticulum. Nature 1994, 370, 373–375. 33. LeRoy, B.; Painter, A.; Sheppard, H.; Popiolek, L.; Samuel-Foo, M.; Andacht, T. Protein
expression profiling of normal and neoplastic canine prostate and bladder tissue. Vet. Comp. Oncol. 2007, 5, 119–130. 34. Muniyan, S.; Chaturvedi, N.; Dwyer, J.; Lagrange, C.; Chaney, W.; Lin, M. Human prostatic Acid
phosphatase: Structure, function and regulation. Int. J. Mol. Sci. 2013, 14, 10438–10464. 35. White, K.; Rodemich, L.; Nyalwidhe, J.; Comunale, M.; Clements, M.; Lance, R.; Schellhammer, P.F.;
Mehta, A.S.; Semmes, O.J.; Drake, R.R. Glycomic characterization of prostate-specific antigen
and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids. J. Proteome Res. Proteomes 2015, 3 Identification of candidate
biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian
cancer patients by glycoproteomic analysis. Proteomics 2010, 10, 470–481. 23. Li, D.; Mallory, T.; Satomura, S. AFP-L3: A new generation of tumor marker for hepatocellular
carcinoma. Clin. Chem. Acta 2001, 313, 15–19. 24. Bryan, R.; Regan, H.; Pirrie, S.; Devall, A.; Cheng, K.; Zeegers, M.; James, N.D.; Knowles, M.A.;
Ward, D.G. Protein shedding in urothelial bladder cancer: Prognostic implications of soluble
urinary EGFR and EpCAM. Br. J. Cancer 2015, 112, 1052–1058. Proteomes 2015, 3 2009, 8, 620–630. 36. Heyderman, E.; Brown, B.; Richardson, T. Epithelial markers in prostatic, bladder, and colorectal
cancer: An immunoperoxidase study of epithelial membrane antigen, carcinoembryonic antigen,
and prostatic acid phosphatase. J. Clin. Pathol. 1984, 37, 1363–1369. 37. Ahmad, S.; Lam, T.; N’Dow, J. Significance of MUC1 in bladder cancer. BJU Int. 2015, 115,
161–162. 38. Simms, M.; Hughes, O.; Limb, M.; Price, M.; Bishop, M. MUC1 mucin as a tumour marker in
bladder cancer. BJU Int. 1999, 84, 350–352. 39. Stojnev, S.; Ristic-Petrovic, A.; Velickovic, L.; Krstic, M.; Bogdanovic, D.; Khanh do, T.; Ristic, A.;
Conic, I.; Stefanovic, V. Prognostic significance of mucin expression in urothelial bladder cancer. Int. J. Clin. Exp. Pathol. 2014, 7, 4945–4958. Proteomes 2015, 3 Proteomes 2015, 3 282 40. Xiang, S.; Zhou, S.; Guan, W.; Hu, Z.; Zhuang, Q.; Ye, Z. Value of quantitative examination of
urine MUC1 in bladder transitional cell carcinoma. Di Yi Jun Yi Da Xue Xue Bao 2005, 25,
998–1000. 41. Miyaoka, Y.; Kato, H.; Ebato, K.; Saito, S.; Miyata, N.; Imamura, T.; Miyajima, A. Retention in
the Golgi apparatus and expression on the cell surface of Cfr/Esl-1/Glg-1/MG-160 are regulated
by two distinct mechanisms. Biochem. J. 2011, 440, 33–41. 42. Antoine, M.; Köhl, R.; Tag, C.; Gressner, A.; Hellerbrand, C.; Kiefer, P. Secreted cysteine-rich
FGF receptor derives from posttranslational processing by furin-like prohormone convertases. Biochem. Biophys. Res. Commun. 2009, 382, 359–364. 43. Uhlen, M.; Oksvold, P.; Fagerberg, L.; Lundberg, E.; Jonasson, K.; Forsberg, M.; Zwahlen, M.;
Kampf, C.; Wester, K.; Hober, S.; et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 2010, 28, 1248–1250. © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/). © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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Species composition and distribution of fishes and prawns in the Amur River channel
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* Кошелев Всеволод Николаевич, кандидат биологических наук, заведующий сектором,
e-mail: scn74@mail.ru; Колпаков Николай Викторович, доктор биологических наук, руководитель
филиала, e-mail: kolpakov_nv@mail.ru.
Koshelev Vsevolod N., Ph.D., head of sector, Khabarovsk branch of VNIRO (KhabarovskNIRO),
13a, Amursky Boulevard, Khabarovsk, 680038, Russia, e-mail: scn74@mail.ru; Kolpakov Nikolai
V., D. Biol., director of branch, Khabarovsk branch of VNIRO (KhabarovskNIRO), 13a, Amursky
Boulevard, Khabarovsk, 680038, Russia, e-mail: kolpakov_nv@mail.ru. Известия ТИНРО Известия ТИНРО 2020 Том 200, вып. 2 УДК [597.2/.5+595.384.12] (282.257.5) В.Н. Кошелев, Н.В. Колпаков*
Хабаровский филиал ВНИРО (ХабаровскНИРО),
680038, г. Хабаровск, Амурский бульвар, 13а ВИДОВОЙ СОСТАВ И РАСПРЕДЕЛЕНИЕ РЫБ И КРЕВЕТОК
В РУСЛЕ НИЖНЕГО АМУРА or 292 Видовой состав и распределение рыб и креветок в русле нижнего Амура 24.0 % of total biomass); other 6 fish species with considerable biomass were lizard gudgeon
Saurogobio dabryi (12.6 %), ussuri catfish T. ussuriensis (9.7 %), amur white-pinned gudgeon
Romanogobio tenuicorpus (7.8 %), amur sturgeon Acipenser schrenckii (5.6 %), amur gudgeon
Gobio cynocephalus (2.4 %), and kaluga Huso dauricus (0.6 %). Distribution density varied
in the range of 0.01–2.57 g/m2 (on average 0.45 ± 0.27 g/m2) for fish and 0.005–0.044 g/m2
(0.021 ± 0.012 g/m2) for prawns. Abundance of both fish and prawns increased multifold to
wards the Amur mouth that correlated with increasing of forage benthos biomass in the same
direction. So, the main portion of fish biomass (82.4 %) was distributed in the Amur River
downstream (40–400 km from the mouth) and the prawn stock was concentrated completely
in its lowermost part (50–150 km from the mouth). iii 24.0 % of total biomass); other 6 fish species with considerable biomass were lizard gudgeon
Saurogobio dabryi (12.6 %), ussuri catfish T. ussuriensis (9.7 %), amur white-pinned gudgeon
Romanogobio tenuicorpus (7.8 %), amur sturgeon Acipenser schrenckii (5.6 %), amur gudgeon
Gobio cynocephalus (2.4 %), and kaluga Huso dauricus (0.6 %). Distribution density varied
in the range of 0.01–2.57 g/m2 (on average 0.45 ± 0.27 g/m2) for fish and 0.005–0.044 g/m2
(0.021 ± 0.012 g/m2) for prawns. Abundance of both fish and prawns increased multifold to
wards the Amur mouth that correlated with increasing of forage benthos biomass in the same
direction. So, the main portion of fish biomass (82.4 %) was distributed in the Amur River
downstream (40–400 km from the mouth) and the prawn stock was concentrated completely
in its lowermost part (50–150 km from the mouth). iii p
(
)
Key words: Amur River, beam-trawl, fish, prawn, fish distribution, fish abundance. Введение Отлов рыб в русле и крупных протоках Амура на протяжении истории ихти
ологических исследований проводили с использованием различных орудий лова:
мальковых ловушек, плавных, ставных и накидных сетей, закидных и ставных
неводов, крючковых снастей [Солдатов, 1915; Пробатов, 1935; Никольский, 1950,
1956; Новомодный и др., 2004]. Однако применение данных орудий в крупных реках
возможно только в мелководной прибрежной зоне, основная же часть русла остается
не охваченной исследованиями по причине больших глубин и высоких скоростей
течения [Zajicek, Wolter, 2018]. Невозможность получить данные о распределении
и численности молоди и взрослых рыб, нагул которых приурочен к руслу реки, не
позволяет сформировать целостное представление о качественном и количественном
составе речного ихтиоцена. р
Оценка в крупных реках численности рыб, за исключением покатной молоди,
методом площадей [Аксютина, 1968] возможна только для закидного невода и на
кидной сети. Использование же для оценки плотности рыб ставных и плавных сетей
имеет множество допущений, требует наличия достоверной промысловой статистики,
проведения трудоемких расчетов [Сечин, 1990; Лобырев, 2008] или экспериментов
по мечению [Пасечник, Шмигирилов, 2008]. Вместе с тем в крупных реках и эстуариях для оценки численности рыб и креве
ток успешно используются тралы, причем предпочтительнее бим-трал, для которого
характерна неизменная величина горизонтального раскрытия [Dettmers et al., 2001;
Wolter, Freyhof, 2004; Herzog et al., 2005; de Souza et al., 2010; Вдовин, Мизюркин,
2011; Szalóky et al., 2014]. Такие работы проводятся и в России, например, в нижнем
течении р. Дон [Живоглядов и др., 2019] и на реках Камчатки [Коваль и др., 2015]. В 2003 г. Хабаровским филиалом ТИНРО-центра (ныне Хабаровский филиал
ВНИРО) была выполнена съемка с помощью бим-трала на р. Амур. Цель нашей
работы — описание видового состава и анализ пространственного распределения
рыб и креветок в русловой части нижнего Амура. ВИДОВОЙ СОСТАВ И РАСПРЕДЕЛЕНИЕ РЫБ И КРЕВЕТОК
В РУСЛЕ НИЖНЕГО АМУРА По результатам траловой съемки, выполненной бим-тралом в сентябре-октябре
2003 г. (57 тралений на глубинах 1,5–23,0 м), описано распределение рыб и креветки
в русле нижней части Амура (40–960 км). Всего поймано 2584 экз. 22 видов рыб и
1077 экз. одного вида беспозвоночных (креветка Palaemon modestus). Среди рыб по числу
видов доминировали представители отрядов Cypriniformes (13 видов) и Siluriformes
(4 вида). Общая численность донных и придонных рыб составила 32,161 млн экз., чис
ленность креветки — 4,887 млн экз. Наиболее многочисленными среди рыб были два
промысловых на Амуре вида: косатка Бражникова Tachysurus brashnikowi (16,26 млн экз.)
и косатка-скрипун T. sinensis (4,32 млн экз.). Численность рыб и креветки многократно
возрастает к устью Амура, что коррелирует с увеличением биомассы кормового бентоса
в том же направлении. Общая биомасса рыб и креветки составила соответственно 583,8
и 8,8 т. Биомасса рыб изменялась в пределах 0,012–2,572 г/м2 (в среднем 0,450 ± 0,270 г/м2),
биомасса креветки была равна 0,005–0,044 г/м2 (0,021 ± 0,012 г/м2). При этом 82,4 %
биомассы рыб было приурочено к нижнему участку (40–400 км от устья), вся биомасса
креветки — к участку 50–150 км от устья. По биомассе в уловах преобладали: косатка-
скрипун (32,9 %), косатка Бражникова (24,0 %), ящерный пескарь Saurogobio dabryi
(12,6 %), косатка-плеть T. ussuriensis (9,7 %), амурский белоперый пескарь Romanogobio
tenuicorpus (7,8 %), амурский осетр Acipenser schrenckii (5,6 %), амурский обыкновенный
пескарь Gobio cynocephalus (2,4 %) и калуга Huso dauricus (0,6 %). Ключевые слова: река Амур, бим-трал, рыбы, креветка, распределение, численность. DOI: 10.26428/1606-9919-2020-200-292-307. Koshelev V.N., Kolpakov N.V. Species composition and distribution of fishes and
prawns in the Amur River channel // Izv. TINRO. — 2020. — Vol. 200, Iss. 2. — P. 292–307.i p
,
Distribution of fishes and prawns in the Amur River (40–960 km from the mouth) is described
on results of the beam-trawl survey conducted in September-October, 2003 (57 trawl stations at
the depth of 1.5–23.0 m). In total, 2584 ind. of 22 fish species and 1077 ind. of 1 invertebrate
species (prawn Palaemon modestus) were caught. The highest species richness was registered
for orders Cypriniformes (13 species) and Siluriformes (4 species). The fish and prawn stocks
were evaluated as 32.161 . 106 ind. (583.8 t) for bottom and near-bottom fish and 4.887 . 106 ind. (8.8 t) for prawns. Two commercial fish species were the most abundant: catfishes Tachysurus
brashnikowi (16.26 . 106 ind. or 32.9 % of total biomass) and T. sinensis (4.32 . 106 ind. Материалы и методы Съемка в р. Амур выполнена в сентябре-октябре 2003 г. на НИС «Профессор
Солдатов» (тип «Ярославец»), в качестве орудия лова использован бим-трал (далее по
тексту трал). За основу при его создании взят мальковый бим-трал Расса, кроме того,
использовались детали конструкций некоторых других тралов [Ахлынов, 1954; Пахору
ков, 1980]. Стальная рама трала имеет размеры 2,5 х 0,9 м (площадь сечения — 2,25 м2)
(рис. 1). Сетной мешок трала двухслойный: наружный слой выполнен из траловой дели
с ячеей 40 х 40 мм, внутренний — из дели с ячеей 10 х 10 мм. Длина сетного мешка 7 м. Буксировка трала осуществлялась по одноваерной схеме на уздечке с двумя поводками. Траления выполнены на 9 участках, расположенных в 40–55 км (I), 65–70 (II), 140–150
(III), 390–397 (IV), 402–407 (V), 519–524 (VI), 590–595 (VII), 865–880 (VIII) и 944–958 км
(IX) от устья (рис. 2). 293 Кошелев В.Н., Колпаков Н.В. Рис. 1. Внешний вид бим-трала, использованного в съемке
Fig. 1. Configuration of beam-trawl used in survey
Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III
а Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII —
бон; VIII — Чепчики; IX — Владимировка
Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III —
l IV
Zh
bt
k
h
l V
K
i
k I l
d VI
Ch
ki I l
d VII Рис. 1. Внешний вид бим-трала, использованного в съемке
Fig. 1. Configuration of beam-trawl used in survey Рис. 1. Внешний вид бим-трала, использованного в съемке
Fig. 1. Configuration of beam-trawl used in survey Рис. 1. Внешний вид бим-трала, использованного в съемке
Fig. 1. Configuration of beam-trawl used in survey Рис. 1. Внешний вид бим-трала, использованного в съемкеi Рис. 1. Внешний вид бим-трала, использованного в съемке
Fig. 1. Configuration of beam-trawl used in survey р
Fig. 1. Configuration of beam-trawl used in survey Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про
тока Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока
Галбон; VIII — Чепчики; IX — Владимировка
Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi
channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galbon
channel; VIII — Chepchiki; IX — Vladimirovka Рис. 2. Материалы и методы Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про
а Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока
бон; VIII — Чепчики; IX — Владимировка Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про
тока Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока
Галбон; VIII — Чепчики; IX — Владимировка
Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi
channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galbon
channel; VIII — Chepchiki; IX — Vladimirovka Рис. 2. Карта-схема района работ: I — Еремейские острова; II — протока Тахта; III — про
тока Прокми; IV — протока Жеребцовская; V — о. Каргинский; VI — о. Ченки; VII — протока
Галбон; VIII — Чепчики; IX — Владимировка
Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi ;
;
д
р
Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prok
channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galb
channel; VIII — Chepchiki; IX — Vladimirovka ;
;
р
Fig. 2. Scheme of surveyed area: I — Yeremeiskiye Isles; II — Takhta channel; III — Prokmi
channel; IV — Zherebtsovskaya channel; V — Karginsky Island; VI — Chenki Island; VII — Galbon
channel; VIII — Chepchiki; IX — Vladimirovka Всего на участке Амура длиной 920 км выполнено 57 тралений общей протя
женностью 115,2 км, суммарная площадь облова составила 287,9 тыс. м2. Скорость
тралений варьировала от 5,5 до 9,0 км/ч (1,5–2,5 м/с), глубины — от 1,5 до 23,0 м. 294 Видовой состав и распределение рыб и креветок в русле нижнего Амура Длина ваера составляла от 4 до 6 глубин. Следует отметить наличие в русле Амура
большого количества «задевов» (стволов деревьев и т.п.), что создавало определенные
сложности при тралениях. Длина ваера составляла от 4 до 6 глубин. Следует отметить наличие в русле Амура
большого количества «задевов» (стволов деревьев и т.п.), что создавало определенные
сложности при тралениях. Все уловы разбирали до вида, определяли число пойманных особей каждого из
них. Биологический анализ рыб проводили по стандартным ихтиологическим мето
дикам [Правдин, 1966]. Материалы и методы У креветок измеряли общую длину тела от конца рострума до
конца тельсона (Lo, n = 30). Массу гидробионтов определяли с точностью ±0,1 г на
весах AND HL. Расчет численности вида вели отдельно для каждого из 9 обловленных участков,
после чего была определена его численность в реке. Численность на каждом из участков
рассчитывали по формуле рассчитывали по формуле N = Sx/(Kq), где S — площадь участка; x — суммарный улов, экз.; K — коэффициент уловистости;
q — площадь обловов на участке. Коэффициент уловистости бим-трала для выловлен
ных видов не определен, поэтому при расчетах он принят равным единице. Биомассу
рыб рассчитывали как произведение численности вида на среднюю массу его особей
в уловах. Точное определение координат районов тралений и скорости буксировки трала
производили с использованием прибора GPS, для измерения глубины воды в ходе тра
ления использовали эхолот «Wide 3D View». Номенклатура приведена в соответствии
с Каталогом рыб Эшмейера [http://researcharchive.calacademy.org/research/]. Результаты и их обсуждение Характеристика условий обитания гидробионтов в Амуре. Исследуемый
участок низовьев р. Амур имеет протяженность 960 км и проходит по территории со
сложным рельефом и геологическим строением. Большую его часть можно отнести к
горной стране со средне- и низкогорным рельефом с большим количеством межгор
ных впадин и равнин [Мордовин, 1996]. Климат низовьев реки обусловлен движением
воздушного потока в зимнее время с континента в сторону океана, а летом — наобо
рот. Зима характеризуется морозной, сухой и солнечной погодой, лето, как правило,
теплое, облачное и дождливое. Наибольшее количество осадков приходится на летний
период — 80–95 % годовой суммы [Мордовин, 1996]. В конце лета нижний участок
Амура подвергается влиянию тропических циклонов, сопровождающихся затяжными
дождями [Крюков и др., 2005]. Среднегодовая температура воздуха составляет в устье
Амура –2,4 оС, в г. Комсомольск-на-Амуре –0,6 оС, у Хабаровска +1,4 оС [Многолет
ние данные…, 1986; Соловьев, 1995; Болдовский, 2006]. Температурные условия в
течение года обусловливают длительность ледостава, который продолжается в Амуре
у Николаевска-на-Амуре в среднем 183 сут, у Хабаровска — 151 сут [Многолетние
данные…, 1986]. Толщина льда в конце зимы, в зависимости от района, варьирует от
0,7 до 1,8 м [Соловьев, 1995]. Река Амур имеет паводочный режим. Основное питание (около 90 %) реки бас
сейна Амура получают от летне-осенних муссонных дождей. Весенние паводки из-за
малоснежности формирует лишь небольшое половодье [Болдовский, 2006]. На теплый
период года (май-октябрь) приходится 87 % годового стока вод, на холодный (ноябрь-
апрель) — 13 % [Жабин и др., 2010]. Самые низкие уровни воды наблюдаются в конце
зимы. Максимальный подъем воды в паводок на Верхнем и Среднем Амуре составляет
10,0–11,0 м, на Нижнем Амуре — 6,0–7,0 м, в устье у Николаевска — 4,3 м [Мордовин,
1996]. Скорости течения на Нижнем Амуре в июне-июле вдоль фарватера составляют
в среднем у поверхности 1,12 м/с, у дна 0,67 м/с, в августе — соответственно 1,18 м/с
и 0,71 м/с [Соловьев, 1974]. Температура воды в разные месяцы на Нижнем Амуре варьирует в широких
пределах (0,1–22,7 оС). Она повышается от устья к границе среднего и нижнего течения 295 Кошелев В.Н., Колпаков Н.В. Кошелев В.Н., Колпаков Н.В. (г. Хабаровск). Средняя сумма тепла (градусо-дни) за год в устье Амура составляет 2389,
у Комсомольска-на-Амуре — 2832, у Хабаровска — 3034 [Многолетние данные…, 1986]. у
ур
у
р
Ширина реки на исследованном участке варьирует от 0,64 до 3,70 км. Глубины
достигают 40 м (р-н пос. Тыр), обычно — 5–10 м. Грунты нижнего течения Амура
представлены участками с песками, илами, песчано-илистыми и гравийно-галечнико
выми фракциями. Результаты и их обсуждение До 35 % (700 км2) площади русла нижнего течения Амура занимают
участки с динамически устойчивыми грунтами — с гравийно-галечниковой фракцией
[Соловьев, Свирский, 1976]. [
,
р
,
]
Кормовая база придонных и донных рыб Амура состоит главным образом из пред
ставителей зообентоса (без моллюсков) [Никольский, 1956]. По данным С.Е. Сиротского
с соавторами [2009] средняя плотность бентосного населения основного русла нижнего
Амура (без моллюсков) в теплое время года составила 2,8 тыс. экз./м2 при средней био
массе 14,7 г/м2. На песчаных биотопах зафиксированы минимальные значения биомассы
бентосных организмов (< 1 г/м2). На песках с примесью иловых отложений биомасса
бентоса достигает 5 г/м2, доминирующими группами зообентоса на этих грунтах явля
ются хирономиды и олигохеты. Максимум биомассы бентосных организмов отмечен на
гравийно-галечниковом субстрате у пос. М. Горький (388 км от устья) — 74,3 г/м2 и у
пос. Нижняя Гавань (180 км) — 56,6 г/м2. Основу сообществ здесь составляют личинки
ручейников. Отмечена общая тенденция увеличения биомассы зообентоса к устью Амура
[Сиротский и др., 2009]. Видовой состав и распределение рыб и креветок. Всего за период работ в
р. Амур выловлено 2584 экз. 22 видов рыб и 1077 экз. одного вида беспозвоночных
(табл. 1). Доминировали в уловах представители отрядов Cypriniformes (13 видов)
и Siluriformes (4 вида). Из встреченных в уловах рыб несомненный интерес с точки
зрения новых знаний, в частности о распределении и численности на протяжении об
следованных ≈1000 км, представляют виды, для которых предпочитаемым биотопом
является русловая часть реки. По литературным данным [Труды Амурской ихтиоло
гической экспедиции…, 1952, 1958; Никольский, 1956; Атлас…, 2002; Кошелев и др.,
2016] к обитателям русла реки и крупных проток (ширина > 200 м) можно отнести 3
вида из рода Tachysurus — китайскую косатку-скрипуна T. sinensis, косатку Бражни
кова T. brashnikowi и косатку-плеть T. ussuriensis, а также 4 вида пескарей — ящерного
Saurogobio dabryi, амурского обыкновенного Gobio cynocephalus, амурского белоперого
Romanogobio tenuicorpus и восьмиусого Gobiobotia pappenheimi. Кроме того, для калуги
Huso dauricus и амурского осетра Acipenser schrenckii река является главным местом
нагула [Никольский, 1956; Крыхтин, Горбач, 1994; Кошелев, 2010]. Таким образом,
из 22 видов рыб 9 донных видов являются типичными обитателями главного русла
Амура. Для остальных рыб, отмеченных в уловах, река — второстепенный биотоп, их
нагул приурочен большей частью к придаточной системе Амура — озерам и притокам. Из трех видов креветок, обитающих в бассейне р. Амур, в наших сборах отмечен
только Palaemon modestus. Этот придонно-пелагический вид встречается как в реках,
так и в озерах [Erchardt, Tiffan, 2016; Барабанщиков, Шаповалов, 2019]. Результаты и их обсуждение В Амуре
распространен от лимана до низовьев р. Биджан, в бассейне р. Уссури, оз. Ханка, в
верховьях Аргуни (озера Далай-нор, Буир-нур и др.) [Бирштейн, Виноградов, 1934;
Куренков, 1950; Боруцкий и др., 1952]. Средняя численность рыб в уловах трала на 9 участках работ варьировала от
390 до 63090 (8980) экз./км2. Вверх по течению реки численность рыб снижалась по
экспоненте (рис. 3), локальные максимумы обилия (63090 и 52180 экз./км2) отмечены
соответственно на участках II (65–70 км от устья) и IV (390–397 км). Креветка в уло
вах встречалась на расстоянии от 50 до 150 км от устья, ее численность составляла
2970–24080 (16260) экз./км2 и вверх по течению также снижалась. Увеличение численности рыб и креветки к устью Амура, по нашему мнению, об
условлено прежде всего состоянием кормовой базы данных видов, у которых в составе 296 Видовой состав и распределение рыб и креветок в русле нижнего Амура 297
Видовой состав и распределение рыб и креветок в русле нижнего Амура
Таблица 1
Видовой состав и численность рыб и беспозвоночных (N) на 9 участках (I–IX) русла Амура по данным траловой съемки (сентябрь-октябрь 2003 г.)
Table 1
Species composition and number of fishes and invertebrates (N) at the 9 parts (I–IX) of the Amur River channel by beam-trawl survey data
(September-October, 2003)
Вид
N, экз./км2
N общая,
млн экз. Стация
I
II
III
IV
V
VI
VII
VIII
IX
Ср. Результаты и их обсуждение значение
Рыбы
Acipenser schrenckii Brandt, 1869
880
1420
0
0
0
0
0
10
0
210
0,211
р
Huso dauricus (Georgi, 1775)
180
0
0
0
0
0
0
0
0
20
0,018
р
Tachysurus sinensis Lacepède, 1803
850
47120
420
710
0
170
0
0
0
2930
4,320
р, о
Tachysurus brashnikowi (Berg, 1907)
1450
60
2000
44180
0
560
1430
0
0
2160
16,26
р, о
Tachysurus argentivittatus (Regan, 1905)
0
0
0
0
0
40
0
0
0
3,4
0,010
р
Tachysurus ussuriensis (Dybowski, 1872)
0
0
0
800
0
560
40
50
80
110
0,435
р
Gobio cynocephalus Dybowski, 1869
450
890
210
360
530
300
170
0
10
180
0,530
п, р, о
Gobiobotia pappenheimi Kreyenberg, 1911
0
0
0
0
0
3570
840
100
0
390
1,480
р
Saurogobio dabryi Bleeker, 1871
3660
6940
2210
3200
3200
2280
1310
30
30
1460
4,430
р, о
Romanogobio tenuicorpus (Mori, 1934)
2810
5340
1680
2490
2130
1760
1180
210
260
1250
3,600
р
Sarcocheilichthys sinensis Bleeker, 1871
30
0
0
0
0
130
0
0
0
10
0,030
р, о
Sarcocheilichthys czerskii (Berg, 1914)
0
120
0
0
0
0
0
0
0
10
0,010
р, о
Hemibarbus maculatus Bleeker, 1871
250
1190
210
180
0
40
0
0
0
120
0,250
р, о
Cyprinus rubrofuscus Lacepède, 1803
0
0
0
0
0
0
0
0
10
3,4
0,001
р, о
Carassius gibelio (Bloch, 1782)
0
0
0
180
530
0
130
0
0
20
0,197
р, о
Hemiculter sp. 0
0
0
0
0
40
210
0
0
20
0,177
р, о
Rhodeus sp. Результаты и их обсуждение 30
0
0
0
0
0
80
0
0
10
0,070
п, р, о
Leuciscus waleckii (Dybowski, 1869)
30
0
0
0
0
40
0
0
0
10
0,012
р, о
Xenocypris macrolepis Bleeker, 1871
50
0
0
0
0
0
0
30
0
10
0,011
р, о
Lota lota (Linnaeus, 1758)
130
0
0
0
0
40
0
0
0
20
0,023
п, р
Pungitius bussei (Warpachowski, 1888)
0
0
110
0
0
0
0
0
0
3,5
0,032
п, р, о
Siniperca chuatsi (Basilewsky, 1855)
0
0
0
90
0
0
0
0
0
3,5
0,029
р, о
Итого рыбы
10780
63090
6840
52180
6400
9550
5390
430
390
8980
32,161
–
Беспозвоночные
Palaemon modestus (Heller, 1862)
24080
2970
7050
0
0
0
0
0
0
16260
4,887
р, о
Примечание. Стации обитания: п — притоки, р — русло Амура, о — пойменные озера. Для некоторых видов жирным шрифтом выделена пред
почитаемая стация. 297 Кошелев В.Н., Колпаков Н.В. Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2
Fig. 3. Variation of fish distribution density along the Amur River channel, ind./km2
N = 43127e-0,004L
R2 = 0,65
0
10000
20000
30000
40000
50000
60000
70000
0
100
200
300
400
500
600
700
800
900
1000
Плотность (N), экз./км2
Расстояние от устья (L), км Плотность (N), экз./км2 Расстояние от устья (L), км Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2
Fig. 3. Variation of fish distribution density along the Amur River channel, ind./km2 Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2
Fig. 3. Variation of fish distribution density along the Amur River channel, in Рис. 3. Изменение численности рыб вдоль русла р. Амур, экз./км2
Fig. 3. Variation of fish distribution density along the Amur River channel, ind./km2 пищи преобладают донные животные [Константинов, 1950; Егорова, 1952; Пикулева,
1952; Никольский, 1956; Кошелев, 2010]. Известно, что биомасса бентоса в нижнем
течении Амура по мере продвижения к устью неуклонно растет [Боруцкий и др., 1952;
Сиротский и др., 2009]. пищи преобладают донные животные [Константинов, 1950; Егорова, 1952; Пикулева,
1952; Никольский, 1956; Кошелев, 2010]. Известно, что биомасса бентоса в нижнем
течении Амура по мере продвижения к устью неуклонно растет [Боруцкий и др., 1952;
Сиротский и др., 2009]. Общая численность донных и придонных рыб в русле Амура по данным трало
вой съемки составила 32,161 млн экз., численность креветки — 4,887 млн экз. (табл. 1). Результаты и их обсуждение Наиболее массовыми были 5 видов рыб (в сумме 93,6 % по численности): косатка
Бражникова (50,6 %), ящерный пескарь (13,8 %), косатка-скрипун (13,4 %), амурский
белоперый (11,2 %) и восьмиусый (4,6 %) пескари (рис. 4). Рис. 4. Соотношение (% по численности) массовых видов рыб в уловах трала в русле
р. Амур (сентябрь-октябрь 2003 г.)
Fig. 4. Percentage of common fish species number in catches of beam-trawl from the Amur
River in September-October of 2003
7VLQHQVLV
7EUDVKQLNRZL
*SDSSHQKHLPL
6GDEU\L
5WHQXLFRUSXV
ɉɪɨɱɢɟ
ɦɥɧɷɤɡ Рис. 4. Соотношение (% по численности) массовых видов рыб в уловах трала в русле
р. Амур (сентябрь-октябрь 2003 г.)
Fig. 4. Percentage of common fish species number in catches of beam-trawl from the Amur
River in September-October of 2003 По нашим расчетам общая биомасса донных и придонных видов рыб в русле
Амура на обследованной площади 2234,52 км2 составила 583,8 т (0,261 г/м2), биомасса
креветки на площади 486,75 км2 — 8,8 т (0,018 г/м2). По участкам биомасса рыб из
менялась в пределах 0,012–2,572 г/м2 (в среднем 0,450 ± 0,270 г/м2), биомасса креветок
была равна 0,005–0,044 г/м2 (0,021 ± 0,012 г/м2) (рис. 5). При этом 82,4 % биомассы рыб
было приурочено к нижнему участку, 40–400 км от устья, вся биомасса креветки — к
участку 50–150 км от устья. 298 Видовой состав и распределение рыб и креветок в русле нижнего Амура
Рис. 5. Изменчивость биомассы рыб (а) и креветки (б) вдоль русла р. Амур (сентябрь-
октябрь 2003 г.)
Fig. 5. Результаты и их обсуждение Variation of fish (а) and prawn (б) biomass in the Amur River in September-October,
2003, by I–IX areas along the channel, g/m2
По биомассе на исследуемом участке доминировали: косатка-скрипун (32,9 %),
косатка Бражникова (24 0 %) ящерный пескарь (12 6 %) косатка-плеть (9 7 %) бе
0,346
2,572
0,629
0,118
0,163
0,067
0,012
0,014
0,109
0,0
0,3
0,6
0,9
1,2
1,5
1,8
2,1
2,4
2,7
I
II
III
IV
V
VI
VII
VIII
IX
Биомасса, г/м2
Участок
0,044
0,005
0,013
0,000
0,010
0,020
0,030
0,040
0,050
I
II
III
IV
V
VI
VII
VIII
IX
Биомасса, г/м2
Участок
б
а Видовой состав и распределение рыб и креветок в русле нижнего Амура Видовой состав и распределение рыб и креветок в русле нижнего Амура 0,346
2,572
0,629
0,118
0,163
0,067
0,012
0,014
0,109
0,0
0,3
0,6
0,9
1,2
1,5
1,8
2,1
2,4
2,7
I
II
III
IV
V
VI
VII
VIII
IX
Биомасса, г/м2
Участок
а а Участок
0,044
0,005
0,013
0,000
0,010
0,020
0,030
0,040
0,050
I
II
III
IV
V
VI
VII
VIII
IX
Биомасса, г/м2
У
б б Участок Рис. 5. Изменчивость биомассы рыб (а) и креветки (б) вдоль русла р. Амур (сентябрь-
октябрь 2003 г.)
Fig. 5. Variation of fish (а) and prawn (б) biomass in the Amur River in September-October,
2003, by I–IX areas along the channel, g/m2 Рис. 5. Изменчивость биомассы рыб (а) и креветки (б) вдоль русла р. Амур (сентябрь-
октябрь 2003 г.)
Fig. 5. Variation of fish (а) and prawn (б) biomass in the Amur River in September-October,
2003, by I–IX areas along the channel, g/m2 ь 2003 г.)
Fig. 5. Variation of fish (а) and prawn (б) biomass in the Amur River in September-October,
by I–IX areas along the channel, g/m2 По биомассе на исследуемом участке доминировали: косатка-скрипун (32,9 %),
косатка Бражникова (24,0 %), ящерный пескарь (12,6 %), косатка-плеть (9,7 %), бе
лоперый пескарь (7,8 %), амурский осетр (5,6 %), амурский обыкновенный пескарь
(2,4 %) и калуга (0,6 %) (рис. 6). По данным траловой съемки, выполненной сходным по конструкции тралом
в оз. Ханка (бассейн р. Амур) в 2018 г., биомасса рыб в придонных слоях в теплый
период года составила 0,199 г/м2, биомасса креветок — 0,047 г/м2 [Барабанщиков,
Шаповалов, 2019]. Это цифры одного порядка с полученными нами данными. Однако 299 Кошелев В.Н., Колпаков Н.В. Рис. 6. Соотношение (% по массе) рыб в траловых уловах в русле р. Амур (сентябрь-
октябрь 2003 г.)
Fig. 6. Характеристика массовых видов. В уловах отмечены оба обитающих в реке Амур
вида осетровых — калуга (n = 7) и амурский осетр (n = 60). Численность амурского
осетра составила 211 тыс. экз., калуги — 18 тыс. экз. В уловах присутствовали только
неполовозрелые особи, у амурского осетра в возрасте 0+…2+ лет (длина 22–47 см), у
калуги в возрасте 0+…1+ лет (30–40 см) (табл. 3). Результаты и их обсуждение Percentage of fish species biomass in catches of beam-trawl from the Amur River in
September-October, 2003
32,9%
24,0%
12,6%
9,7%
7,8%
5,6%
2,4%
0,6%
4,4%
T. sinensis
T. brashnikowi
S. dabryi
T. ussuriensis
R. tenuicorpus
A. schrenckii
G. cynocephalus
H. dauricus
Прочие
583,8 т 32,9%
24,0%
12,6%
9,7%
7,8%
5,6%
2,4%
0,6%
4,4%
T. sinensis
T. brashnikowi
S. dabryi
T. ussuriensis
R. tenuicorpus
A. schrenckii
G. cynocephalus
H. dauricus
Прочие
583,8 т 12,6% Рис. 6. Соотношение (% по массе) рыб в траловых уловах в русле р. Амур (сентябрь-
октябрь 2003 г.)
Fig. 6. Percentage of fish species biomass in catches of beam-trawl from the Amur River in
September-October, 2003 следует отметить довольно существенные различия в составе наиболее массовых
видов. В оз. Ханка в уловах рыб по биомассе, так же как и в русле р. Амур, преоб
ладала косатка Бражникова (60,1 %), вместе с тем здесь существенную долю уловов
составляли молодь горбушек Chanodichthys spp. (17,9 %) и уклея Culter alburnus
(5,8 %), а также интродуцент китайская лапша-рыба Protosalanx sinensis (8,8 %) и
уссурийская востробрюшка Hemiculter lucidus (4,7 %). Биомасса рыб на Нижнем Дону по данным съемки бим-тралом составила от 8,01
до 12,27 г/м2 [Живоглядов и др., 2019], что существенно выше наших оценок. Правда,
в последнем случае для бим-трала со сходными характеристиками (1,9 х 0,6 м) ис
пользован коэффициент уловистости 0,1. С учетом этой особенности методического
подхода биомасса рыб в нижнем течении Дона (0,8–1,2 г/м2) будет в 3–6 раз выше,
чем в среднем в русле Амура и в оз. Ханка, но вполне сопоставима с биомассой рыб
в нижнем течении Амура (табл. 2). Наши данные также весьма близки к оценкам, по
лученным для нижнего течения рек Пенжина и Таловка (северо-западная Камчатка)
[Коваль и др., 2015] (табл. 2). 300
Таблица 2
Биомасса рыб в ряде водоемов России (коэффициент уловистости 1)
Table 2
Fish Fish biomass in some water bodies of Russia (catchability coefficient 1.0)
Водоем
Биомасса рыб,
г/м2
Орудие отлова
Источник данных
Р. Дон
0,8–1,2
Бим-трал
Живоглядов и др., 2019
Р. Пенжина (30–70 км
от устья)
0,9
Бим-трал, закидной
невод
Коваль и др., 2015
Р. Таловка (25–45 км
от устья)
0,7
Русло р. Амур: 40–960 км
0,3
Бим-трал
Наши данные
40–400 км
0,6
40–70 км
1,5
Оз. Ханка
0,2
Бим-трал
Барабанщиков, Шаповалов, 2019
Характеристика массовых видов. В уловах отмечены оба обитающих в реке Амур
вида осетровых — калуга (n = 7) и амурский осетр (n = 60). Численность амурского
осетра составила 211 тыс. Результаты и их обсуждение экз., калуги — 18 тыс. экз. В уловах присутствовали только
неполовозрелые особи, у амурского осетра в возрасте 0+…2+ лет (длина 22–47 см), у
калуги в возрасте 0+…1+ лет (30–40 см) (табл. 3). Таблица
Биомасса рыб в ряде водоемов России (коэффициент уловистости 1)
T bl Биомасса рыб в ряде водоемов России (коэффициент уловистости 1) Table 2
Fish Fish biomass in some water bodies of Russia (catchability coefficient 1.0)
Водоем
Биомасса рыб,
г/м2
Орудие отлова
Источник данных
Р. Дон
0,8–1,2
Бим-трал
Живоглядов и др., 2019
Р. Пенжина (30–70 км
от устья)
0,9
Бим-трал, закидной
невод
Коваль и др., 2015
Р. Таловка (25–45 км
от устья)
0,7
Русло р. Амур: 40–960 км
0,3
Бим-трал
Наши данные
40–400 км
0,6
40–70 км
1,5
Оз. Ханка
0,2
Бим-трал
Барабанщиков, Шаповалов, 2019 Характеристика массовых видов. В уловах отмечены оба обитающих в реке Амур
вида осетровых — калуга (n = 7) и амурский осетр (n = 60). Численность амурского
осетра составила 211 тыс. экз., калуги — 18 тыс. экз. В уловах присутствовали только
неполовозрелые особи, у амурского осетра в возрасте 0+…2+ лет (длина 22–47 см), у
калуги в возрасте 0+…1+ лет (30–40 см) (табл. 3). 300 Видовой состав и распределение рыб и креветок в русле нижнего Амура Таблица 3
Биологические показатели массовых видов рыб в уловах
Table 3
Biological characters of most common fish species in the beam-trawl catches
Вид
Длина тела, см
Масса, г
n
Калуга
33,30 ± 1,53
30–40
186,4 ± 28,7
130–313
7
Амурский осетр
31,60 ± 0,84
22–47
153,6 ± 13,6
43–474
60
Косатка-скрипун
12,3 ± 1,4
4,5–19,0
44,4 ± 15,8
1,7–158,0
11
Косатка Бражникова
6,8 ± 0,5
3,2–16,5
8,6 ± 1,9
0,6–77,8
50
Амурский обыкновенный пескарь
11,9 ± 0,3
10,6–14,0
25,6 ± 2,9
15,7–39,0
9
Белоперый пескарь
9,7 ± 0,2
6,8–13,6
12,7 ± 0,9
3,7–32,5
55
Ящерный пескарь
10,5 ± 0,5
2,4–18,4
16,6 ± 2,0
0,1–60,5
73
Восьмиусый пескарь
3,8 ± 0,1
2,5–5,8
0,90 ± 0,04
0,2–2,7
108
Примечание. Над чертой — пределы изменчивости параметра, под чертой — среднее
значение параметра и ошибка средней. Т
Биологические показатели массовых видов рыб в уловах Данные высококормные
участки были зафиксированы в диапазоне глубин 8–20 м, что сходно с обследованными
глубинами на II и IV участках (7–17 м). Анализ питания косатки-скрипуна и косатки
Бражникова показал, что на этом участке реки ручейники составляют 98–100 % их
рациона [Никольский, 1956]. Можно полагать, что высокое обилие этих косаток в
нижнем течении Амура приурочено именно к локальным участкам с массовыми по
селениями ручейников. ру
Косатка-плеть (0,435 млн экз.) отмечена от района с. Владимировка (20-й км
Среднего Амура) до протоки Жеребцовской (398-й км Нижнего Амура). Ее распростра
нение полностью совпадает с определенным по материалам предыдущих исследований. По-прежнему северная граница распространения данного вида проходит примерно в
одном районе. По данным Г.В. Никольского [1956] это с. Сухановка (370-й км Ниж
него Амура), по нашим данным это район протоки Жеребцовской (398-й км Нижнего
Амура). В уловах присутствовали только половозрелые особи (> 20 см) длиной от 22
до 48 см и массой от 107 до 514 г. ру
Косатка-плеть (0,435 млн экз.) отмечена от района с. Владимировка (20-й км
Среднего Амура) до протоки Жеребцовской (398-й км Нижнего Амура). Ее распростра
нение полностью совпадает с определенным по материалам предыдущих исследований. нение полностью совпадает с определенным по материалам предыдущих исследований. По-прежнему северная граница распространения данного вида проходит примерно в
одном районе. По данным Г.В. Никольского [1956] это с. Сухановка (370-й км Ниж
него Амура), по нашим данным это район протоки Жеребцовской (398-й км Нижнего
Амура). В уловах присутствовали только половозрелые особи (> 20 см) длиной от 22
до 48 см и массой от 107 до 514 г. Такие некрупные рыбы, как пескари, не являются объектами промысла в рос
сийских водах Амура, в отличие от КНР, где повсеместно встречаются на рыбных
рынках. В связи с отсутствием промысла изучение пескарей Амура было эпизоди
ческим и большей частью прошло в период Амурской экспедиции (1945–1949 гг.)
[Никольский, 1956; Труды Амурской ихтиологической экспедиции…, 1958]. По
нашим данным 3 из наиболее массовых видов пескарей — ящерный (4,43 млн экз.),
белоперый (3,60 млн экз.) и амурский обыкновенный (0,53 млн экз.) — широко
распространены в русловой части нижнего течения Амура (см. табл. 1). Размерно-
массовые характеристики этих видов приведены в табл. 3. Восьмиусый пескарь (1,48 млн экз.) отмечен в уловах от района с. Владимировка
(23–28-й км Среднего Амура) до района о.Ченки (528-й км Нижнего Амура). Основной
улов, 87 из 117 экз. (74,3 %), пришелся на участок в районе о. Ченки. Т
Биологические показатели массовых видов рыб в уловах Biological characters of most common fish species in the beam-trawl catches Примечание. Над чертой — пределы изменчивости параметра, под чертой — среднее
ие параметра и ошибка средней. Из 67 экз. осетровых 60 отловлены на двух самых нижних участках I и II (со
ответственно 45–50 и 65–70 км от устья). Исходя из полученных данных можно
полагать, что оба вида в первые годы жизни скатываются на нагул в предустьевой
участок Амура. Впоследствии в возрасте 3–5 лет они осваивают солоноватые воды,
выходя для нагула на высококормные участки Амурского лимана [Кошелев, 2006]. За
все время изучения амурских осетровых в литературе имеются сведения о поимке 6
мальков (сеголеток) амурского осетра и 27 сеголеток калуги [Солдатов, 1915; Соин,
1951; Юхименко, 1963]. Таким образом, данные о численности молоди начальных
возрастных групп осетровых в р. Амур являются первыми в истории изучения
этих видов. Более ранние [Крыхтин, 1979] и поздние работы [Кошелев и др., 2016],
судя по размерному составу уловов плавных сетей, охватывали наблюдениями рыб
крупнее и старше. Наиболее многочисленными в уловах в нижнем течении Амура были косатка
Бражникова (16,42 млн экз.) и китайская косатка-скрипун (4,32 млн экз.) (см. табл. 1). Косатка Бражникова отмечена на 6 из 9 обследованных участков на глубинах от 2 до
17 м. По мнению Г.В. Никольского [1956], данный вид косаток широко распространен
в среднем и нижнем течении Амура. Плотность ее скоплений варьировала от 0,6 экз./га
в районе протоки Тахта до 441,8 экз./га в районе протоки Жеребцовской. В уловах
встречались как молодь, так и половозрелые особи (см. табл. 3). Косатка-скрипун
присутствовала в уловах почти на 500-километровом участке русла Нижнего Амура
от о. Ченки (524-й км) до района Еремейских островов (50-й км). По мнению Г.В. Ни
кольского [1956], это самая многочисленная косатка нижнего и среднего течения Амура,
что не подтверждается нашими данными (см. табл. 1). Плотность скоплений данного
вида варьировала от 1,7 экз./га в районе о. Ченки до 471,2 экз./га в районе протоки
Тахта. Косатка-скрипун отмечена на глубинах от 3 до 16 м. Формирование плотных скоплений этих двух косаток (табл. 1) на II и IV участ
ках, по-видимому, обусловлено комплексом благоприятных факторов, к основным из
которых можно отнести наличие агрегаций зообентоса и гидрологические условия
(скорости течения и глубины). В нижней части Амура (0–400 км) отмечено несколь
ко мест с биомассой бентоса (основу которого составляют ручейники), во много раз 301 Кошелев В.Н., Колпаков Н.В. превышающей средние показатели [Сиротский и др., 2009]. Т
Биологические показатели массовых видов рыб в уловах Все восьмиусые
пескари пойманы на глубинах до 10 м, а большинство (74,3 %) — на глубинах от 2 до 7 м. у
у
Креветка Palaemon modestus встречалась только в уловах на 100-километровом
участке реки от протоки Прокми (145-й км) до района Еремейских островов (50-й км). Распределение креветок неоднородно, как уже было отмечено, по мере приближения к
устью Амура происходило увеличение уловов. Все креветки пойманы на глубинах от
1,5 до 10,0 м, основная масса — от 3,0 до 6,0 м. В уловах присутствовали как молодь,
так и половозрелые особи. р
Биологические показатели креветки P. modestus в траловых уловах: длина —
35,0–63,0 (в среднем 51,3 ± 1,5) мм, масса — 0,35–3,45 (1,81 ± 1,50) г, n = 30. Анализ итогов работы на р. Амур свидетельствует, что сконструированный сотруд
никами Хабаровского ТИНРО бим-трал позволяет проводить работы по отлову рыб и
беспозвоночных на большей части русла Амура и в его крупных протоках. Данный трал
постоянно идет по дну, не всплывая в толщу воды. Тем самым выполняется основное
условие лова донных рыб и креветок — постоянный контакт трала с дном [Кушнарен
ко, 1975]. Данный трал устойчив к «задевам», перескакивая или выворачивая их. Трал
имеет высокую горизонтальную устойчивость и не переворачивается. Более длинная
рама по сравнению с мальковым бим-тралом Расса обеспечивает, соответственно, и
большую (в 2,5 раза) общую площадь облова при одинаковой длине траления. Особен
ности конструкции позволяют работать по одноваерной схеме, используя только одну
лебедку. Трал достаточно прост и надежен в эксплуатации. Данный трал возможно
рекомендовать к использованию на других крупных реках России. Благодарности Авторы выражают благодарность за помощь в сборе и обработке материала со
трудникам Хабаровского филиала ТИНРО С.А. Иванову и Ж.С. Литовченко. Соблюдение этических стандартов Все применимые международные, национальные и/или институциональные
принципы использования животных были соблюдены. Информация о всех пойманных
рыбах была включена в статью. Библиографические ссылки на все использованные в
работе данные других авторов оформлены в соответствии с правилами данного издания. Информация о вкладе авторов В.Н. Кошелев — сбор биологического материала, систематизация первичных
данных, подготовка первой версии статьи, Н.В. Колпаков — систематизация первичных
данных, редактирование и подготовка окончательного варианта рукописи. Заключение По данным съемки с использованием бим-трала осенью 2003 г. (сентябрь-октябрь)
на нижнем участке русла р. Амур (40–960 км) в уловах встречались 22 вида рыб и 1 вид 302 Видовой состав и распределение рыб и креветок в русле нижнего Амура беспозвоночных. Наибольшим числом видов были представлены отряды Cypriniformes
(13 видов) и Siluriformes (4 вида). Суммарная численность донных и придонных рыб
на обследованном участке составила 32,161 млн экз., численность креветки Palaemon
modestus — 4,887 млн экз. Самыми массовыми среди рыб стали промысловые виды
косаток — косатка Бражникова (16,26 млн экз.) и косатка-скрипун (4,32 млн экз.). Вы
явлен значительный рост численности рыб и креветок по мере приближения к устью
Амура, что связано с увеличением биомассы кормового бентоса в том же направлении. В зависимости от участка, биомасса рыб варьировала в пределах от 0,012 до 2,572 г/м2 (в
среднем 0,450 ± 0,270 г/м2). Сходная ситуация с креветкой — 0,005–0,044 г/м2 (в среднем
0,021 ± 0,012 г/м2). Общая биомасса рыб и креветки составила соответственно 583,8 и
8,8 т. Подавляющая часть биомассы рыб (82,4 %) была сконцентрирована на нижнем
участке реки (40–400 км от устья), вся биомасса креветок — на участке 50–150 км
от устья. Среди рыб лидерами по биомассе стали косатка-скрипун (32,9 %), косатка
Бражникова (24,0 %), ящерный пескарь (12,6 %) и косатка-плеть (9,7 %). Опробованный
в съемке 2003 г. бим-трал позволяет успешно решить проблему количественного учета
донных и придонных рыб Амура на большей части его русла и в крупных протоках. Финансирование работы Исследование не имело спонсорской поддержки. Список литературы Аксютина З.М. Элементы математической оценки результатов наблюдений в биологи
ческих и рыбохозяйственных исследованиях : моногр. — М. : Пищ. пром-сть, 1968. — 288 с. Атлас пресноводных рыб России / под ред. Ю.С. Решетникова. — М. : Наука, 2002. —
Т. 1. — 379 с. Ахлынов И.Я. Устройство трала и техника тралового лова : моногр. — М. : Пищепром-
издат, 1954. — 84 с. Барабанщиков Е.И., Шаповалов М.Е. Распределение и динамика количественных
показателей дальневосточных пресноводных креветок (сем. Palaemonidae) в оз. Ханка в летне-
осенний период 2018 года // Чтения памяти Владимира Яковлевича Леванидова. — 2019. — Вып. 8. — C. 23–27. doi.org/10.25221/levanidov.08.03. Бирштейн Я.А., Виноградов Л.Г. Пресноводные Decapoda СССР и их географическое
распространение // Зоол. журн. — 1934. — Т. 13, вып. 1. — С. 39–70. Болдовский Н.В. Региональная гидрогеология юга Дальнего Востока России : учеб. по
ие. — Хабаровск : Тихоокенск. гос. ун-т, 2006. — 101 с. Боруцкий Е.В., Ключарева О.А., Никольский Г.В. Донные беспозвоночные (зообен
тос) Амура и их роль в питании амурских рыб // Тр. Амурской ихтиологической экспедиции
1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — С. 5–139. 303 Кошелев В.Н., Колпаков Н.В. Вдовин А.Н., Мизюркин М.А. Сравнение оценок обилия рыб по данным учетных работ
лами разных конструкций // Вопр. рыб-ва. — 2011. — Т. 12, № 1(45). — С. 150–161. Егорова М.Н. Материалы по питанию косатки-плети (Liocassis ussuriensis Dyb.) и косатки
Герценштейна (Liocassis herzensteini Berg) в бассейне Амура // Тр. Амурской ихтиологической
экспедиции 1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — С. 449–490. Жабин И.А., Абросимова А.А., Дубина В.А., Некрасов Д.А. Влияние стока р. Амур на
гидрологические условия Амурского лимана и Сахалинского залива Охотского моря в период
весенне-летнего паводка // Метеорол. и гидрол. — 2010. — № 4. — С. 93–100. Живоглядов А.А., Живоглядова Л.А., Жердев Н.А. и др. Результаты комплексных ис
следований сообществ Нижнего Дона в 2017 г. : препринт / Researchgate. — 2019. — 7 с. DOI:
10.13140/RG.2.2.15295.15521. Коваль М.В., Горин С.Л., Калугин А.А. Экологическая характеристика сообщества мо
лоди рыб и нектобентоса гиперприливного эстуария рек Пенжина и Таловка (северо-западная
Камчатка) в августе 2014 г. // Исслед. водн. биол. ресурсов Камчатки и сев.-зап. части Тихого
океана. — 2015. — Вып. 37. — С. 164–191. DOI: 10.15853/2072-8212.2015.37.164-191. Константинов А.С. Хирономиды бассейна р. Амур и их роль в питании амурских рыб // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 147–286. Список литературы h
k
d
(
б Константинов А.С. Хирономиды бассейна р. Амур и их роль в питании амурских рыб // Тр. Амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 147–286. Кошелев В.Н. Амурский осетр Acipenser schrenckii Brandt, 1869 (распределение, биоло
гия искусственное воспроизводство) : дис
канд биол наук
Хабаровск 2010
188 с ур
д ц
,
Кошелев В.Н. Амурский осетр Acipenser schrenckii Brandt, 1869 (распределение, биоло
гия, искусственное воспроизводство) : дис. … канд. биол. наук. — Хабаровск, 2010. — 188 с. у
р
)
у
р
Кошелев В.Н. Изучение распределения молоди калуги и амурского осетра в лимане
Амура в 2005 г. // Ресурсы и экологические проблемы Дальнего Востока : мат-лы межрегион. науч.-практ. конф. — Хабаровск : ДВГГУ, 2006. — С. 180–184. Кошелев В.Н., Шмигирилов А.П., Рубан Г.И. Распределение, численность и размер
ная структура популяций калуги Acipenser dauricus и амурского осетра A. schrenckii в нижнем
Амуре и Амурском лимане // Вопр. ихтиол. — 2016. — Т. 56, № 2. — С. 156–162. DOI: 10.7868/
S0042875216020090. Крюков В.Г., Воронов Б.А., Гаврилов А.В., Макаров А.В. Река Амур: проблемы и пути
их решения (результаты деятельности Координационного комитета по устойчивому развитию
бассейна р. Амур) : моногр. — Хабаровск : Приамурское географическое общество, 2005. — 153 с. Крыхтин М.Л. Современное состояние и перспективы развития осетрового хозяйства в
бассейне р. Амур // Биологические основы развития осетрового хозяйства в водоемах СССР. —
М. : Наука, 1979. — С. 68–74. у
Крыхтин М.Л., Горбач Э.И. Осетровые рыбы Дальнего Востока // Экономическая жизнь
Дальнего Востока. — 1994. — Т. 1, № 3. — С. 86–91. Куренков И.И. К биологии дальневосточных пресноводных креветок // Тр. Амурской
ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 379–390. Кушнаренко А.И. Поведение молоди рыб у сетного полотна и в зоне действия малькового
трала // Вопр. ихтиол. — 1975. — Т. 15, вып. 5(94). — С. 871–878. р
р
(
)
Лобырев Ф.С. Оценивание численности рыб на основе моделирования работы жаберных
сетей : автореф. дис. … канд. биол. наук. — М. : МГУ им. М.В. Ломоносова, 2008. — 22 с. р ф д
д
у
,
Многолетние данные о режиме и ресурсах поверхностных вод суши. Т. 1, вып. 19 :
Бассейны Амура (без бассейнов Шилки, Амгуни, Амазара, Уссури) и Уды / ред. И.С. Якорь. —
М. : Гидрометеоиздат, 1986. — 412 с. р ф
у
Многолетние данные о режиме и ресурсах поверхностных вод суши. Список литературы Т. 1, вып. 1
Бассейны Амура (без бассейнов Шилки, Амгуни, Амазара, Уссури) и Уды / ред. И.С. Якорь М. : Гидрометеоиздат, 1986. 412 с. Мордовин А.М. Годовой и сезонный сток рек бассейна Амура. — Хабаровск, 1996. — 37 с. Никольский Г.В. Амурская ихтиологическая экспедиция 1945–1949 гг. // Тр. Амурской
ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1950. — Т. 1. — С. 1–18. д ц
,
Никольский Г.В. Рыбы бассейна Амура : моногр. — М. : АН СССР, 1956. — 553 с. Новомодный ГВ
Золотухин С Ф
Шаров П О Рыбы Амура: богатство и кризис : д ц
,
Никольский Г.В. Рыбы бассейна Амура : моногр. — М. : АН СССР, 1956. — 553 с Новомодный Г.В., Золотухин С.Ф., Шаров П.О. Рыбы Амура: богатство и кризи
аналит. обзор. — Владивосток : Апельсин, 2004. — 63 с. Пасечник О.И., Шмигирилов А.П. Оценка численности амурской кеты Oncorhynchus
keta (Walbaum, 1792) по результатам мечения // Чтения памяти Владимира Яковлевича Лева
нидова. — Владивосток : Дальнаука, 2008. — Вып. 4. — С. 294–303. Пахоруков А.М. Изучение распределения молоди рыб в водохранилищах и озерах :
моногр. — М. : Наука, 1980. — 65 с. Пикулева В.А. Питание косатки-скрипуна (Psevdobagrus fulvidraco Rich.) и малой косатки
(Liocassis brashnikovi Berg) в бассейне Амура // Тр. Амурской ихтиологической экспедиции
1945–1949 гг. — М. : МОИП, 1952. — Т. 3. — С. 435–448. ,
Правдин И.Ф. Руководство по изучению рыб (преимущественно пресноводных) :
моногр. — 4-е изд., перераб. и доп. — М. : Пищепромиздат, 1966. — 375 с. 304 Видовой состав и распределение рыб и креветок в русле нижнего Амура Пробатов А.Н. Материалы по изучению осетровых рыб Амура // Учен. зап. Пермского
та. — 1935. — Т. 1, вып. 1. — С. 33–72. Сечин Ю.Т. Методические указания по оценке численности рыб в пресноводных водо
емах. — М. : ВНИИПРХ, 1990. — 50 с. Сиротский С.Е., Макарченко Е.А., Макарченко М.А. Характеристика бассейна реки
Амур по составу зообентоса // Вопр. рыб-ва. — 2009. — Т. 10, № 3(39). — С. 453–467. Соин С.Г. Материлы по развитию осетровых р. Амур // Тр. Амурской ихтиологической
экспедиции 1945–1949 гг. — М. : МОИП, 1951. — Т. 2. — С. 223–232. Солдатов В.К. Обзор исследований, произведенных на Амуре в 1909–1913 гг.; Исследо
вание осетровых Амура : Материалы к познанию русского рыболовства. — Петроград : тип. В.Ф. Киршбаума (отд-ние), 1915. — Т. 3, вып. 12. — 415 с. р
у
(
),
,
Соловьев И.А. Список литературы 64. — P. 632–642. doi.org/10.1111/j.1095-8649. 2004.00327.x.i Zajicek P., Wolter Ch. The gain of additional sampling methods for the fish-based assessment
of large rivers // Fish. Res. — 2018. — Vol. 197. — P. 15–24. Список литературы Амуролиманский русловой процесс и водные пути : моногр. — Влади
восток : ДВО РАН, 1995. — 271 с. Соловьев И.А. Русловой процесс и водные пути Амурского лимана : моногр. — Влади
восток, 1974. — 290 с. Соловьев И.А., Свирский В.Г. Гидрологическая обстановка Нижнего Амура и ее роль в
воспроизводстве осетра и калуги // Биология рыб Дальнего Востока. — Владивосток : ДВГУ,
1976. — С. 70–74. Труды амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1952. —
Т. 3. — 512 с. Труды амурской ихтиологической экспедиции 1945–1949 гг. — М. : МОИП, 1958. —
Т. 4. — 358 с. Юхименко С.С. Питание амурского осетра и калуги в нижнем течении Амура // Вопр. ихтиол. — 1963. — Т. 3, вып. 2(27). — С. 311–318. de Souza M.R., Fagundes L., von Seckendorff R.W. et al. Construction characteristics of a
beam trawl for catching young shrimps in estuaries // Rev. Bras. Eng. Pesca. — 2010. — Vol. 5, №
3. — P. 42–50. Dettmers J.M., Gutreuter S., Wahl D.H., Soluk D.A. Patterns and abundance in main channels
of the upper Mississippi River system // Can. J. Fish. Aquat. Sci. — 2001. — Vol. 58. — P. 933–942. Erchardt J.M., Tiffan K.F. Ecology of nonnative Siberian prawn (Palaemon modestus) in the
l
S
k Ri
W hi
t
USA // A
ti E
l
2016
V l 50 №4
P 607 621 Dettmers J.M., Gutreuter S., Wahl D.H., Soluk D.A. Patterns and abundance in main channels
of the upper Mississippi River system // Can. J. Fish. Aquat. Sci. — 2001. — Vol. 58. — P. 933–942. y
Erchardt J.M., Tiffan K.F. Ecology of nonnative Siberian prawn (Palaemon modestus) in the
lower Snake River, Washington, USA // Aquatic Ecology. — 2016. — Vol. 50, № 4. — P. 607–621.i Herzog D.P., Barko V.A., Scheibe J.S. et al. Efficacy of a benthic trawl for sampling small-bod
ied fishes in large river systems // North American Journal of Fisheries Management. — 2005. — Vol. 25. — P. 594–603. Ái Szalóky Z., György Á.I., Tóth B. et al. Application of an electrified benthic frame trawl for
sampling fish in a very large European river (the Danube River) — Is offshore monitoring necessary? //
Fish. Res. — 2014. — Vol. 151. — P. 12–19. doi.org/10.1016/j.fishres.2013.12.004.i g
ji
Wolter C., Freyhof J. Diel distribution patterns of fishes in a temperate large lowland rive
J. Fish Biol. — 2004. — Vol. References Aksyutina, Z.M., Elementy matematicheskoi otsenki rezul’tatov nablyudenii v biologicheskikh
i rybokhozyaistvennykh issledovaniyakh (Elements of Mathematical Evaluation of the Results of Observations in Biological and Fishery Research), Moscow: Pishchevaya Promyshlennost’, 1968. Atlas presnovodnykh ryb Rossii (Atlas of Freshwater Fishes in Russia), Reshetnikov, Yu.S.,
Moscow: Nauka, 2002, vol. 1.i ,
,
,
Akhlynov, I.Ya., Ustrojstvo trala i tekhnika tralovogo lova (Trawl device and trawl fishing
technique), Moscow: Pishchepromizdat, 1954. Barabanshchikov, Ye.I. and Shapovalov, M.Ye., Distribution and dynamics of quantitative
indicators of Far Eastern freshwater shrimp (family Palaemonidae) in Lake Hanka in the summer-
autumn period of 2018, in Chteniya pamyati Vladimira Yakovlevicha Levanidova (Vladimir Yakov
levich Levanidov’s Biennial Memorial Meetings), Vladivostok: Dal’nauka, 2019, vol. 8, pp. 23–27. Birshtein, Y.A. and Vinogradov, L.G., Freshwater Decapoda of the USSR and their geographi
cal distribution, Zool. Zh., 1934, vol. 13, no. 1, pp. 39–70. pp
Boldovsky, N.V., Regional’naya gidrogeologiya yuga Dal’nego Vostoka Rossii (Regional
hydrogeology of the south of the Russian Far East), Khabarovsk: Tikhookeanskiy. gos. un-t, 2006. 305 Кошелев В.Н., Колпаков Н.В. Borutsky, E.V., Klyuchareva, O.A., and Nikolsky, G.V., Benthic invertebrates (zoobenthos) of
Amur and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949
gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 5–139. Borutsky, E.V., Klyuchareva, O.A., and Nikolsky, G.V., Benthic invertebrates (zoobenthos Borutsky, E.V., Klyuchareva, O.A., and Nikolsky, G.V., Benthic invertebrates (zoobenthos
Amur and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–19 y
y
y
(
)
Amur and their role in the nutrition of Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949
gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 5–139.i gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 5–139. Vdovin, A.N. and Mizyurkin, M.A. The comparison of estimations of fishes’ abundance, on
data of registration works carried out by trawls of different construction, Vopr. Rybolov., 2011, vol. 12, no. 1(45), pp. 150–161. Egorova, M.N., Nutritional information for killer whales (Liocassis ussuriensis Dyb.) And Her
zenstein killer whales (Liocassis herzensteini Berg) in the Amur basin, Tr. Amurskoy ikhtiologicheskoy
ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 449–490.l Zhabin, I.A., Abrosimova, A.A., Dubina, V.A., and Nekrasov, D.A., Influence of the Amur
River runoff on the hydrological conditions of the Amur Liman and Sakhalin Bay (Sea of Okhotsk)
during the spring-summer flood, Russ. Meteorol. Hydrol., 2010, vol. 35, no. 4, pp. 295–300. References g
p
gl
y
pp
Zhivoglyadov, A.A., Zhivoglyadova, L.А., Zherdev, N.A., Guskova, O.S., Luzhnyak, O.L.,
and Shlyakhova, N.A., Results of complex investigation of the Lower Don communities in 2017, in
Researchgate, 2019. doi 10.13140/RG.2.2.15295.15521 Koval, M.V., Gorin, S.L., and Kalugin, A.A., Ecological characteristic of nectobenthos and
juvenile fish community of hypertidal estuary of Penzhina and Talovka Rivers (Northwest Kamchatka)
in August 2014, Issled. Vodn. Biol. Resur. Kamchatki i Sev.-Zapadn. Chasti Tikhogo Okeana, 2015,
vol. 37, pp. 164–191. doi 10.15853/2072-8212.2015.37.164-191 Konstantinov, A.S., Chironomids of the river basin Cupid and their role in the nutrition of
Amur fish, Tr. Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1950, vol. 1, pp. 147–286. Koshelev, V.N., Amur sturgeon Acipenser schrenckii Brandt, 1869 (distribution, biology, arti
ficial reproduction), Cand. Sci. (Biol.) Dissertation, Khabarovsk, 2010. Koshelev, V.N., A study of the distribution of juvenile Kaluga and Amur sturgeon in the Amur
estuary in 2005, in Mat-ly Mezhregion. Nauchno-Pract. Conf. “Resursy i ekologicheskiye problemy
Dal’nego Vostoka” (Materials of the Inter-Regional Sci. Pract. Conf. “Resources and environmental
problems of the Far East”), Khabarovsk: DVGGU, 2006, pp. 180–184. Koshelev, V.N., Shmigirilov, A.P., and Ruban, G.I., Distribution, abundance, and size structure
of Amur kaluga Acipenser dauricus and Amur sturgeon A. schrenckii in the Lower Amur and Amur
Estuary, Vopr. Ikhtiol., 2016, vol. 56, no. 2, pp. 235–241. y
p
pp
Kryukov, V.G., Voronov, B.A., Gavrilov, A.V., and Makarov, A.V., Reka Amur: problemy i
puti ikh resheniya (rezul’taty deyatel’nosti Koordinatsionnogo komiteta po ustoychivomu razvitiyu
basseyna r. Amur) (Amur River: problems and solutions (results of the Coordination Committee for
Sustainable Development of the Amur River Basin)), Khabarovsk: Priamurskoye geograficheskoye
obshchestvo, 2005. Krykhtin, M.L., The current state and development prospects of sturgeon in the river basin. Amur, in Biologicheskiye osnovy razvitiya osetrovogo khozyaystva v vodoyemakh SSSR, Moscow:
Nauka, 1979, pp. 68–74.i Krykhtin, M.L. and Gorbach, E.I., Sturgeon fish of the Far East, in Ekonomicheskaya zhizn’
Dal’nego Vostoka, 1994, vol. 1, no. 3, pp. 86–91. Kurenkov, I.I., To the biology of Far Eastern freshwater shrimp, Tr. Amurskoy ikhtiologicheskoy
ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1950, vol. 1, pp. 379–390.i Kushnarenko, A.I., The behavior of juvenile fish near the net and in the zone of activity of the
fry trawl, Vopr. Ikhtiol., 2075, vol. 15, no. 5(94), pp. References (Proceedings of the Amur ich
ological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. (Proceedings of the Amur ich
thyological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. (Proceedings of the Amur ich g
p
)
py
Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. (Proceedings of the Amur ich
ological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1958, vol. 4. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945 1949 gg. (Proceedings o
thyological expedition 1945–1949), Moscow: Mosk. O-vo Ispyt. Prir., 1958, vol. 4. Yukhimenko, S.S., Food of the Amur sturgeon and Kaluga in the lower reaches of the Amur,
Vopr. Ikhtiol., 1963, vol. 3, no. 2(27), pp. 311–318. Vopr. Ikhtiol., 1963, vol. 3, no. 2(27), pp. 311–318. de Souza, M.R., Fagundes, L., von Seckendorff, R.W., Tomas, A.W.G., Tutui, S.L., Casarini,
L.M., Construction characteristics of a beam trawl for catching young shrimps in estuaries, Rev. Bras. de Souza, M.R., Fagundes, L., von Seckendorff, R.W., Tomas, A.W.G., Tutui, S.L., Casarini,
L.M., Construction characteristics of a beam trawl for catching young shrimps in estuaries, Rev. Bras. Eng. Pesca, 2010, vol. 5, no. 3, pp. 42–50. Dettmers, J.M., Gutreuter, S., Wahl, D.H., and Soluk, D.A., Patterns and abundance in main
nnels of the upper Mississippi River system, Can. J. Fish. Aquat. Sci., 2001, vol. 58, pp. 933–942. Erchardt, J.M. and Tiffan, K.F., Ecology of nonnative Siberian prawn (Palaemon modestus)
in the lower Snake River, Washington, USA, Aquatic Ecology, 2016, vol. 50, no. 4, pp. 607–621.i Herzog, D.P., Barko, V.A., Scheibe, J.S., Hrabik, R.A., and Ostendorf, D.E., Efficacy of
a benthic trawl for sampling small-bodied fishes in large river systems, North American Journal of
Fisheries Management, 2005, vol. 25, pp. 594–603. Á Szalóky, Z., György, Á.I., Tóth, B., Sevcsik, A., Specziár, A., Csanyi, B., Szekeres, J., and
Erős, T., Application of an electrified benthic frame trawl for sampling fish in a very large European
river (the Danube River) — Is offshore monitoring necessary?, Fish. Res., 2014, vol. 151, pp. 12–19. doi.org/10.1016/j.fishres.2013.12.004i i
Wolter, C. and Freyhof, J., Diel distribution patterns of fishes in a temperate large lowland
river, J. Fish Biol., 2004, vol. 64, pp. 632–642. doi.org/10.1111/j.1095-8649.2004.00327.xi ,
,
,
, pp
g
j
Zajicek, P. and Wolter, Ch., The gain of additional sampling methods for the fish-based as
sessment of large rivers, Fish. Res., 2018, vol. 197, pp. 15–24. References 871–878.i y
p
(
) pp
Lobyrev, F.S., Estimation of fish abundance based on the modeling of gill nets, Extended Ab
stract of Cand. Sci. (Biol.) Dissertation, Moscow: Mosk. Gos. Univ., 2008. Mnogoletniye dannyye o rezhime i resursakh poverkhnostnykh vod sushi (Long-term data on
the regime and resources of land surface water, vol. 1, issue 19: Amur basins (without Shilka, Amguni, Lobyrev, F.S., Estimation of fish abundance based on the modeling of gill nets, Extended Ab
stract of Cand. Sci. (Biol.) Dissertation, Moscow: Mosk. Gos. Univ., 2008. Mnogoletniye dannyye o rezhime i resursakh poverkhnostnykh vod sushi (Long-term data on
the regime and resources of land surface water, vol. 1, issue 19: Amur basins (without Shilka, Amguni,
Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986. the regime and resources of land surface water, vol. 1, issue 19: Amur basins (without Shilka, Amguni,
Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986.l t e eg
e a d esou ces o a d su ace wate , vo . , ssue 9:
u bas s (w t out S
a,
gu ,
Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986.l Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986. Amazar, Ussuri) and Uda), Yakor’, I.S., ed., Moscow: Gidrometeoizdat, 1986. Mordovin, A.M., Godovoy i sezonnyy stok rek basseyna Amura (The annual and seasonal flow
of the rivers of the Amur basin), Khabarovsk, 1996. Mordovin, A.M., Godovoy i sezonnyy stok rek basseyna Amura (The annual and seasonal flow
of the rivers of the Amur basin), Khabarovsk, 1996. Nikolsky, G.V., Amur ichthyological expedition 1945–1949, Tr. Amurskoy ikhtiologicheskoy
ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1950, vol. 1, pp. 1–18.i Nikolsky, G.V., Ryby bassejna Amura (Amur basin fish), Moscow: Аkad. Nauk SSSR, 1956. Novomodny, G.V., Zolotukhin, S.F., and Sharov, P.O., Ryby Amura: bogatstvo i krizis (Cupid
Fish: Wealth and Crisis), Vladivostok: Apel’sin, 2004. )
p
Pasechnik, O.I. and Shmigirilov, A.P., Estimation of the number of Amur chum salmon On
corhynchus keta (Walbaum, 1792) based on tagging results, in Chteniya pamyati Vladimira Yakov 306 Видовой состав и распределение рыб и креветок в русле нижнего Амура Видовой состав и распределение рыб и креветок в русле нижнего Амура levicha Levanidova (Vladimir Yakovlevich Levanidov’s Biennial Memorial Meetings), Vladiv
Dal’nauka, 2008, vol. 4, pp. 294–303. cha Levanidova (Vladimir Yakovlevich Levanidov’s Biennial Memorial Meetings), Vladivostok:
’nauka, 2008, vol. 4, pp. 294–303. References Pakhorukov, A.M., Izucheniye raspredeleniya molodi ryb v vodokhranilishchakh i ozorakh
(Study of the distribution of juvenile fish in reservoirs and lakes), Moscow: Nauka, 1980. Pikuleva, V.A., Feeding of a killer whale (Psevdobagrus fulvidraco Rich.) And a small killer
whale (Liocassis brashnikovi Berg) in the Amur basin, Tr. Amurskoy ikhtiologicheskoy ekspeditsii
1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 435–448. whale (Liocassis brashnikovi Berg) in the Amur basin, Tr. Amurskoy ikhtiologicheskoy ekspeditsii
1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 435–448. (
g)
,
y
g
y
p
1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1952, vol. 3, pp. 435–448. Pravdin, I.F., Rukovodstvo po izucheniyu ryb (preimushchestvenno presnovodnykh) (Guide to
the Study of Fish (Mainly Freshwater)), 4th ed., Moscow: Pishchevaya Promyshlennost’, 1966. P
b
A N M
i l
h
d
f A
fi h
U h
P
k Probatov, A.N., Materials on the study of Amur sturgeon fishes, Uchen. zap. Permskogo un-ta,
5, vol. 1, no. 1, pp. 33–72. Sechin, Yu.T., Metodicheskiye ukazaniya po otsenke chislennosti ryb v presnovodnykh vodo-
yemakh (Guidelines for assessing the number of fish in freshwater bodies), Moscow: Vses. Nauchno-
Issled. Inst. Presnovodn. Rybn. Khoz., 1990. ssled. Inst. Presnovodn. Rybn. Khoz., 1990. Sirotsky, S.E., Makarchenko, E.A., and Makarchenko, M.A., Water quality of the Am
River basin based on zoobentos composition, Vopr. Rybolov., 2009, vol. 10, no. 3(39), pp. 453–46 Soin, S.G., Materials for the development of sturgeon p. Amur, Tr. Amurskoy ikhtiologiches
ekspeditsii 1945–1949 gg., Moscow: Mosk. O-vo Ispyt. Prir., 1951, vol. 2, pp. 223–232. Soldatov, V.K., A review of studies conducted on the Amur River in 1909-1913; The study of
sturgeon Amur, in Materialy k poznaniyu russkogo rybolovstva (Materials for the knowledge of Rus
sian fisheries), Petrograd: tip. V.F. Kirshbauma (otd-niye), 1915, vol. 3, issue 12. Soloviev, I.A., Amurolimanskiy ruslovoy protsess i vodnyye puti (Amurolimanian channel process
and waterways), Vladivostok: Dal’nevost. Otd. Ross. Akad. Nauk, 1995. Soloviev, I.A., Ruslovoy protsess i vodnyye puti Amurskogo limana (Channel process and wa
ways of the Amur estuary), Vladivostok, 1974. Soloviev, I.A. and Svirsky, V.G., The hydrological situation of the Lower Amur and its role in
the reproduction of sturgeon and Kaluga, in Biologiya ryb Dal’nego Vostoka (Biology of fish of the
Far East), Vladivostok: Dal’nevost. Gos. Univ., 1976, pp. 70–74. East), Vladivostok: Dal nevost. Gos. Univ., 1976, pp. 70 74. Trudy Amurskoy ikhtiologicheskoy ekspeditsii 1945–1949 gg. References Поступила в редакцию 9.12.2019 г. После доработки 7.04.2020 г. Принята к публикации 20.05.2020 г. 307
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Polyomavirus T Antigen Induces<i>APOBEC3B</i>Expression Using an LXCXE-Dependent and TP53-Independent Mechanism
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Download date: 2024-10-24 Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and
TP53-Independent Mechanism
Starrett, G.J.; Serebrenik, Artur A.; Roelofs, P.A.; McCann, Jennifer L.; Verhalen, Brandy; Jarvis,
Matthew C.; Span, P.N.; Harris, R.S. 2019, Article / Letter to editor (Mbio, 10, 1, (2019), article e02690-18)
Doi link to publisher: https://doi.org/10.1128/mBio.02690-18 Polyomavirus T Antigen Induces APOBEC3B Expression Using an LXCXE-Dependent and
TP53-Independent Mechanism
Starrett, G.J.; Serebrenik, Artur A.; Roelofs, P.A.; McCann, Jennifer L.; Verhalen, Brandy; Jarvis,
Matthew C.; Span, P.N.; Harris, R.S. 2019, Article / Letter to editor (Mbio, 10, 1, (2019), article e02690-18)
Doi link to publisher: https://doi.org/10.1128/mBio.02690-18 Polyomavirus T Antigen Induces APOBEC3B Expression Using
an LXCXE-Dependent and TP53-Independent Mechanism Gabriel J. Starrett,a Artur A. Serebrenik,a Pieter A. Roelofs,b Jennifer L. McCann,a Brandy Verhalen,c Matthew C. Jarvis,a
Teneale A. Stewart,a Emily K. Law,a Annabel Krupp,d Mengxi Jiang,c John W. M. Martens,e Ellen Cahir-McFarland,d
Paul N. Span,b Reuben S. Harrisa,f on April 15, 2019 b
http://mbio.asm.org/
Downloaded from on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from aDepartment of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, Institute for Molecular Virology, University of Minnesota, Minneapolis,
Minnesota, USA bDepartment of Radiation Oncology, Radiotherapy and OncoImmunology Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands
cDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
dDepartment of Neuroimmunology, Biogen, Cambridge, Massachusetts, USA
eDepartment of Medical Oncology Cancer Genomics Netherlands Erasmus MC Cancer Institute Erasmus University Medical Center Rotterdam The Netherlands bDepartment of Radiation Oncology, Radiotherapy and OncoImmunology Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands
cDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
dDepartment of Neuroimmunology Biogen Cambridge Massachusetts USA on April 15, 2019 by guest
http://mbio.asm.org/
from eDepartment of Medical Oncology, Cancer Genomics Netherlands, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands
fHoward Hughes Medical Institute, University of Minnesota, Minneapolis, Minnesota, USA ABSTRACT
APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial
innate antiviral functions. However, misregulated APOBEC3B can also be detrimental
by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of mul-
tiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B
overexpression, perhaps to their own benefit, but little is known about the cellular
mechanisms hijacked by these viruses to do so. Here we investigate the molecular
mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First,
we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and
partially localized to virus replication centers. Second, truncated T antigen (truncT) is
sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not
the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in
combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated in-
duction of APOBEC3B. Fourth, CDK4/6 inhibition by palbociclib is also insufficient to
suppress truncT-mediated induction of APOBEC3B. Last, global gene expression anal-
yses in a wide range of human cancers show significant associations between ex-
pression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. January/February 2019
Volume 10
Issue 1
e02690-18 Citation Starrett GJ, Serebrenik AA, Roelofs PA,
McCann JL, Verhalen B, Jarvis MC, Stewart TA,
Law EK, Krupp A, Jiang M, Martens JWM, Cahir-
McFarland E, Span PN, Harris RS. 2019.
Polyomavirus T antigen induces APOBEC3B
expression using an LXCXE-dependent and
TP53-independent mechanism. mBio
10:e02690-18. https://doi.org/10.1128/mBio
.02690-18. Note: Note:
To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). RESEARCH ARTICLE
Host-Microbe Biology
crossm RESEARCH ARTICLE
Host-Microbe Biology Starrett et al. G
enetic diversity is key to virus replication, pathogenesis, and transmission, and
particularly for escape from adaptive immune responses in vertebrate species
(1–3). Each virus has evolved to maintain an optimized level of genetic diversity for its
own unique life cycle through various mechanisms, with some viruses having high
mutation rates and others much lower mutation rates, notably the dsDNA viruses (4, 5). Recently, it has been concluded that the genome compositions of multiple DNA tumor
viruses, including high-risk human papillomavirus (HPV) types and BK polyomavirus
(BKPyV), have been shaped by long-term interactions with the innate, antiviral APOBEC
deaminases (6–9). It has also been suggested that acutely occurring mutations by these
enzymes in the major capsid gene of polyomaviruses promote antibody escape during
polyomavirus-associated nephropathy and progressive multifocal leukoencephalopa-
thy (10). The overlap between these disease variants and oncogenic enzymes is striking
especially in light of growing evidence linking BKPyV infection and a subset of urothe-
lial carcinomas with high levels of APOBEC-signature mutations (11). G g
g
Several APOBEC enzymes, including APOBEC3B (A3B), bind 5=-TC dinucleotide mo-
tifs in single-stranded DNA and catalyze the hydrolytic conversion of cytosine to uracil
(12, 13). Left unrepaired, uracil lesions can serve as the templates for new DNA synthesis
and directly result in C-to-T mutations. Alternatively, if the uracil base is excised by
cellular uracil DNA glycosylase 2 (UNG2), then the resulting abasic site becomes
noninstructional and may trigger cellular DNA polymerases to insert an adenine
opposite the lesion, except for REV1, which tends to incorporate either adenine or
cytosine. Thus, APOBEC-catalyzed DNA deamination of 5=-TC motifs results in both
C-to-T and C-to-G mutations (a signature frequently expanded to include the 3’-
nucleobases A and T and referred to in the context of trinucleotide motifs 5=-TCA and
5=-TCT). An additional hallmark of virus mutagenesis by APOBEC enzymes is a bias
toward occurring on the template of lagging-strand DNA replication (14–16). A likely
mechanistic relationship with single-stranded DNA replication intermediates is sup-
ported by similar correlations in model yeast and Escherichia coli experiments (17, 18). Human cells have the potential to express up to nine active DNA cytosine deami-
nases (AID, APOBEC1, and A3A/B/C/D/F/G/H) (19–22). Seven of these enzymes prefer
5=-TC motifs in single-stranded DNA, whereas AID uniquely prefers 5=-RC and
APOBEC3G (A3G) prefers 5=-CC. Starrett et al. A3B is the most likely APOBEC family member to
contribute to the mutagenesis and evolution of small DNA tumor viruses because it is
specifically upregulated by viral oncoproteins. For high-risk HPV types, the oncopro-
teins E6 and E7 have been implicated through various pathways (23–26). For polyo-
maviruses, including JC, BK, and Merkel cell (JCPyV, BKPyV, and MCPyV, respectively),
the large T antigen (TAg) is sufficient for A3B upregulation through a yet-to-be
determined mechanism (6). However, the considerable functional overlap of these
proteins, RB inactivation by E7 and TAg and p53 inactivation by E6 and TAg, may
indicate limited pathways for A3B modulation by viruses (27, 28). Here we investigate
the molecular mechanism by which polyomaviruses promote the transcriptional up-
regulation of A3B with results converging on the cellular RB/E2F pathway, which is
often deregulated in cancer. January/February 2019
Volume 10
Issue 1
e02690-18 Polyomavirus T Antigen Induces APOBEC3B Expression Using
an LXCXE-Dependent and TP53-Independent Mechanism These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B
transcription, yet they also suggest that the polyomavirus RB-binding motif has at
least one additional function in addition to RB inactivation for triggering APOBEC3B
upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many
different cancers and correlates with elevated frequencies of C-to-T and C-to-G mu-
tations in 5=-TC motifs, oncogene activation, acquired drug resistance, and poor clin-
ical outcomes. The mechanisms responsible for APOBEC3B overexpression are not
fully understood. Here, we show that the polyomavirus truncated T antigen (truncT)
triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in
turn likely modulates the binding of E2F family transcription factors to promote
APOBEC3B expression. This work strengthens the mechanistic linkage between active
cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this muta-
tional mechanism damages cellular genomes, viruses may leverage it to promote
evolution, immune escape, and pathogenesis. The cellular portion of the mechanism
may also be relevant to nonviral cancers, where genetic mechanisms often activate
the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution. KEYWORDS APOBEC3B, RB/E2F pathway, polyomavirus, virus evolution January/February 2019
Volume 10
Issue 1
e02690-18 ®
mbio.asm.org
1 mbio.asm.org
1 ® Starrett et al. Starrett et al. RESULTS Visualization of endogenous APOBEC3B protein in polyomavirus-infected cells. A3B induction by polyomaviruses has been shown at the mRNA level by RT-qPCR and
at the protein level by immunoblotting in primary renal proximal epithelial cells
(RPTECs) (6). To extend these results to other relevant cell types, RT-qPCR and immu-
nofluorescent microscopy were used to ask whether polyomavirus infection causes a
general pan-nuclear upregulation of A3B enzyme and/or localization to discrete sub-
nuclear regions such as virus replication centers. Immortalized human kidney
[HuK(i)G10] cells were infected with BKPyV (Dunlop strain) and JCPyV (MAD1 strain) and
subjected to analyses at various days postinfection (dpi). Infected cells have enlarged
nuclei and robust expression of TAg and VP1 at 3 to 5 dpi (Fig. 1A). A3B expression was
more variable but still clearly and significantly increased after infection with either virus mbio.asm.org
2 January/February 2019
Volume 10
Issue 1
e02690-18 ® Polyomavirus T Antigen Induces APOBEC3B Expression F
E
C
D
A
B
G
0
1000
2000
3000
4000
0
200
400
600
TAg
A3B
0
1000
2000
3000
0
100
200
300
400
TAg
EdU
p < 0.0001
p < 0.0001
Spearman’s rho = 0.42
Spearman’s rho = 0.73
DAPI
TAg
EdU
A3B
Merge
20μm
A3B expression
relative to TBP
1 dpi
5 dpi
Mock
BKPyV
Mock
BKPyV
Mock
BKPyV
Mock
BKPyV
1dpi
5dpi
TAg
Percent positive cells
VP1
A3B
Mock
JCPyV (Mad1)
TAg
VP1
DAPI
100μm
A3B
Merge
0
0.5
1.0
1.5
p=0.041
p=0.036
p=0.050
p=0.022
p=0.014
BKPyV
JCPyV
Mock
6dpi (HuK(i)G10)
BKPyV
BKPyV
JCPyV
0
20
40
60
80
100
10
2
4
6
8
0
Percent A3B positive cells
3
5
7
9
Days post infection
FIG 1 Visualization and quantification of A3B expression in PyV-infected cells. (A and B) Immunofluorescent images and quantification of TAg, VP1, and A3B
in BKPyV-infected HuK(i)G10 cells at 1 and 5 dpi (significance determined using Welch’s two-tailed t test; n 2 biological replicates). (C) Time course of A3B
mRNA levels in JCPyV (Mad1 strain) versus mock-infected HuK(i)G10 cells. (D) RT-qPCR quantification of A3B transcripts in mock-, BKPyV-, and JCPyV
(Mad1)-infected HuK(i)G10 cells at 6 dpi (significance determined by Welch’s two-tailed t test; n 3 technical replicates). (E) High-resolution immunofluorescent
microscopy images of DAPI, A3B, EdU, and TAg in HuK(i)G10 cells infected with JCPyV (Mad1 strain). RESULTS (F and G) Correlation coefficients and P values for EdU and
A3B levels versus T antigen intensity in 100 cell images from a single experiment similar to that in panel E. on April 15, 2019 by g
http://mbio.asm.org/
Downloaded from C
D
A3B expression
relative to TBP
Mock
JCPyV (Mad1)
0
0.5
1.0
1.5
p=0.041
p=0.036
BKPyV
JCPyV
Mock
6dpi (HuK(i)G10)
10
2
4
6
8
0
Percent A3B positive cells
3
5
7
9
Days post infection D
A3B expression
relative to TBP
0
0.5
1.0
1.5
p=0.041
p=0.036
BKPyV
JCPyV
Mock
6dpi (HuK(i)G10) A
Mock
BKPyV
Mock
BKPyV
1dpi
5dpi
TAg
VP1
DAPI
100μm
A3B
Merge
BKPyV D A C A
Mock
BKPyV
1dpi
TAg
VP1
DAPI
100μm
A3B
Merge
BKPyV E
DAPI
TAg
EdU
A3B
Merge
20μm
JCPyV E on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from Merge G
0
1000
2000
3000
4000
0
200
400
600
TAg
A3B
0
p < 0.0001
Spearman’s rho = 0.42 F
G
0
1000
2000
3000
0
100
200
300
400
TAg
EdU
p < 0.0001
Spearman’s rho = 0.73 B
1 dpi
5 dpi
Mock
BKPyV
Mock
BKPyV
TAg
Percent positive cells
VP1
A3B
p=0.050
p=0.022
p=0.014
BKPyV
0
20
40
60
80
100 G B F FIG 1 Visualization and quantification of A3B expression in PyV-infected cells. (A and B) Immunofluorescent images and quantification of TAg, VP1, and A3B
in BKPyV-infected HuK(i)G10 cells at 1 and 5 dpi (significance determined using Welch’s two-tailed t test; n 2 biological replicates). (C) Time course of A3B
mRNA levels in JCPyV (Mad1 strain) versus mock-infected HuK(i)G10 cells. (D) RT-qPCR quantification of A3B transcripts in mock-, BKPyV-, and JCPyV
(Mad1)-infected HuK(i)G10 cells at 6 dpi (significance determined by Welch’s two-tailed t test; n 3 technical replicates). (E) High-resolution immunofluorescent
microscopy images of DAPI, A3B, EdU, and TAg in HuK(i)G10 cells infected with JCPyV (Mad1 strain). (F and G) Correlation coefficients and P values for EdU and
A3B levels versus T antigen intensity in 100 cell images from a single experiment similar to that in panel E. compared to mock-infected controls (Fig. 1A to D). Generally, JCPyV is regarded to have
slower replication dynamics than BKPyV (Dunlop), so initial JCPyV infections were run
out in a time course showing peak A3B expression at 7 dpi (Fig. 1C). January/February 2019
Volume 10
Issue 1
e02690-18 RESULTS A
J
LXCXE
J
LXCXE
TPPK NLS
DBD
Helicase
HR-AH
TPPK NLS
p53
Rb
BK LTAg
BK truncTAg
105 LFCHEDMF
105 ****KK**
Rb-binding mutant
C A3B
Merge
truncT
truncT
pRb-
TAg
C
50μm
50μm A C Merge Rb-binding mutant B
37 kDa
37 kDa
37 kDa
75 kDa
100 kDa
75 kDa
100 kDa
15 kDa
20 kDa
15 kDa
20 kDa
Mock
Empty
TAg
TAg-pRb-
truncT
truncT-pRb-
A3G
A3B
TAg
GAPDH
UNG2
TAg
truncT
truncT
Longer
exposures
Shorter
exposures B on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from Mock
Empty
truncT
truncT pRb-
D
37 kDa
37 kDa
100 kDa
20 kDa
TAg
A3G
A3B
truncT
GAPDH Mock
Empty
truncT
truncT pRb-
E
0
0.02
0.04
0.06
0.08
0.10
A3B mRNA relative to TBP
p=2.05x10-5
p=0.0049
p=0.016 D E FIG 2 RB-binding domain is necessary for A3B induction by polyomavirus T antigen. (A) Diagram of BKPyV T antigen isoforms and the LXCXE mutant used
here. (B) Immunoblots for the indicated proteins in RPTECs transduced with a lentiviral vector expressing LTAg, truncT, the indicated mutants, or nothing
(empty). Mock-transduced cells were analyzed in parallel as an additional control. (C) Immunofluorescent microscopy images for truncT, truncT LXCXE mutant,
and A3B in transduced RPTECs. (D and E) Immunoblots and RT-qPCR results for MCF10A cells transduced with the indicated constructs as in panel B (mean and
SEM shown in panel E; n 3 biological replicates; P value determined by Welch’s two-tailed t test). APOBEC3B upregulation by polyomavirus large T antigen requires the canon-
ical RB-interacting motif LXCXE. Based on the results above and our previous studies
(6), multiple polyomaviruses have the conserved capacity to upregulate A3B expression
in primary and immortalized kidney epithelial cells through the functions of large (L)
TAg. To investigate the LTAg domains responsible for A3B induction, and thus also
implicate associated cellular factors, we tested a naturally occurring splice variant of
BKPyV LTAg, known as truncT, which lacks the DNA-binding and helicase domains
essential for p53 neutralization (30–32) (schematic in Fig. 2A). In parallel, we also
assessed derivatives of LTAg and truncT with a disrupted LXCXE motif, which is required
for inhibiting the tumor suppressor protein RB1 as originally shown for SV40 TAg (33). RESULTS Across these
experiments, JCPyV-infected HuK(i)G10 cells showed a greater differential expression of
A3B mRNA and protein compared to mock-treated cells (Fig. 1B to D). JCPyV-infected cells were also analyzed 7 dpi by high-resolution immunofluorescent
microscopy for expression of A3B and viral proteins and for formation of virus replica-
tion foci. Cells were stained for DAPI, TAg, A3B, and EdU with virus replication centers
appearing as brightly stained puncta positive for both TAg and EdU (representative
images in Fig. 1A and E) (29). In infected cells, A3B is strongly induced with a
pan-nuclear staining pattern that is sometimes coincident with EdU-positive virus
replication foci. Incorporation of EdU into active replication foci is highlighted by strong
positive correlations with TAg stain intensity, as expected, whereas A3B showed weaker
but still significantly positive correlations (Fig. 1F and G). These data indicate that A3B
upregulation may be a general property of polyomavirus infection and that A3B may
access at least a subset of virus replication centers. mbio.asm.org
3 ® Starrett et al. Mock
Empty
truncT
truncT pRb-
Mock
Empty
truncT
truncT pRb-
A
B
D
E
J
LXCXE
J
LXCXE
TPPK NLS
DBD
Helicase
HR-AH
TPPK NLS
p53
Rb
BK LTAg
BK truncTAg
105 LFCHEDMF
105 ****KK**
Rb-binding mutant
A3B
Merge
truncT
truncT
pRb-
TAg
C
50μm
50μm
0
0.02
0.04
0.06
0.08
0.10
37 kDa
37 kDa
100 kDa
20 kDa
TAg
A3G
A3B
truncT
GAPDH
A3B mRNA relative to TBP
37 kDa
37 kDa
37 kDa
75 kDa
100 kDa
75 kDa
100 kDa
15 kDa
20 kDa
15 kDa
20 kDa
Mock
Empty
TAg
TAg-pRb-
truncT
truncT-pRb-
A3G
A3B
TAg
GAPDH
UNG2
TAg
truncT
truncT
Longer
exposures
Shorter
exposures
p=2.05x10-5
p=0.0049
p=0.016
FIG 2 RB-binding domain is necessary for A3B induction by polyomavirus T antigen. (A) Diagram of BKPyV T antigen isoforms and the LXCXE mutant used
here. (B) Immunoblots for the indicated proteins in RPTECs transduced with a lentiviral vector expressing LTAg, truncT, the indicated mutants, or nothing
(empty). Mock-transduced cells were analyzed in parallel as an additional control. (C) Immunofluorescent microscopy images for truncT, truncT LXCXE mutant,
and A3B in transduced RPTECs. (D and E) Immunoblots and RT-qPCR results for MCF10A cells transduced with the indicated constructs as in panel B (mean and
SEM shown in panel E; n 3 biological replicates; P value determined by Welch’s two-tailed t test). January/February 2019
Volume 10
Issue 1
e02690-18 RESULTS MCF10A
β-Actin
p53
WT
55 kDa
43 kDa
17 kDa
p21
ΔTP53
C MCF-7L
DMSO
Nutlin, 5 µ M
PMA
Nutlin + PMA
B
A3B
TP53
P21
MDM2
SLC7a11
0
0.2
0.4
0.6
0.8
1.0
10
20
30
40
* *
*
*
*
*
*
*
* *
* mRNA relative to TBP
MCF10A
A
A3B
TP53
P21
MDM2
SLC7a11
0
1
2
3
10
20
30
40
* *
*
*
*
*
*
*
*
* DMSO
PMA
DMSO
PMA
0
0.02
0.04
0.06
0.08
0.10
0.5
1.0
1.5
2.0
2.5
A3B mRNA relative to TBP
WT
ΔTP53
MCF10A
D D B A on April 15, 2019 b
http://mbio.asm.org/
Downloaded from on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from FIG 3 Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells
treated with DMSO, 5 M nutlin, PMA, or nutlin PMA. Statistically significant changes by Student’s t test (P 0.05) are noted by an asterisk (mean and SEM;
n 3 technical replicates). (C) Immunoblots of WT and TP53 KO MCF10A cell lines. (D) RT-qPCR results showing the effects of PMA treatment on A3B mRNA
levels in WT and TP53 KO MCF10A cell lines. on April 15, 2019 by guest
http://mbio.asm.org/
aded from Immunofluorescent microscopy images also show truncT-mediated induction of nu-
clear A3B but not by the RB-binding mutant derivative (Fig. 2C). To date, many aspects of A3B regulation and function have been determined using
normal-like and cancerous mammary epithelial cell lines due to higher capacities for
genetic manipulation over primary cells and greater relevance to cancer (35, 36). To ask
whether TAg induction of A3B also occurs in one of these more tractable systems, the
normal-like mammary epithelial cell line MCF10A was transduced with constructs
expressing BKPyV truncT or the RB-binding mutant and analyzed as described above. Both immunoblotting and RT-qPCR yielded similar results with truncT but not the
RB-binding mutant causing A3B induction (Fig. 2D and E). Thus, polyomavirus T antigen
appears to possess a conserved, LXCXE-dependent capacity to induce A3B in different
epithelial cell types. TP53 inactivation is dispensable for APOBEC3B induction. The aforementioned
data comparing LTAg and truncT simultaneously implicate RB1 and demonstrate that
p53 inhibition is not required for A3B induction because truncT completely lacks the
p53 binding domain (Fig. 2). RESULTS RPTECs were transduced with lentiviruses expressing an empty multiple cloning site as
a negative control, BKPyV LTAg as a positive control, BKPyV truncT, and RB-binding site
mutant derivatives; incubated 3 days; and assessed by immunoblotting and fluores-
cence microscopy. Mock-transduced cells express low levels of A3G, and transduction
with empty lentivirus causes a modest increase in this protein and also raises A3B levels
to faintly detectable levels (Fig. 2B). In contrast, both LTAg and truncT induce expres-
sion of A3B and UNG2, a known target of the RB-E2F axis (34), and all induction for both
of these proteins is eliminated by two amino acid substitutions shown to abrogate RB
binding in SV40 TAg (LFCHED to LFCHKK) (33) (Fig. 2B). The LTAg and truncT mutants
invariably migrate faster than the corresponding wild-type proteins during SDS-PAGE,
which is likely due to a charge differential caused by the two amino acid substitutions. mbio.asm.org
4 Polyomavirus T Antigen Induces APOBEC3B Expression ® DMSO
PMA
DMSO
PMA
0
0.02
0.04
0.06
0.08
0.10
0.5
1.0
1.5
2.0
2.5
A3B mRNA relative to TBP
WT
ΔTP53
MCF10A
MCF10A
mRNA relative to TBP
MCF10A
MCF-7L
DMSO
Nutlin, 5 µ M
PMA
Nutlin + PMA
β-Actin
p53
WT
55 kDa
43 kDa
17 kDa
p21
ΔTP53
A
B
D
C
A3B
TP53
P21
MDM2
SLC7a11
A3B
TP53
P21
MDM2
SLC7a11
0
1
2
3
10
20
30
40
0
0.2
0.4
0.6
0.8
1.0
10
20
30
40
* *
*
*
*
*
*
*
*
*
* *
*
*
*
*
*
*
* *
*
FIG 3 Inactivation of p53 does not affect A3B expression. (A and B) Bar plots of RT-qPCR measurements of relevant genes in MCF10A (A) and MCF7L (B) cells
treated with DMSO, 5 M nutlin, PMA, or nutlin PMA. Statistically significant changes by Student’s t test (P 0.05) are noted by an asterisk (mean and SEM;
n 3 technical replicates). (C) Immunoblots of WT and TP53 KO MCF10A cell lines. (D) RT-qPCR results showing the effects of PMA treatment on A3B mRNA
levels in WT and TP53 KO MCF10A cell lines. January/February 2019
Volume 10
Issue 1
e02690-18 RESULTS 0
1
2
3
4
Fold change expression
RB1
RBL1
RBL2
0
5
10
15
A3B
UNG2
CCNE2
A
RPTE
RB1
RBL1
RBL2
+
-
-
+
+
-
+
+
+
-
-
-
+
-
+
-
+
-
-
+
+
-
-
+
siRNA
*
*
**
**
**
**
**
** ** **
**
**
**
**
+
+ B
siRNA
130
95
130
95
RB1
RBL1
-
-
+
-
-
+
55
43
RB1
RBL1
A3B
TUB
40
35
kDa B A Fold change expression on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from on April 15, 2019 by guest
http://mbio.asm.org/
aded from RB1
RBL1
RBL2
A3B
UNG2
CCNE2
0
1
2
3
4
Fold change expression
0
5
10
15
C
MCF10A
RB1
RBL1
RBL2
+
-
-
+
+
-
+
+
+
-
-
-
+
-
+
-
+
-
-
+
+
-
-
+
siRNA
**
*
*
**
+
+
+
*
*
*
*
*
*
*
*
** D
siRNA
55
43
130
95
-
-
+
-
RB1
RBL1
40
35
kDa
RB1
TUB
A3B D C Fold change expression siRNA
55
43
130
95
-
-
-
+
RB1
RBL1
40
35
kDa
TUB
A3B
RBL1 FIG 4 Modulation of RB family genes and A3B regulation. (A and C) Bar plots of RT-qPCR quantification of RB-family mRNAs
(top) and predicted responsive genes, A3B, UNG2, and CCNE2 (bottom), in RPTEC and MCF10A cells with siRNA-mediated KD
of RB-family genes. P values for each siRNA combination compared to control were calculated using Welch’s two-tailed t test
and were indicated using the following symbols: , P 0.1; *, P 0.05; **, P 0.01; n 3 biological replicates. (B and D)
Immunoblots for A3B, RB1, and RBL1 in RPTEC and MCF10A cells following treatment with the indicated siRNA. tional activities. To investigate the roles of RB1, RBL1, and RBL2 in A3B transcriptional
regulation, a series of knockdown experiments was done with siRNAs targeting each of
these factors in RPTECs and MCF10A cells (Fig. 4A to D). RT-qPCR showed that 75%
knockdown was achieved for each targeted gene (upper panels in Fig. 4A and C). As
controls, CCNE2 was upregulated upon RB1 knockdown and UNG2 was moderately
upregulated by RBL2 knockdown (lower panels in Fig. 4A and C). RESULTS 0
1
2
3
4
Fold change expression
RB1
RBL1
RBL2
0
5
10
15
A3B
UNG2
CCNE2
RB1
RBL1
RBL2
A3B
UNG2
CCNE2
0
1
2
3
4
Fold change expression
0
5
10
15
A
C
B
D
MCF10A
RPTE
RB1
RBL1
RBL2
+
-
-
+
+
-
+
+
+
-
-
-
+
-
+
-
+
-
-
+
+
-
-
+
siRNA
RB1
RBL1
RBL2
+
-
-
+
+
-
+
+
+
-
-
-
+
-
+
-
+
-
-
+
+
-
-
+
siRNA
siRNA
siRNA
siRNA
*
*
**
**
**
**
**
** ** **
**
**
**
**
+
+
**
*
*
**
+
+
+
*
*
*
*
*
*
*
*
**
130
95
130
95
RB1
RBL1
-
-
+
-
-
+
55
43
55
43
55
43
RB1
RBL1
A3B
TUB
40
35
130
95
130
95
-
-
+
-
RB1
RBL1
-
-
-
+
RB1
RBL1
40
35
40
35
kDa
kDa
kDa
RB1
TUB
A3B
TUB
A3B
RBL1
FIG 4 Modulation of RB family genes and A3B regulation. (A and C) Bar plots of RT-qPCR quantification of RB-family mRNAs
(top) and predicted responsive genes, A3B, UNG2, and CCNE2 (bottom), in RPTEC and MCF10A cells with siRNA-mediated KD
of RB-family genes. P values for each siRNA combination compared to control were calculated using Welch’s two-tailed t test
and were indicated using the following symbols: , P 0.1; *, P 0.05; **, P 0.01; n 3 biological replicates. (B and D)
Immunoblots for A3B, RB1, and RBL1 in RPTEC and MCF10A cells following treatment with the indicated siRNA. RESULTS To further ask whether p53 inactivation might influence
A3B gene expression, we quantified A3B mRNA levels in two cell lines that have been
used to study A3B regulation, MCF10A and the human estrogen receptor-positive
breast cancer cell line MCF-7L (above and references 35 to 38). Each cell line was
treated with either DMSO or 5 M nutlin, which is a drug that protects p53 from
MDM2-mediated degradation (39). As controls, mRNA levels were quantified for two
genes activated by p53 (P21, MDM2) (40–43) and one gene repressed by p53 (SLC7A11)
(44, 45). Respectively, the expression of these genes was derepressed or repressed by
nutlin treatment (Fig. 3A and B). In comparison, neither steady-state nor PMA-induced
A3B mRNA levels were changed by nutlin (Fig. 3A and B). Moreover, Cas9-mediated
knockout of TP53 in MCF10A cells also caused no significant effect on basal or
PMA-induced A3B expression levels (Fig. 3C and D). These data combine to indicate that
p53 by itself has no significant role in the either the PMA-induced pathway or
basal-state transcriptional regulation of A3B, discouraging our original hypothesis (23)
and conflicting with published data (38) (see Discussion). RB-family knockdown is insufficient to induce APOBEC3B expression. RB1 is argu-
ably the most widely studied target of the LXCXE motif of viral proteins such as HPV E7,
adenovirus E1A, and polyomavirus LTAg (27, 31, 32, 46). However, the related pocket
proteins RBL1 (p107) and RBL2 (p130) also have an LXCXE-binding motif, are similarly
targeted and inactivated by LTAg and truncT, and may be involved in A3B regulation
(47–50). The aforementioned viral proteins bind to the hypophosphorylated forms of
RB1, RBL1, and RBL2, which inhibits phosphorylation by cyclin-dependent kinases
(CDKs) and leads to an accelerated cell cycle in part by deregulation of E2F transcrip- mbio.asm.org
5 January/February 2019
Volume 10
Issue 1
e02690-18 ® Starrett et al. January/February 2019
Volume 10
Issue 1
e02690-18 RESULTS (C) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A
ll
d
h
lb
l b f
d h
d
h
/
l
/
l P
f
d
h
p
p
g 0
0.5
1.0
1.5
CCNE2
MCF10A
0
1
4
MCF7
0
1
4
T47D
0
1
4
HCC1143
0
1
4
HCC1954
0
1
4
SUM225CWN
0
1
4
HCC1937
0
1
4
HCC1395
0
1
4
HCC1599
0
1
4
HCC202
0
1
4
Hs578T
0
1
4
Fold chan
Hours post palbociclib treatment
[Palbo] on April 15, 2019 by guest
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Downloaded from A3B
CCNE2
Fold change expression
0
0.5
1.0
1.5
2.0
0
5
10
15
20
No PMA
PMA-treated
0
0.5
1.0
1.5
C
0
1
3
0
1
3
ociclib treatment
Hours post PMA treatment
[Palbo] A3B
CCNE2
Fold change expression
,
0
0.5
1.0
1.5
2.0
2.5
0
10
20
30
No PMA
3h PMA-pretreated
0
0.5
1.0
1.5
2.0
B
0
1
3
0
1
3
Hours post palbociclib treatment
p
p
[Palbo] C B 1
3
0
1
Hours post palbociclib treatment Hours post PMA treatment Hours post PMA treatment FIG 5 Palbociclib treatment of cancer cell lines and A3B expression. (A) RT-qPCR quantification of A3B and CCNE2 mRNA expression in the indicated cell lines
treated with 0, 0.5, or 2.5 M palbociclib for 0, 1, or 4 h. (B) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0 ng/ml or
25 ng/ml PMA for 3 h prior to treatment with 0, 0.5, or 2.5 M palbociclib for 0, 1, and 3 h. (C) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A
cells pretreated with 0, 0.5, or 2.5 M palbociclib for 30 min and then treated with 0 ng/ml or 25 ng/ml PMA for 0, 1, and 3 h. scriptional. These results also suggest that truncT may have at least one additional
activity mediated by its LXCXE motif that contributes to A3B upregulation. Pharmacological inhibition of CKD4/6 does not alter APOBEC3B expression. Pal-
bociclib is a selective inhibitor of CDK4 and CDK6, which are kinases that function
normally to phosphorylate pRB, prevent binding to E2F transcription factors, and
stimulate the expression of many genes involved in cell cycle progression (51–53). To
corroborate the knockdown experiments above, we treated a panel of transformed cell
lines with palbociclib and quantified mRNA expression levels over time. RESULTS Polyomavirus T Antigen Induces APOBEC3B Expression
®
on April 15, 2019
http://mbio.asm.org/
Downloaded from ® Polyomavirus T Antigen Induces APOBEC3B Expression 0
0.5
1.0
1.5
A3B
0
0.5
1.0
1.5
CCNE2
MCF10A
0
1
4
MCF7
0
1
4
T47D
0
1
4
HCC1143
0
1
4
HCC1954
0
1
4
SUM225CWN
0
1
4
HCC1937
0
1
4
HCC1395
0
1
4
HCC1599
0
1
4
HCC202
0
1
4
Hs578T
0
1
4
Fold change expression
A
Hours post palbociclib treatment
[Palbo] 0
0.5
1.0
1.5
A3B
1 5
CCNE2
nge expression
A A 0
0.5
1.0
1.5
CCNE2
A3B
CCNE2
A3B
CCNE2
MCF10A
0
1
4
MCF7
0
1
4
T47D
0
1
4
HCC1143
0
1
4
HCC1954
0
1
4
SUM225CWN
0
1
4
HCC1937
0
1
4
HCC1395
0
1
4
HCC1599
0
1
4
HCC202
0
1
4
Hs578T
0
1
4
Fold change
Fold change expression
Fold change expression
,
0
0.5
1.0
1.5
2.0
2.5
0
10
20
30
No PMA
3h PMA-pretreated
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
5
10
15
20
No PMA
PMA-treated
0
0.5
1.0
1.5
B
C
0
1
3
0
1
3
0
1
3
0
1
3
Hours post palbociclib treatment
Hours post palbociclib treatment
Hours post PMA treatment
[Palbo]
[Palbo]
[Palbo]
FIG 5 Palbociclib treatment of cancer cell lines and A3B expression. (A) RT-qPCR quantification of A3B and CCNE2 mRNA expression in the indicated cell lines
treated with 0, 0.5, or 2.5 M palbociclib for 0, 1, or 4 h. (B) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0 ng/ml or
25 ng/ml PMA for 3 h prior to treatment with 0, 0.5, or 2.5 M palbociclib for 0, 1, and 3 h. RESULTS However, no combi-
nation of siRNAs resulted in significant upregulation of A3B mRNA levels (lower panels
in Fig. 4A and C). Knockdown of RB1 and RBL1 was validated at the protein level, but
RBL2 could not be clearly discerned with available commercial antibodies (Fig. 4B and
D). In contrast to the RT-qPCR results, protein-level A3B expression did appear to be
elevated upon RBL1 knockdown. These results suggest that depletion of each RB family
member alone or in combination is insufficient to significantly upregulate A3B mRNA
levels, at least in these two different normal-like cell types where A3B is induced by TAg
and truncT. However, the observed upregulation at the protein level in RBL1-depleted
cells raises the possibility of an additional layer of regulation that may be posttran- mbio.asm.org
6 January/February 2019
Volume 10
Issue 1
e02690-18 0
0.5
1.0
1.5
A3B
0
0.5
1.0
1.5
CCNE2
A3B
CCNE2
A3B
CCNE2
MCF10A
0
1
4
MCF7
0
1
4
T47D
0
1
4
HCC1143
0
1
4
HCC1954
0
1
4
SUM225CWN
0
1
4
HCC1937
0
1
4
HCC1395
0
1
4
HCC1599
0
1
4
HCC202
0
1
4
Hs578T
0
1
4
Fold change expression
Fold change expression
Fold change expression
,
0
0.5
1.0
1.5
2.0
2.5
0
10
20
30
No PMA
3h PMA-pretreated
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
5
10
15
20
No PMA
PMA-treated
0
0.5
1.0
1.5
A
B
C
0
1
3
0
1
3
0
1
3
0
1
3
Hours post palbociclib treatment
Hours post palbociclib treatment
Hours post PMA treatment
[Palbo]
[Palbo]
[Palbo]
FIG 5 Palbociclib treatment of cancer cell lines and A3B expression. (A) RT-qPCR quantification of A3B and CCNE2 mRNA expression in the indicated cell lines
treated with 0, 0.5, or 2.5 M palbociclib for 0, 1, or 4 h. (B) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A cells pretreated with 0 ng/ml or
25 ng/ml PMA for 3 h prior to treatment with 0, 0.5, or 2.5 M palbociclib for 0, 1, and 3 h. (C) RT-qPCR quantification of A3B and CCNE2 expression in MCF10A
cells pretreated with 0, 0.5, or 2.5 M palbociclib for 30 min and then treated with 0 ng/ml or 25 ng/ml PMA for 0, 1, and 3 h. January/February 2019
Volume 10
Issue 1
e02690-18 RESULTS This panel of
cell lines was constructed based on A3B expression, ranging from low to high (54, 55),
TP53 status, and ability to phosphorylate RB. As a positive control for palbociclib
efficacy, we analyzed expression of CCNE2, which encodes cyclin E2, promotes entry
into S phase, and is a known CDK4/6-RB-regulated gene (56, 57). The majority of cell
lines showed a dose- and time-responsive decrease in CCNE2 mRNA expression
(Fig. 5A). This effect was minimal in HCC1937 and HCC1599 cells, which are known to
display decreased RB phosphorylation (58, 59). In contrast, none of the palbociclib-
treated cell lines showed a reproducible or significant change in A3B mRNA expression. In addition, MCF10A cells were treated with PMA to induce A3B mRNA expression by
the PKC/ncNF-B pathway, and again, palbociclib had little effect (palbociclib added mbio.asm.org
7 January/February 2019
Volume 10
Issue 1
e02690-18 ® Starrett et al. post- or pre-PMA addition in Fig. 5B and C). These results combine to indicate that the
kinase activity of CDK4 and CDK6 is dispensable for A3B expression in multiple different
cell lines. Tumor transcriptome analyses support involvement of the RB pathway in
APOBEC3B regulation. We next used bioinformatics approaches to mine TCGA data
and assess global correlates with A3B mRNA expression in human tumors. First, we
conducted pathway analysis using all genes with significant positive correlations
between A3B expression in the TCGA breast tumor cohort. This analysis revealed that
11 of the top 20 significantly enriched upstream transcription factors contributing to
this expression pattern are part of the CDK4/6-cyclin D-RB-E2F axis (green-labeled
genes in Fig. 6A). These regulatory factors were either significantly activated or inhib-
ited, generally corresponding with known functions, with the net outcome being
accelerated cell cycling (respectively, red and blue bars in Fig. 6A). Upon closer pairwise
examination of effectors in this signal transduction pathway, A3B mRNA expression has
the strongest positive correlations with expression of RBL1, E2F1, E2F2, E2F7, and E2F8
(Fig. 6B). Last, we expanded this expression correlation analysis to include 22 different tumor
types in TCGA and all 11 APOBEC family members. This global approach further
highlighted strong correlations between A3B and expression of E2F1, E2F2, E2F7, and
E2F8 and indicated that the association between A3B, this signal transduction pathway,
and the cell cycle is evident in many cancer types (Fig. 6C). RESULTS Heat map intensities also
indicated that A3B is the only APOBEC family member that positively and globally
correlates with activation of the RB-E2F axis. To further reconcile our experimental and
bioinformatics data sets, we used the meta-analysis regulatory data from TargetGen-
eReg (http://www.targetgenereg.org/) to compare the regulation of A3B with known
cell cycle-related genes (60). These results, summarized in Table 1, further indicate that
the A3B mRNA expression profile is consistent with that of a cell cycle-regulated gene
that becomes upregulated during the G2/M phase, which is similar to FOXM1 and
distinct from UNG2, CCNE2, and A3C (the last being an APOBEC3 family member likely
to be regulated by p53). January/February 2019
Volume 10
Issue 1
e02690-18 DISCUSSION RB1
Spearman: -0.173
q=1.74x10-4
5
6
7
8
9
10
11
12
13
3
4
5
6
7
8
9
10
11
E2F1
Spearman: 0.552
q=3.98x10-74
E2F4
Spearman: 0.228
q=2.32x10-9
E2F7
Spearman: 0.429
q=9.74x10-41
12
13
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
3
4
5
6
7
8
9
10
11
12
0
1
2
A3
mRNA expression (RSEM Log2)
-4 -2
0
2
4
6
8 10 12 14
B 0
20
40
60
E2F4
TP53
E2F1
RB1
TBX2
CCND1
CDKN2A
E2F3
FOXM1
TCF3
MITF
FOXO3
E2F2
RBL1
SMARCB1
YY1
NUPR1
KDM5B
E2F6
E2F7
Activated
Neutral
Inhibited
Enrichment (-log p-value)
A RB1
Spearman: -0.173
q=1.74x10-4
-4 -2
0
2
4
6
8 10 12 14
5
6
7
8
9
10
11
12
13
3
4
5
6
7
8
9
10
11
E2F1
Spearman: 0.552
q=3.98x10-74
E2F2
Spearman: 0.573
q=2.57x10-81
E2F3
Spearman: 0.301
q=3.74x10-18
E2F4
Spearman: 0.228
q=2.32x10-9
E2F5
Spearman: 0.223
q=8.20x10-9
E2F7
Spearman: 0.429
q=9.74x10-41
E2F6
Spearman: 0.115
q=0.953
E2F8
Spearman: 0.534
q=1.98x10-68
12
13
3
4
5
6
7
8
9
10
11
12
13
2
4
6
8
10
12
14
0
2
4
6
8
10
12
6
7
8
9
10
11
12
6
7
8
9
10
11
12
13
14
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
3
4
5
6
7
8
9
10
11
12
0
1
2
A3B mRNA expression (RSEM Log2)
mRNA expression (RSEM Log2)
Spearman: 0.353
q=2.89x10-26
0
2
4
6
8
Spearman: -0.134
q=0.0248
7
8
9
RBL1
RBL2
10
12
10
11
12
13
-4 -2
0
2
4
6
8 10 12 14
-4 -2
0
2
4
6
8 10 12 14
B B A A mRNA expression (RSEM Log2) on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from on April 15, 2019 by guest
http://mbio.asm.org/
aded from A3B mRNA expression (RSEM Log2) AICDA
A1
A2
A3A
A3B
A3C
A3D
A3F
A3G
A3H
A4
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
GBM
GBM
GBM
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
−0.3
0.0
0.3
0.6
Spearman’s correlation
C
FIG 6
Evidence for A3B regulation by the RB/E2F pathway in tumors. DISCUSSION (A) Top 20 hits from IPA enrichment analysis of upstream
transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. Negative logs
of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific
transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway
with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation
coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal
clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. A2
A3A
A3D
A3H
GBM
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
C C A1
A3C
A3G
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
GBM
−0.3
0.0
0.3
0.6
Spearman’s correlation AICDA
A3B
A3F
A4
OV
RCA
NSC
LCA
ESC
IRP
UAD
USC
EAD
AML
SCA
BM
GG
IHC
AAD
RAD
ARC
TAD
GCT
HCA
CEC
CM A3G A4 FIG 6
Evidence for A3B regulation by the RB/E2F pathway in tumors. (A) Top 20 hits from IPA enrichment analysis of upstream
transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. Negative logs
of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific
transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway
with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation
coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal
clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. January/February 2019
Volume 10
Issue 1
e02690-18 DISCUSSION Negative logs
of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific
transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway
with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation
coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal
clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. DISCUSSION In this study, we investigate the mechanism of A3B upregulation by polyomavirus T
antigen through analyses of separation-of-function mutants, genetic knockdowns,
pharmacologic treatments, and transcriptomic data. We use high-resolution fluores-
cence microscopy to show that polyomavirus infection causes A3B upregulation and
protein accumulation in the nuclear compartment with the potential to access viral
replication foci. Second, we show that the LXCXE motif of LTAg and truncT, which is
well known to inhibit the tumor suppressor RB, is essential for A3B upregulation,
whereas the p53-binding domain is dispensable. Further investigation into this path-
way using genetic and pharmacologic treatments indicate that solely perturbing RB
family members (RB, RBL1, and RBL2) or kinases responsible for their phosphorylation
(CDK4 and CDK6) is insufficient to cause A3B mRNA upregulation. However, bioinfor-
matics analyses of tumor expression data show strong global correlations between A3B
mRNA expression and expression of other genes regulated by the RB-E2F signaling
pathway, including several members of the E2F family of transcription factors. Additional analysis of cell cycle-regulatory networks using the TargetGeneReg da-
tabase suggest that A3B might be a late cell cycle gene repressed by the RB/E2F family
members associated with the DREAM complex in quiescence and activated by other
transcription factors. Suppression by the DREAM complex is supported by the mild
upregulation of A3B protein observed upon RBL1 knockdown in this study. One
putative activating transcription factor is FOXM1, which was significantly enriched as an
upstream regulator of A3B and is known to activate genes in late G2/M (61). Interest-
ingly, in lymphoblastoid B cells, FOXM1 has been reported to frequently cooccupy
NF-B binding sites and form protein complexes with NF-B transcription factors, which
have been implicated in A3B regulation (36, 62). DISCUSSION Taken together, these results indicate mbio.asm.org
8 ® Polyomavirus T Antigen Induces APOBEC3B Expression RB1
Spearman: -0.173
q=1.74x10-4
-4 -2
0
2
4
6
8 10 12 14
5
6
7
8
9
10
11
12
13
3
4
5
6
7
8
9
10
11
E2F1
Spearman: 0.552
q=3.98x10-74
E2F2
Spearman: 0.573
q=2.57x10-81
E2F3
Spearman: 0.301
q=3.74x10-18
E2F4
Spearman: 0.228
q=2.32x10-9
E2F5
Spearman: 0.223
q=8.20x10-9
E2F7
Spearman: 0.429
q=9.74x10-41
E2F6
Spearman: 0.115
q=0.953
E2F8
Spearman: 0.534
q=1.98x10-68
12
13
3
4
5
6
7
8
9
10
11
12
13
2
4
6
8
10
12
14
0
2
4
6
8
10
12
6
7
8
9
10
11
12
6
7
8
9
10
11
12
13
14
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
3
4
5
6
7
8
9
10
11
12
0
1
2
A3B mRNA expression (RSEM Log2)
mRNA expression (RSEM Log2)
Spearman: 0.353
q=2.89x10-26
0
2
4
6
8
Spearman: -0.134
q=0.0248
7
8
9
RBL1
RBL2
10
12
10
11
12
13
-4 -2
0
2
4
6
8 10 12 14
-4 -2
0
2
4
6
8 10 12 14
AICDA
A1
A2
A3A
A3B
A3C
A3D
A3F
A3G
A3H
A4
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
GBM
GBM
GBM
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
OV
BRCA
HNSC
BLCA
CESC
KIRP
LUAD
LUSC
READ
LAML
ESCA
LGG
LIHC
PAAD
PRAD
SARC
STAD
TGCT
THCA
UCEC
SKCM
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
E2F1
E2F2
E2F3
E2F4
E2F5
E2F6
E2F7
E2F8
RB1
RBL1
RBL2
−0.3
0.0
0.3
0.6
0
20
40
60
E2F4
TP53
E2F1
RB1
TBX2
CCND1
CDKN2A
E2F3
FOXM1
TCF3
MITF
FOXO3
E2F2
RBL1
SMARCB1
YY1
NUPR1
KDM5B
E2F6
E2F7
Activated
Neutral
Inhibited
Enrichment (-log p-value)
Spearman’s correlation
A
C
B
FIG 6
Evidence for A3B regulation by the RB/E2F pathway in tumors. (A) Top 20 hits from IPA enrichment analysis of upstream
transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. DISCUSSION FIG 6
Evidence for A3B regulation by the RB/E2F pathway in tumors. (A) Top 20 hits from IPA enrichment analysis of upstream
transcriptional regulators of A3B in TCGA breast cancer with RB-pathway/cell cycle-related genes highlighted in green. Negative logs
of enrichment P values are shown in the bar graph on the right colored by predicted activation (red) or inhibition (blue) of the specific
transcription factor. (B) Scatter plots showing the correlation between A3B mRNA levels and transcription factors in the RB pathway
with Spearman’s correlation coefficient and q value reported in the lower left corner of each subpanel. (C) Spearman’s correlation
coefficient values for all APOBEC-family members against RB pathway transcription factors across 22 cancers ordered by hierarchal
clustering. Negative correlations are shown in blue, positive correlations are shown in red, and no data is represented by grey. mbio.asm.org
9 January/February 2019
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e02690-18 ® Starrett et al. TABLE 1 Cell cycle analysis of A3B and other cell cycle-regulated genes
Type of value
Value for gene:
APOBEC3B
APOBEC3C
CCNE2
FOXM1
UNG2
Chromosome
22
22
8
12
12
p53 expression score
4
14
14
17
14
No. of cell cycle data sets
2
1
5
2
5
G1/S or G2/M
G2/M
0
G1/S
G2/M
G1/S
p53 target
No
Yes
No
No
No
Cell cycle gene
Yes
No
Yes
Yes
Yes
DREAM target
Yes
No
No
Yes
Yes
MMB-FoxM1 target
No
No
No
No
No
RB-E2F target
No
No
Yes
No
Yes TABLE 1 Cell cycle analysis of A3B and other cell cycle-regulated genes on April 15, 2019 by guest
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Downloaded from that the RB/E2F pathway, which is commonly modified in cancer, likely contributes to
A3B overexpression observed in virus infections and in different tumor types, but
additional unknown signals are also likely to be required for full induction (model in
Fig. 7). on April 15, 2019 by guest
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aded from Our original studies with HPV and A3B led us to propose a model in which p53
represses A3B transcription and that p53 inactivation by the viral oncoprotein E6 or by
genetic mutation results in derepression of A3B transcription (23). This transcription
repression model is consistent with strong correlations in tumors and cell lines between
A3B overexpression and TP53 inactivation (54). A recent study confirmed these corre-
lations and used pharmacologic and genetic approaches to provide further support for
such a model (38). mbio.asm.org
10 DISCUSSION However, three different results in the cell-based systems presented
here do not support a dominant role for p53 in A3B repression. Specifically, the p53
binding domain of BKPyV is dispensable for A3B upregulation, nutlin treatment has no
effect on basal or induced A3B expression, and p53 knockout alone fails to induce A3B
expression (Fig. 2 and 3). Moreover, another recent study showed that p53 inactivation
renders cells more permissive for A3B mutagenesis (55). Therefore, we disfavor a
transcriptional role and now favor a “tolerance model” in which p53 inactivation
(genetic, epigenetic, or viral) is required for cells to be able to tolerate the increased
levels of DNA damage caused by A3B overexpression. This model also explains why
somatic TP53 mutations were identified as a significant global correlate with A3B
overexpression in cancer (11). The RB-E2F signaling axis is one of the most frequently mutated pathways in cancer,
which contributes to several hallmarks of cancer by deregulating the cell cycle (63). RB p21
CyclinD1
Rb
CDK4/6
Deleted/Suppressed
Amplified/Overexpressed
PyV LTAg
PyV truncT
p107
p130
P
P
P
p53
PyV LTAg
Palbociclib
ncNFκB
A3B
TEAD
Other regulators
FIG 7 Model for A3B transcriptional regulation. Schematic of the cell cycle-related proteins affected by
T antigen and drug treatments used in this study with implications for A3B transcriptional regulation. Solid lines represent established processes, and dashed lines represent regulatory interactions/pathways
implicated by our studies. Deleted/Suppressed
Amplified/Overexpressed FIG 7 Model for A3B transcriptional regulation. Schematic of the cell cycle-related proteins affected by
T antigen and drug treatments used in this study with implications for A3B transcriptional regulation. Solid lines represent established processes, and dashed lines represent regulatory interactions/pathways
implicated by our studies. FIG 7 Model for A3B transcriptional regulation. Schematic of the cell cycle-related proteins affected by
T antigen and drug treatments used in this study with implications for A3B transcriptional regulation. Solid lines represent established processes, and dashed lines represent regulatory interactions/pathways
implicated by our studies. mbio.asm.org
10 January/February 2019
Volume 10
Issue 1
e02690-18 ® Polyomavirus T Antigen Induces APOBEC3B Expression inactivation is also a common target for viral genes in order to promote the survival of
infected cells. The integration and continued expression of viral oncogenes in the host
genome are common characteristics of virus-associated tumors. MATERIALS AND METHODS Cell lines, culture conditions, and lentivirus production. Primary renal proximal tubule epithelial
cells (RPTECs; Lonza) were grown in REGM (Lonza). MCF10A cells were grown in MEGM (Lonza)
containing penicillin (100 U/ml) and streptomycin (100 g/ml). HuK(i)G10 cells were grown in RenaLife
epithelial medium (Lifeline Cell Technologies) with 5% FBS. MCF7 and derivative cell lines were grown
in Richter’s modification medium containing 5% fetal bovine serum, penicillin (100 U/ml), streptomycin
(100 g/ml), and 11.25 nM recombinant human insulin. All cell lines were grown at 37°C in a 5% CO2
incubator. Lentiviruses expressing TAg and mutant derivatives were produced in 293T cells and trans-
duced into RPTECs as described previously (23). Antibodies. Large T and truncT forms of BKPyV T antigen were detected using pAb416 (30). JCPyV
large T antigen was detected using PAB2000 (68). A Harris lab custom rabbit anti-human A3B monoclonal
antibody, 5210.87.13, was used in immunoblotting assays and in high-resolution immunofluorescent
microscopy experiments with JCPyV-infected HuK(i)G10 cells (69). Santa Cruz sc-130688 was used for A3B
quantification and lower-resolution microscopy of BKPyV- and JCPyV-infected HuK(i)G10 cells (69). UNG2
was detected using the Abcam antibody ab23926. RB1 was detected using Santa Cruz sc-102, and RBL1
was detected using Cell Signaling 89798, whereas RBL2 could not be detected in immunoblots with
Abcam antibody ab71143. TP53 (p53) was detected using clone DU-1 (Santa Cruz SC-126), p21 using CST
clone 1201 (CST no. 2947), and beta-actin using CST clone 13E5 (CST no. 4970). RNA isolation, RT-qPCR, and immunoblots. Total RNA was harvested by removal of medium and
resuspension in TRIzol (Thermo Fisher), and purification was done per the manufacturer’s protocol. RT-qPCR was used to quantify A3B and UNG2 transcripts in siRNA experiments as described previously
(11, 23) and these methods were adapted for CCNE2. Protein lysates from virus and siRNA experiments
were harvested at 3 or 7 dpi or postransduction, quantified, and immunoblotted as described previously
(70). Data were plotted and t tests were calculated using GraphPad Prism 7. Immunofluorescent microscopy experiments. HuK(i)G10 kidney cells were seeded at 6,000 cells/
well in a 96-well plate. Twenty-four hours later, infection with JCPyV was performed as described, and
then cells were collected 7 dpi. DISCUSSION For example, HPV-
positive tumors frequently express the viral E7 oncoprotein, which also has an LXCXE
motif, and this is thought to be critical for tumor development (64, 65). These tumors
also tend to have a high burden of APOBEC-associated mutations on the DNA strand
that serves as the template for lagging-strand replication, which is synthesized during
the S phase of the cell cycle (11, 14, 17). Although some of these effects have been
explained by perturbations to the p53 pathway, other pathways affected by E6 have
also been shown to alter A3B gene expression, such as those regulated by the TEAD and
ZNF384 transcription factors (23, 25, 36, 38). It is therefore not surprising in hindsight
that expression of the antiviral enzyme A3B is also induced upon disruption of the
RB-E2F pathway. This idea is supported by a study showing that high-risk HPV E7 is
capable of upregulating A3B (26). An additional study also found that elevated A3B
expression is significantly correlated with proliferative features in breast cancer (66). Last, the LXCXE motif may be acting through another cellular signaling pathway. For
instance, the LXCXE of various viral proteins triggers activation of the antiviral cGAS-
STING pathway, which could also contribute to A3B transcriptional regulation (67). on April 15, 2019 by guest
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aded from All of these results combine to indicate that A3B transcriptional regulation is
complex and governed by multiple pathways and different transcription factors. Per-
haps linkage to the cell cycle evolved to prevent potentially oncogenic mutations of the
host genome during normal cellular DNA replication or, alternatively, to maximize
antiviral responses during particularly susceptible cell cycle stages. Further experiments
using mutant viral oncogenes as molecular probes, such as T antigen, E6, and E7, are
likely to continue to provide valuable insights into the regulation of A3B and lead to a
greater understanding of its roles in tumorigenesis, virus evolution, and antiviral
immunity. January/February 2019
Volume 10
Issue 1
e02690-18 MATERIALS AND METHODS Infected cells were incubated with EdU (Click-iT Plus EdU Alexa Fluor 647
imaging kit; Thermo Fisher Scientific) for 15 min and incubated with CSK buffer (10 mM HEPES-KOH, pH
7.4; 300 mM sucrose; 100 mM NaCl; 3 mM KCl; 0.5% Triton X-100) (71) for 2 min on ice. Cells were then
fixed in 4% PFA for 10 min followed by permeabilization with 0.5% Triton X-100 for 20 min. For EdU
detection, the Click-iT reagent was added for 30 min in the dark according to the manufacturer’s protocol
and washed three times with PBS. Samples were incubated with BlockAid blocking solution (Thermo
Fisher) for 1 h at room temperature. T antigen, VP1, and A3B staining was performed using the
aforementioned antibodies at 1:1,000, 1:1,000, and 1:100 (1:50 for sc-130688) dilutions in BlockAid,
respectively, overnight at 4°C followed by staining with the secondary antibodies for 1 h at room mbio.asm.org
11 ® Starrett et al. temperature. Images were acquired on the Opera Phenix (PerkinElmer) with the confocal 63 water
objective. Immunofluorescence in RPTE cells was performed as described using the above-mentioned
antibodies and imaged on the Invitrogen EVOS FL Imaging System (72). siRNA and expression construct transfection. siRNAs targeting RB1 (J-003296-23; Dharmacon),
RBL1 (SI02629921; Qiagen), and RBL2 (sc-29425; Santa Cruz) and fluorescein-conjugated nontargeting
control siRNA (sc-36869; Santa Cruz) were purchased and diluted to a working concentration of 20 M. A final concentration of 20 nM was used for all targets in RPTECs, and 40 nM was used in MCF10A cells. siRNAs were delivered to cells using Lipofectamine RNAiMax (Thermo Fisher) as described previously (73). Drug treatment. Cells were treated with 5 M nutlin (Sigma) for 24 h, and after 18 h of treatment,
25 ng/ml phorbol myristate acetate (PMA; Sigma) was added for the final 6 h prior to RNA extraction. MCF10A cells were treated only with PMA or DMSO, and RNA was isolated 6 h after treatment. For the
palbociclib experiments, MCF10A and MCF7 (p53 WT and low A3B expression); HCC1937 and HCC1395
(low A3B expression); T47D, HCC1954, and Hs578T (intermediate A3B expression); and HCC1599,
HCC1143, SUM-225-CWN, and HCC202 (high A3B expression) cells were cultured in 6-well plates (Costar
3516; Corning Incorporated) until 70% confluence. Palbociclib (S1116; Selleckchem) was stored as a 5 mM
solution in H2O and added to cells at concentrations of 0 M (H2O control), 0.5 M, and 2.5 M. MATERIALS AND METHODS No
palbociclib was added to cells of the 0-h time point, which instead was transferred to ice prior to RNA
isolation. RNA was also isolated at 0, 1, and 4 h post-palbociclib administration (total RNA purification kit
37500; Norgen), and cDNA was synthesized with 500 ng RNA (iScript 170-8891; Bio-Rad). RT-qPCR assays
for A3B and CCNE2 were performed using the C1000 Thermal Cycler (Bio-Rad). For pretreatment with
PMA (tlrl-pma; InvivoGen; 1-mg/ml stock in DMSO), cells were treated with 0 ng/ml (DMSO) or 25 ng/ml
PMA for 3 h, followed by treatment with 0 M, 0.5 M, and 2.5 M palbociclib. RNA was isolated 0, 1, and
3 h after addition of palbociclib and processed as described above. For pretreatment with palbociclib,
cells were treated with 0 M, 0.5 M, and 2.5 M palbociclib for 30 min, followed by treatment with
0 ng/ml (DMSO) or 25 ng/ml PMA. RNA was isolated 0, 1, and 3 h after addition of PMA and processed
as described above. on April 15, 2019 by guest
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Downloaded from on April 15, 2019 by guest
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d from Bioinformatics. TCGA expression data were downloaded from the Broad GDAC Firehose as of
January 2016. Expression correlations against A3B by all other genes in the breast cancer cohort were
calculated, and significant correlates were used to determine significant activation or inhibition of
upstream regulators using Ingenuity Pathway Analysis (Qiagen). All other Spearman correlations and P
values were calculated, and heat maps were plotted using the R statistical environment. P values were
adjusted for multiple comparisons using the Bonferroni correction, and resulting q values were reported. Cell cycle data were acquired from http://www.targetgenereg.org/ in May 2018. ACKNOWLEDGMENTS We thank James A. DeCaprio, Diako Ebrahimi, and N. Alpay Temiz for valuable
discussions and comments. We thank James A. DeCaprio, Diako Ebrahimi, and N. Alpay Temiz for valuable
discussions and comments. This work was supported in part by Biogen and by grants from the National
Institutes of Health (NIAID R01 AI123162 to M.J., NCI R21 CA206309 to R.S.H., and NIAID
R37 to R.S.H.). J.W.M.M., P.N.S., and R.S.H. received funding from the Dutch Cancer
Society (KWF grant no. EMCR-2016-10270). Partial salary support for T.A.S. was provided
by the Susan G. Komen Foundation, support for G.J.S. and J.L.M. was provided by a
Graduate Research Fellowship from the National Science Foundation, and support for
M.C.J. was provided by a training grant from the NCI (T32 CA009138). R.S.H. is the
Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished
McKnight University Professor, and an Investigator of the Howard Hughes Medical
Institute. R.S.H. is a cofounder, shareholder, and consultant for ApoGen Biotechnologies Inc. Biogen sponsored a portion of the work in the Harris laboratory. 1. Escalera-Zamudio M, Rojas-Anaya E, Kolokotronis SO, Taboada B, Loza-
Rubio E, Méndez-Ojeda ML, Arias CF, Osterrieder N, Greenwood AD.
2016. Bats, primates, and the evolutionary origins and diversification of
mammalian gammaherpesviruses. mBio 7:e01425-16. https://doi.org/10
.1128/mBio.01425-16. January/February 2019
Volume 10
Issue 1
e02690-18 2. Cuevas JM, Geller R, Garijo R, López-Aldeguer J, Sanjuán R. 2015. Ex-
tremely high mutation rate of HIV-1 in vivo. PLoS Biol 13:e1002251.
https://doi.org/10.1371/journal.pbio.1002251. Polyomavirus T Antigen Induces APOBEC3B Expression Crystal
structure of APOBEC3A bound to single-stranded DNA reveals structural
basis for cytidine deamination and specificity. Nat Commun 8:15024. https://doi.org/10.1038/ncomms15024. 32. Ludlow JW, DeCaprio JA, Huang CM, Lee WH, Paucha E, Livingston DM. 1989. SV40 large T antigen binds preferentially to an underphosphory-
lated member of the retinoblastoma susceptibility gene product family. Cell 56:57–65. https://doi.org/10.1016/0092-8674(89)90983-5. Cell 56:57–65. https://doi.org/10.1016/0092-8674(89)90983-5. 33. Christensen JB, Imperiale MJ. 1995. Inactivation of the retinoblastoma
susceptibility protein is not sufficient for the transforming function of
the conserved region 2-like domain of simian virus 40 large T antigen. J
Virol 69:3945–3948. on April 15, 2019 by guest
http://mbio.asm.org/
from 14. Seplyarskiy VB, Soldatov RA, Popadin KY, Antonarakis SE, Bazykin GA,
Nikolaev SI. 2016. APOBEC-induced mutations in human cancers are
strongly enriched on the lagging DNA strand during replication. Ge-
nome Res 26:174–182. https://doi.org/10.1101/gr.197046.115. 34. Haug T, Skorpen F, Aas PA, Malm V, Skjelbred C, Krokan HE. 1998. Regulation of expression of nuclear and mitochondrial forms of human
uracil-DNA glycosylase. Nucleic Acids Res 26:1449–1457. 15. Haradhvala NJ, Polak P, Stojanov P, Covington KR, Shinbrot E, Hess JM,
Rheinbay E, Kim J, Maruvka YE, Braunstein LZ, Kamburov A, Hanawalt PC,
Wheeler DA, Koren A, Lawrence MS, Getz G. 2016. Mutational strand
asymmetries in cancer genomes reveal mechanisms of DNA damage
and repair. Cell 164:538–549. https://doi.org/10.1016/j.cell.2015.12.050. 35. Law EK, Sieuwerts AM, LaPara K, Leonard B, Starrett GJ, Molan AM, Temiz
NA, Vogel RI, Meijer-van Gelder ME, Sweep FCGJ, Span PN, Foekens JA,
Martens JWM, Yee D, Harris RS. 2016. The DNA cytosine deaminase
APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer. Sci Adv 2:e1601737. https://doi.org/10.1126/sciadv.1601737. 16. Morganella S, Alexandrov LB, Glodzik D, Zou X, Davies H, Staaf J,
Sieuwerts AM, Brinkman AB, Martin S, Ramakrishna M, Butler A, Kim H-Y,
Borg Å, Sotiriou C, Futreal PA, Campbell PJ, Span PN, Van Laere S,
Lakhani SR, Eyfjord JE, Thompson AM, Stunnenberg HG, van de Vijver
MJ, Martens JWM, Børresen-Dale A-L, Richardson AL, Kong G, Thomas G,
Sale J, Rada C, Stratton MR, Birney E, Nik-Zainal S. 2016. The topography
of mutational processes in breast cancer genomes. Nat Commun
7:11383. https://doi.org/10.1038/ncomms11383. 36. Leonard B, McCann JL, Starrett GJ, Kosyakovsky L, Luengas EM, Molan
AM, Burns MB, McDougle RM, Parker PJ, Brown WL, Harris RS. 2015. The
PKC/NF-kB signaling pathway induces APOBEC3B expression in multiple
human cancers. Cancer Res 75:4538–4547. https://doi.org/10.1158/0008
-5472.CAN-15-2171-T. 37. Polyomavirus T Antigen Induces APOBEC3B Expression Santiago ML, Pyeon D. 2015. APOBEC3A functions as a restriction factor
of human papillomavirus. J Virol 89:688–702. https://doi.org/10.1128/JVI
.02383-14. 9. Vartanian J, Guétard D, Henry M, Wain-Hobson S. 2008. Evidence for
editing of human papillomavirus DNA by APOBEC3 in benign and
precancerous lesions. Science 320:230–233. https://doi.org/10.1126/
science.1153201. 27. 27. Barbosa MS, Edmonds C, Fisher C, Schiller JT, Lowy DR, Vousden KH. 1990. The region of the HPV E7 oncoprotein homologous to adenovirus
E1a and Sv40 large T antigen contains separate domains for Rb binding
and casein kinase II phosphorylation. EMBO J 9:153–160. 10. Peretti A, Geoghegan EM, Pastrana DV, Smola S, Feld P, Sauter M, Lohse
S, Ramesh M, Lim ES, Wang D, Borgogna C, FitzGerald PC, Bliskovsky V,
Starrett GJ, Law EK, Harris RS, Killian JK, Zhu J, Pineda M, Meltzer PS,
Boldorini R, Gariglio M, Buck CB. 2018. Characterization of BK polyoma-
viruses from kidney transplant recipients suggests a role for APOBEC3 in
driving in-host virus evolution. Cell Host Microbe 23:628–635.e7. https://doi.org/10.1016/j.chom.2018.04.005. and casein kinase II phosphorylation. EMBO J 9:153–160. 28. Mietz JA, Unger T, Huibregtse JM, Howley PM. 1992. The transcriptional
transactivation function of wild-type p53 is inhibited by SV40 large
T-antigen and by HPV-16 E6 oncoprotein. EMBO J 11:5013–5020. 29. Low J, Humes HD, Szczypka M, Imperiale M. 2004. BKV and SV40
infection of human kidney tubular epithelial cells in vitro. Virology
323:182–188. https://doi.org/10.1016/j.virol.2004.03.027. g
j
11. Burns MB, Temiz NA, Harris RS. 2013. Evidence for APOBEC3B mutagen-
esis in multiple human cancers. Nat Genet 45:977–983. https://doi.org/
10.1038/ng.2701. 30. Abend JR, Joseph AE, Das D, Campbell-Cecen DB, Imperiale MJ. 2009. A
truncated T antigen expressed from an alternatively spliced BK virus
early mRNA. J Gen Virol 90:1238–1245. https://doi.org/10.1099/vir.0
.009159-0. 12. Shi K, Carpenter MA, Banerjee S, Shaban NM, Kurahashi K, Salamango DJ,
McCann JL, Starrett GJ, Duffy JV, Demir Ö, Amaro RE, Harki DA, Harris RS,
Aihara H. 2017. Structural basis for targeted DNA cytosine deamination
and mutagenesis by APOBEC3A and APOBEC3B. Nat Struct Mol Biol
24:131–139. https://doi.org/10.1038/nsmb.3344. on April 15, 2019 b
http://mbio.asm.org/
Downloaded from on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from 31. DeCaprio JA, Garcea RL. 2013. A cornucopia of human polyomaviruses. Nat Rev Microbiol 11:264–276. https://doi.org/10.1038/nrmicro2992. 13. Kouno T, Silvas TV, Hilbert BJ, Shandilya SMD, Bohn MF, Kelch BA, Royer
WE, Somasundaran M, Kurt Yilmaz N, Matsuo H, Schiffer CA. 2017. Polyomavirus T Antigen Induces APOBEC3B Expression Sieuwerts AM, Willis S, Burns MB, Look MP, Meijer-Van Gelder ME, Schlicker
A, Heideman MR, Jacobs H, Wessels L, Leyland-Jones B, Gray KP, Foekens JA,
Harris RS, Martens JWM. 2014. Elevated APOBEC3B correlates with poor
outcomes for estrogen-receptor-positive breast cancers. Horm Cancer
5:405–413. https://doi.org/10.1007/s12672-014-0196-8. 17. Hoopes JI, Cortez LM, Mertz TM, Malc EP, Mieczkowski PA, Roberts SA. 2016. APOBEC3A and APOBEC3B preferentially deaminate the lagging
strand template during DNA replication. Cell Rep 14:1273–1282. https://
doi.org/10.1016/j.celrep.2016.01.021. pril 15, 2019 by guest 18. Bhagwat AS, Hao W, Townes JP, Lee H, Tang H, Foster PL. 2016. Strand-
biased cytosine deamination at the replication fork causes cytosine to
thymine mutations in Escherichia coli. Proc Natl Acad Sci U S A 113:
2176–2181. https://doi.org/10.1073/pnas.1522325113. 38. Periyasamy M, Singh AK, Gemma C, Kranjec C, Farzan R, Leach DA,
Navaratnam N, Pálinkás HL, Vertessy BG, Fenton TR, Doorbar J, Fuller-
Pace F, Meek DW, Coombes RC, Buluwela L, Ali S. 2017. P53 controls
expression of the DNA deaminase APOBEC3B to limit its potential
mutagenic activity in cancer cells. Nucleic Acids Res 45:11056–11069. https://doi.org/10.1093/nar/gkx721. g
19. Harris RS, Dudley JP. 2015. APOBECs and virus restriction. Virology
479-480:131–145. https://doi.org/10.1016/j.virol.2015.03.012. 39. Vassilev LT. 2004. Small-molecule antagonists of p53-MDM2 binding:
research tools and potential therapeutics. Cell Cycle 3:417–419. https://
doi.org/10.4161/cc.3.4.801. 20. Conticello SG. 2008. The AID/APOBEC family of nucleic acid mutators. Genome Biol 9:229. https://doi.org/10.1186/gb-2008-9-6-229. 21. Vieira VC, Soares MA. 2013. The role of cytidine deaminases on innate
immune responses against human viral infections. Biomed Res Int
2013:1. https://doi.org/10.1155/2013/683095. 40. Lahav G, Rosenfeld N, Sigal A, Geva-Zatorsky N, Levine AJ, Elowitz MB,
Alon U. 2004. Dynamics of the p53-Mdm2 feedback loop in individual
cells. Nat Genet 36:147–150. https://doi.org/10.1038/ng1293. 22. Venkatesan S, Rosenthal R, Kanu N, McGranahan N, Bartek J, Quezada SA,
Hare J, Harris RS, Swanton C. 2018. Perspective: APOBEC mutagenesis in
drug resistance and immune escape in HIV and cancer evolution. Ann
Oncol 29:563–572. https://doi.org/10.1093/annonc/mdy003. 41. Barak Y, Juven T, Haffner R, Oren M. 1993. Mdm2 expression is induced
by wild type p53 activity. EMBO J 12:461–468. 42. El-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jack-
man J, Pietenpol JA, Burrell M, Hill DE, Wang Y, Wiman KG, Mercer WE,
Kastan MB, Kohn KW, Elledge SJ, Kinzler KW, Vogelstein B. 1994. WAF1/
CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res
54:1169–1174. 23. REFERENCES in viruses: patterns and determinants. Nat Rev Genet 9:267–276. https://
doi.org/10.1038/nrg2323. 5. Sanjuán R, Domingo-Calap P. 2016. Mechanisms of viral mutation. Cell
Mol Life Sci 73:4433–4448. https://doi.org/10.1007/s00018-016-2299-6. 5. Sanjuán R, Domingo-Calap P. 2016. Mechanisms of viral mutation. Cell
Mol Life Sci 73:4433–4448. https://doi.org/10.1007/s00018-016-2299-6. 6. Verhalen B, Starrett GJ, Harris RS, Jiang M. 2016. Functional upregulation
of the DNA cytosine deaminase APOBEC3B by polyomaviruses. J Virol
90:6379–6386. https://doi.org/10.1128/JVI.00771-16. 2. Cuevas JM, Geller R, Garijo R, López-Aldeguer J, Sanjuán R. 2015. Ex-
tremely high mutation rate of HIV-1 in vivo. PLoS Biol 13:e1002251. https://doi.org/10.1371/journal.pbio.1002251. 7. Warren CJ, Van Doorslaer K, Pandey A, Espinosa JM, Pyeon D. 2015. Role
of the host restriction factor APOBEC3 on papillomavirus evolution. Virus
Evol 1:vev015. https://doi.org/10.1093/ve/vev015. 3. Buck CB, Van Doorslaer K, Peretti A, Geoghegan EM, Tisza MJ, An P, Katz
JP, Pipas JM, McBride AA, Camus AC, McDermott AJ, Dill JA, Delwart E,
Ng TFF, Farkas K, Austin C, Kraberger S, Davison W, Pastrana DV, Varsani
A. 2016. The ancient evolutionary history of polyomaviruses. PLoS Pat-
hog 12:e1005574. https://doi.org/10.1371/journal.ppat.1005574. 8. Wang Z, Wakae K, Kitamura K, Aoyama S, Liu G, Koura M, Monjurul AM,
Kukimoto I, Muramatsu M. 2014. APOBEC3 deaminases induce hyper-
mutation in human papillomavirus 16 DNA upon beta interferon stim-
ulation. J Virol 88:1308–1317. https://doi.org/10.1128/JVI.03091-13. hog 12:e1005574. https://doi.org/10.1371/journal.ppat.100 y S, Shackelton LA, Holmes EC. 2008. Rates of evolutionary c 4. Duffy S, Shackelton LA, Holmes EC. 2008. Rates of evolutionary change mbio.asm.org
12 mbio.asm.org
12 mbio.asm.org
12 ® Polyomavirus T Antigen Induces APOBEC3B Expression Polyomavirus T Antigen Induces APOBEC3B Expression Vieira VC, Leonard B, White EA, Starrett GJ, Temiz NA, Lorenz LD, Lee D,
Soares MA, Lambert PF, Howley PM, Harris RS, Harris S. 2014. Human
papillomavirus E6 triggers upregulation of the antiviral and cancer
genomic DNA deaminase APOBEC3B. mBio 5:e02234-14. https://doi.org/
10.1128/mBio.02234-14. 43. Seoane J, Le H-V, Massagué J. 2002. Cell culture Myc suppression of the p21
Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA
damage. Nature 419:729–734. https://doi.org/10.1038/nature01119. 24. Mori S, Takeuchi T, Ishii Y, Kukimoto I. 2015. Identification of APOBEC3B
promoter elements responsible for activation by human papillomavirus
type 16 E6. Biochem Biophys Res Commun 460:555–560. https://doi.org/
10.1016/j.bbrc.2015.03.068. 44. Chen D, Tavana O, Chu B, Erber L, Chen Y, Baer R, Gu W. 2017. NRF2 is
a major target of ARF in p53-independent tumor suppression. Mol Cell
68:224–232.e4. https://doi.org/10.1016/j.molcel.2017.09.009. 10.1016/j.bbrc.2015.03.068. 25. Mori S, Takeuchi T, Ishii Y, Yugawa T, Kiyono T, Nishina H, Kukimoto I. 2017. Human papillomavirus 16 E6 upregulates APOBEC3B via the TEAD
transcription factor. J Virol 91:e02413-16. https://doi.org/10.1128/JVI
.02413-16. 45. Jiang L, Kon N, Li T, Wang SJ, Su T, Hibshoosh H, Baer R, Gu W. 2015. Ferroptosis as a p53-mediated activity during tumour suppression. Na-
ture 520:57–62. https://doi.org/10.1038/nature14344. 26. Warren CJ, Xu T, Guo K, Griffin LM, Westrich JA, Lee D, Lambert PF, 46. Whyte P, Buchkovich KJ, Horowitz JM, Friend SH, Raybuck M, Weinberg January/February 2019
Volume 10
Issue 1
e02690-18 ® Starrett et al. cycle gene regulatory networks. Nucleic Acids Res 44:6070–6086. https://doi.org/10.1093/nar/gkw523. RA, Harlow E. 1988. Association between an oncogene and an anti-
oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene
product. Nature 336:124–129. https://doi.org/10.1038/334124a0. product. Nature 336:124–129. https://doi.org/10.1038/3341 61. Laoukili J, Kooistra MRH, Brás A, Kauw J, Kerkhoven RM, Morrison A,
Clevers H, Medema RH. 2005. FoxM1 is required for execution of the
mitotic programme and chromosome stability. Nat Cell Biol 7:126–136. https://doi.org/10.1038/ncb1217. 47. Caracciolo V, Reiss K, Khalili K, De Falco G, Giordano A. 2006. Role of the
interaction between large T antigen and Rb family members in the
oncogenicity of JC virus. Oncogene 25:5294–5301. https://doi.org/10
.1038/sj.onc.1209681. 62. Zhao B, Barrera LA, Ersing I, Willox B, Schmidt SCS, Greenfeld H, Zhou H,
Mollo SB, Shi TT, Takasaki K, Jiang S, Cahir-McFarland E, Kellis M, Bulyk
ML, Kieff E, Gewurz BE. 2014. The NF-B genomic landscape in lympho-
blastoid B cells. Cell Rep 8:1595–1606. https://doi.org/10.1016/j.celrep
.2014.07.037. 48. Henley SA, Dick FA. 2012. The retinoblastoma family of proteins and
their regulatory functions in the mammalian cell division cycle. Cell Div
7:10. https://doi.org/10.1186/1747-1028-7-10. p
g
49. Classon M, Dyson N. 2001. P107 and P130: versatile proteins with
interesting pockets. Exp Cell Res 264:135–147. https://doi.org/10.1006/
excr.2000.5135. 63. Sherr CJ, Beach D, Shapiro GI. 2016. Targeting CDK4 and CDK6: from
discovery to therapy. Cancer Discov 6:353–367. https://doi.org/10.1158/
2159-8290.CD-15-0894. 50. Harris KF, Christensen JB, Imperiale MJ. 1996. BK virus large T antigen:
interactions with the retinoblastoma family of tumor suppressor pro-
teins and effects on cellular growth control. J Virol 70:2378–2386. 64. 10.1016/j.bbrc.2015.03.068. Mirabello L, Yeager M, Yu K, Clifford GM, Xiao Y, Zhu B, Cullen M, Boland
JF, Wentzensen N, Nelson CW, Raine-Bennett T, Chen Z, Bass S, Song L,
Yang Q, Steinberg M, Burdett L, Dean M, Roberson D, Mitchell J, Lorey T,
Franceschi S, Castle PE, Walker J, Zuna R, Kreimer AR, Beachler DC,
Hildesheim A, Gonzalez P, Porras C, Burk RD, Schiffman M. 2017. HPV16
E7 genetic conservation is critical to carcinogenesis. Cell 170:
1164–1174.e6. https://doi.org/10.1016/j.cell.2017.08.001. on April 15, 2019 by guest
http://mbio.asm.org/
Downloaded from on April 15, 2019 b
http://mbio.asm.org/
Downloaded from 51. Whittaker SR, Mallinger A, Workman P, Clarke PA. 2017. Inhibitors of
cyclin-dependent kinases as cancer therapeutics. Pharmacol Ther 173:
83–105. https://doi.org/10.1016/j.pharmthera.2017.02.008. 52. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. 2015. The history and
future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev
Drug Discov 14:130–146. https://doi.org/10.1038/nrd4504. 1164–1174.e6. https://doi.org/10.1016/j.cell.2017.08.001. 65. Zhen S, Hua L, Takahashi Y, Narita S, Liu YH, Li Y. 2014. In vitro and in
vivo growth suppression of human papillomavirus 16-positive cervical
cancer cells by CRISPR/Cas9. Biochem Biophys Res Commun 450:
1422–1426. https://doi.org/10.1016/j.bbrc.2014.07.014. 53. O’Leary B, Finn RS, Turner NC. 2016. Treating cancer with selective
CDK4/6 inhibitors. Nat Rev Clin Oncol 13:417–430. https://doi.org/10
.1038/nrclinonc.2016.26. on April 15, 2019 by guest
http://mbio.asm.org/
from 54. Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B,
Refsland EW, Kotandeniya D, Tretyakova N, Nikas JB, Yee D, Temiz NA,
Donohue DE, McDougle RM, Brown WL, Law EK, Harris RS. 2013. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature
494:366–370. https://doi.org/10.1038/nature11881. 66. Cescon DW, Haibe-Kains B, Mak TW. 2015. APOBEC3B expression in
breast cancer reflects cellular proliferation, while a deletion polymor-
phism is associated with immune activation. Proc Natl Acad Sci U S A
112:2841–2846. https://doi.org/10.1073/pnas.1424869112. 67. Lau L, Gray EE, Brunette RL, Stetson DB. 2015. DNA tumor virus onco-
genes antagonize the cGAS-STING DNA-sensing pathway. Science 350:
568–571. https://doi.org/10.1126/science.aab3291. 55. Nikkilä J, Kumar R, Campbell J, Brandsma I, Pemberton HN, Wallberg F,
Nagy K, Scheer I, Vertessy BG, Serebrenik AA, Monni V, Harris RS, Pettitt
SJ, Ashworth A, Lord CJ. 2017. Elevated APOBEC3B expression drives a
kataegic-like mutation signature and replication stress-related therapeu-
tic vulnerabilities in p53-defective cells. Br J Cancer 117:113–123. https://
doi.org/10.1038/bjc.2017.133. 68. Knowles WA, Sharp IR, Efstratiou L, Hand JF, Gardner SD. 1991. 10.1016/j.bbrc.2015.03.068. Prepa-
ration of monoclonal antibodies to JC virus and their use in the diag-
nosis of progressive multifocal leucoencephalopathy. J Med Virol 34:
127–131. https://doi.org/10.1002/jmv.1890340211. 56. Otto T, Sicinski P. 2017. Cell cycle proteins as promising targets in cancer
therapy. Nat Rev Cancer 17:93–115. https://doi.org/10.1038/nrc.2016
.138. 69. Leonard B, Starrett GJ, Maurer MJ, Oberg A, Van Bockstal M, Van Dorpe
J, De Wever O, Helleman J, Sieuwerts AM, Berns EMJJ, Martens JWM,
Anderson B, Brown WL, Kalli KR, Kaufmann SH, Harris RS. 2016. APOBEC3G expression correlates with T cell infiltration and improved
clinical outcomes in high-grade serous ovarian carcinoma. Clin Cancer
Res 22:4746–4755. https://doi.org/10.1158/1078-0432.CCR-15-2910. 57. Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade M, Trachet E, Albassam
M, Zheng X, Leopold WR, Pryer NK, Toogood PL. 2004. Specific inhibition
of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor
activity in human tumor xenografts. Mol Cancer Ther 3:1427–1438. pril 15, 2019 by guest 70. Jiang M, Abend JR, Tsai B, Imperiale MJ. 2009. Early events during BK
virus entry and disassembly. J Virol 83:1350–1358. https://doi.org/10
.1128/JVI.02169-08. 58. Raspé E, Coulonval K, Pita JM, Paternot S, Rothé F, Twyffels L, Brohée S,
Craciun L, Larsimont D, Kruys V, Sandras F, Salmon I, Van Laere S, Piccart M,
Ignatiadis M, Sotiriou C, Roger PP. 2017. CDK4 phosphorylation status and
a linked gene expression profile predict sensitivity to palbociclib. EMBO Mol
Med 9:1052–1066. https://doi.org/10.15252/emmm.201607084. 71. Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M. 2009. Mechanism of
genomic instability in cells infected with the high-risk human papillo-
maviruses. PLoS Pathog 5:e1000397. https://doi.org/10.1371/journal
.ppat.1000397. 59. Stoeck A, Lejnine S, Truong A, Pan L, Wang H, Zang C, Yuan J, Ware C,
MacLean J, Garrett-Engele PW, Kluk M, Laskey J, Haines BB, Moskaluk C,
Zawel L, Fawell S, Gilliland G, Zhang T, Kremer BE, Knoechel B, Bernstein
BE, Pear WS, Liu XS, Aster JC, Sathyanarayanan S. 2014. Discovery of
biomarkers predictive of GSI response in triple-negative breast cancer
and adenoid cystic carcinoma. Cancer Discov 4:1154–1167. https://doi
.org/10.1158/2159-8290.CD-13-0830. 72. Jiang M, Entezami P, Gamez M, Stamminger T, Imperiale MJ. 2011. Functional reorganization of promyelocytic leukemia nuclear bodies
during BK virus infection. mBio 2:e00281-10. 73. Jiang M, Zhao L, Gamez M, Imperiale MJ. 2012. Roles of ATM and
ATR-mediated DNA damage responses during lytic BK polyomavirus
infection. PLoS Pathog 8:e1002898. https://doi.org/10.1371/journal.ppat
.1002898. 60. Fischer M, Grossmann P, Padi M, DeCaprio JA. 2016. January/February 2019
Volume 10
Issue 1
e02690-18 10.1016/j.bbrc.2015.03.068. Integration of TP53,
DREAM, MMB-FOXM1 and RB-E2F target gene analyses identifies cell mbio.asm.org
14 January/February 2019
Volume 10
Issue 1
e02690-18
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https://openalex.org/W4293062697
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https://zenodo.org/record/7002276/files/Influence_of_Position_of_Rice_Anthers_At_Plating_on_Callusing_and_Plant_Regeneration.pdf
|
English
| null |
Influence of Position of Rice Anthers At Plating on Callusing and Plant Regeneration
|
Zenodo (CERN European Organization for Nuclear Research)
| 1,987
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cc-by
| 637
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Influence of position of rice anthers at
plating on callusing and plant
regeneration Callusing response of anthers. 1 =Anther positioned on edge callusing from upper lohc. 2 =One anthe-r on
edge at left and the nthcrllat at right. both callusing. 3 =Anther llat.caHusing from hoth lobe~. 4 =Anther flat. callusing from only one lobe. 5 =Anther (JtJ edge, callusing fr{lnl upper and lower lobes. (l =Anther on edge. callusing from lower lohc only. Note calli from lower lobes growing into the medium. S. T Men:r and F./. Zapata, Tissue Culture
Lahora/ory. Plalll Breeding
Department. I I? Rl S. T Men:r and F./. Zapata, Tissue Culture
Lahora/ory. Plalll Breeding
Department. I I? Rl We studied the effect of anther
orientation at plating on callusing
response and plant regeneration in
japonica rice variety Taipei 309. Callus
induction from anthers was according to
standard I R R I procedure. Anthers were plated on semisolid EIO
(modified B5 medium. each liter
containing I mg 2.4-D. 0.5 mg BAP. 0.5
mg lA A. 20 g sucrose, and 5 g glucose) . Sixty anthers were inoculated at random
(specific positioning of anthers is
extremely difficult because l)f their small
size). Microscopic examination showed
that anthers were naturally positioned
approximately half on edge (one lobe
touching the medium) and half nat
(both lobes in contact with the medium). Callusing response of anthers. 1 =Anther positioned on edge callusing from upper lohc. 2 =One anthe-r on
edge at left and the nthcrllat at right. both callusing. 3 =Anther llat.caHusing from hoth lobe~. 4 =Anther flat. callusing from only one lobe. 5 =Anther (JtJ edge, callusing fr{lnl upper and lower lobes. (l =Anther on edge. callusing from lower lohc only. Note calli from lower lobes growing into the medium. Plates were incubated at 26-29 °C in
dim light and scored for callusing 40 d
alter plating. Some calli of both nat and
edge-oriented anthers were transferred
for regeneration in MS medium (I mg
each NAA and kinetin/liter and 30 g
sucrose/liter) under continuous light. Percentage of green plant regeneration
was recorded. About 15% of the calli transferred ror
regeneration produced green or grcr!1
and albino plantlets. or the 2,160 anthers plated, 518 (24%)
produced calli. Of the anthers with calli,
60% were plated on edge (see figure) and
40% nat. Most of the anthers plated nat
callused on both lobes, but callusing on
one lobe was also observed. Most
anthers plated on edge produced callus
on the upper lobe. Published by the International Rice Research Institute, P. 0. Box 933, Manila, Philippines
Genetic Evaluation and utilization
TISSUE CULTURE Published by the International Rice Research Institute, P. 0. Box 933, Manila, Philippines
Genetic Evaluation and utilization
TISSUE CULTURE Published by the International Rice Research Institute, P. 0. Box 933, Manila, Philippin Influence of position of rice anthers at
plating on callusing and plant
regeneration Only rarely did
anthers plated on edge produce callus
from both lobes or from the lower lobe
only. Observations m<.~dc on rice varieties
IRS, Basmati 370, and Pankaj showed
the same anther callusing response. No special anther inoculation;
technique is needed 10 get satisf:ictory
callusing response in rice cxcept'\o sec
I
I
. . •
t 1at ant 1ers are not buned 111 the
medium. D Observations m<.~dc on rice varieties
IRS, Basmati 370, and Pankaj showed
the same anther callusing response. Most of the anthers dehisccd within 5
d of plating. liberating a large part of
the pollen grains on the medium. None
of the liberated pollen grains developed
into calli. Calli from pollen grains held
within the anther started appearing
about 25 d after inoculation. No special anther inoculation;
technique is needed 10 get satisf:ictory
callusing response in rice cxcept'\o sec
I
I
. . •
t 1at ant 1ers are not buned 111 the
medium. D IRRN 12:4 (August 1987)
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https://openalex.org/W4318700676
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https://biblio.ugent.be/publication/01GTM0MMAJ5M120CW3XCHYPTNT/file/01GZXFBKJBZ8PV00WWCEJ566PD.pdf
|
English
| null |
Unexpected complexity of the ammonia monooxygenase in archaea
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The ISME journal
| 2,023
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cc-by
| 12,891
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INTRODUCTION diverse enzymes of the CuMMO (copper-dependent membrane
monooxygenase) protein family, with a few notable exceptions. Crystal structures [22–26] and cryo-EM structures [27, 28] of
particulate methane monooxygenase (pMMO) from five methano-
trophs have consistently confirmed a three-polypeptide protomer
(subunits-A, -B and -C) arranged in a trimer of α3β3γ3 configuration
with at least two conserved metal sites in each protomer. Even so,
the elucidation of the active site has remained ambiguous. It was
first proposed to reside in the PmoB subunit of pMMO [29]. More
recently, a cryo-EM analysis supports the active site primarily
being coordinated by PmoA [27], while differing amino acid
conservation in Verrucomicrobia [30], a recent spectroscopic
analysis [31], and mutagenesis of a hydrocarbon monooxygenase
[32], suggest its localization in the PmoC subunit. Nitrification, the conversion of ammonium to nitrate, is a
crucial step in the global nitrogen cycle solely performed by
microorganisms. The process has attracted particular attention
due to its agricultural and environmental relevance. The first
and rate limiting [1] step of nitrification is the oxidation of
ammonia via the integral membrane protein complex ammo-
nia monooxygenase (AMO) [2, 3]. While ammonia-oxidizing
bacteria (AOB) were first discovered over 125 years ago [4]
and have been extensively studied, this biological process was
also detected in the archaeal domain in the last 20 years
[5–7]. Ammonia-oxidizing archaea (AOA) have gained broad
attention as they are widespread in nature and are more
abundant than their bacterial counterparts in most terrestrial
and
marine
environments,
indicating
important
roles
in
nitrogen cycling [8–14]. Their central nitrogen and carbon
metabolism, however, is distinct from that of AOB [15–18]. In
particular, subunits of the AMO complex show only about 40%
identity
to
those
of
bacteria
[19]
and
archaeal
proteins
catalyzing the second step in ammonia oxidation, i.e. the
conversion of hydroxylamine to nitrite, are still unknown
[19–21]. These differences imply important functional differ-
entiation in their environmental roles that have yet to be
unraveled. Although no AMO structure has been determined experimen-
tally, homology modeling for the AMO of the bacterium
Nitrosomonas europaea using pMMO as a template supported
a homotrimeric structure as well as conservation of the CuB and
CuC copper sites [33]. The archaeal AMO complex is the most
distantly related of all CuMMO proteins [34, 35] and very little is
known so far about its structure or
function. Received: 8 July 2022 Revised: 9 January 2023 Accepted: 12 January 2023
Published online: 31 January 2023
1Archaea Biology and Ecogenomics Unit, Department of Functional and Evolutionary Ecology, University of Vienna, Vienna, Austria. 2Mass Spectrometry Facility, Max Perutz Labs,
Vienna BioCenter (VBC), Vienna, Austria. 3VIB Center for Inflammation Center and Department of Biochemistry & Microbiology, Ghent University, Ghent, Belgium. 4Molecular
Systems Biology Unit, Department of Functional and Evolutionary Ecology, University of Vienna, Vienna, Austria. 5Department of Biochemistry and Cell Biology, Max Perutz Labs,
University of Vienna, Vienna, Austria. ✉email: christa.schleper@univie.ac.at ARTICLE
OPEN
Unexpected complexity of the ammonia monooxygenase in
archaea
Logan H. Hodgskiss
1, Michael Melcher1, Melina Kerou
1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides
3,
Stefanie Wienkoop4, Markus Hartl2,5 and Christa Schleper
1✉ Logan H. Hodgskiss
1, Michael Melcher1, Melina Kerou
1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides
3,
Stefanie Wienkoop4, Markus Hartl2,5 and Christa Schleper
1✉ 1, Michael Melcher1, Melina Kerou
1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides
3
Markus Hartl2,5 and Christa Schleper
1✉ Logan H. Hodgskiss
1, Michael Melcher1, Melina Kerou
1, Weiqiang Chen2, Rafael I. Ponce-Toledo1, Savvas N. Savvides
3,
Stefanie Wienkoop4, Markus Hartl2,5 and Christa Schleper
1✉ © The Author(s) 2023, corrected publication 2023 © The Author(s) 2023, corrected publication 2023 © The Author(s) 2023, corrected publication 2023 Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental
knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally
abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis
and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted
AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated,
and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches
support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals
clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this
ecologically important nitrification complex. The ISME Journal (2023) 17:588–599; https://doi.org/10.1038/s41396-023-01367-3 The ISME Journal (2023) 17:588–599; https://doi.org/10.1038/s41396-023-01367-3 www.nature.com/ismej Received: 8 July 2022 Revised: 9 January 2023 Accepted: 12 January 2023
Published online: 31 January 2023 MATERIALS AND METHODS
Reactor growth Ladders for each gel are represented at the bottom of the respe
ournal (2023) 17:588 – 599 1 2
3
4
5
6
7 8
9
11
13
15
17
18
19 20
21
22
1236
1048
720
480
242
146
66
kDa
A
Relative intensity
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
Gel band number
AMO Complex of N. viennensis
AmoA
AmoB
AmoC4/C6
AmoX
NVIE_004540
NVIE_004550 A
Relative intensity
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
Gel band number
AMO Complex of N. viennensis
AmoA
AmoB
AmoC4/C6
AmoX
NVIE_004540
NVIE_004550 A 1 2
2
3
4
5
6
7 8
9
11
13
15
17
18
19 20
21
22
1236
1048
720
480
242
146
66
kDa
1
3
5
7 9
11 13
17 18 19 20
22
23
24
25
26
27
1236 1048
720
480
242
146
66
kDa
B
Relative in
0.0
0.1
0.2
0.3
0.4
0.5
0.6
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
Gel band number
Relative intensity
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Gel band number
AMO Complex of N. cavascurensis
AmoA
AmoB
AmoC
AmoX (NCAV_0491)
NCAV_0488
NCAV_0486
Fig. 1
Relative intensity patterns of AMO subunits in BN-PAGE gels. Relative abundance of iBAQ normalized intensities of known and
putative AMO subunits. iBAQ intensities for each protein are normalized to the highest detected intensity of that protein to create a relative
abundance profile for each protein. A Patterns of AMO intensity in N. viennensis. B Patterns of AMO intensity in N. cavascurensis. Bands selected
to be cut and analyzed via mass spectroscopy are indicated by numbered brackets from left (top of gel) to right (bottom of gel) and
correspond to numbers on the x-axis of respective plots. Ladders for each gel are represented at the bottom of the respective panels. MATERIALS AND METHODS
Reactor growth and chemical cross-linking. Beside the three known AmoABC
proteins, three additional potential subunits were identified and
one of the six predicted AmoC proteins in N. viennensis was
recognized as the primary homolog in the protein complex. In
addition, the overall subunit composition of the AMO complex
was
confirmed
in
the
distantly
related
thermophilic
AOA
Nitrosocaldus cavascurensis. g
Nitrososphaera viennensis was grown as a continuous culture in 2 L bioreactors
(Eppendorf) filled with 1.5 L of freshwater medium (FWM) [37, 38] with modified
trace element solution [5], 7.5 µM FeNaEDTA, 2 mM NH4Cl, and 1 mM pyruvate
at 42 °C and pH 7.5. Carbonate was supplied by gassing the reactors with a 98%
air 2% CO2 mixture. The applied dilution rates ranged from 0.035 to 0.07 h−1. 1 2
2
3
4
5
6
7 8
9
11
13
15
17
18
19 20
21
22
1236
1048
720
480
242
146
66
kDa
1
3
5
7 9
11 13
17 18 19 20
22
23
24
25
26
27
1236 1048
720
480
242
146
66
kDa
A
B
Relative intensity
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22
Gel band number
AMO Complex of N. viennensis
AmoA
AmoB
AmoC4/C6
AmoX
NVIE_004540
NVIE_004550
Relative intensity
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Gel band number
AMO Complex of N. cavascurensis
AmoA
AmoB
AmoC
AmoX (NCAV_0491)
NCAV_0488
NCAV_0486
ative intensity patterns of AMO subunits in BN-PAGE gels. Relative abundance of iBAQ normalized intensit
MO subunits. iBAQ intensities for each protein are normalized to the highest detected intensity of that protein t
e profile for each protein. A Patterns of AMO intensity in N. viennensis. B Patterns of AMO intensity in N. cavascurens
and analyzed via mass spectroscopy are indicated by numbered brackets from left (top of gel) to right (bo
d to numbers on the x-axis of respective plots. INTRODUCTION Based on
comparative metagenomics alone, it has been suggested that
an additional subunit might be present in the complex, termed
AmoX [15, 36]. Due to the difficulty of growing nitrifying organisms and the
inherent problems with isolating membrane proteins, no struc-
tural studies have been successfully carried out for any AMO
complex, bacterial or archaeal. This holds true for most of the To gain insights into the overall architecture of the archaeal
AMO complex, membrane protein fractions from the well
characterized soil AOA, Nitrososphaera viennensis, were analyzed
biochemically using native gel electrophoresis, mass spectrometry, L.H. Hodgskiss et al. 589 MATERIALS AND METHODS
Reactor growth 1 2
3
4
5
6
7 8
9
11
13
15
17
18
19 20
21
22
1236
1048
720
480
242
146
66
kDa B
Relative intensity
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Gel band number
AMO Complex of N. cavascurensis
AmoA
AmoB
AmoC
AmoX (NCAV_0491)
NCAV_0488
NCAV_0486 B 0.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Gel band number 1 12 13 14 15 16
Gel band number 2
1
3
5
7 9
11 13
17 18 19 20
22
23
24
25
26
27
1236 1048
720
480
242
146
66
kDa 242 Fig. 1
Relative intensity patterns of AMO subunits in BN-PAGE gels. Relative abundance of iBAQ normalized intensities of known and
putative AMO subunits. iBAQ intensities for each protein are normalized to the highest detected intensity of that protein to create a relative
abundance profile for each protein. A Patterns of AMO intensity in N. viennensis. B Patterns of AMO intensity in N. cavascurensis. Bands selected
to be cut and analyzed via mass spectroscopy are indicated by numbered brackets from left (top of gel) to right (bottom of gel) and
correspond to numbers on the x-axis of respective plots. Ladders for each gel are represented at the bottom of the respective panels. The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. MATERIALS AND METHODS
Reactor growth 590 C
B
X
A
170
kDa
130
100
70
55
40
35
25
15
10
Most Abundant Proteins (% iBAQ Intensity of Band)
(F1) SlaA (90%)
(F3) NVIE_028570 (28%), SlaA (26%)
(F5) NVIE_024150 (56%)
(F6) AmoA (63%)
Ladder
(F7) AmoC (81%)
(F8) NVIE_021780 (31.9%), AmoB (12%)
(F9) NVIE_004540 (47%), NVIE_004550 (35%), AmoX (8%)
SimplyBlue SafeStain
96%
4%
<0.05%
AmoC6/C4
AmoC1/C2
AmoC3
Composition of AmoC Peptides
(unique peptides identified) C
B
X
A
170
kDa
130
100
70
55
40
35
25
15
10
Most Abundant Proteins (% iBAQ Intensity of Band)
(F1) SlaA (90%)
(F3) NVIE_028570 (28%), SlaA (26%)
(F5) NVIE_024150 (56%)
(F6) AmoA (63%)
Ladder
Ladder
Ladder
(F7) AmoC (81%)
(F8) NVIE_021780 (31.9%), AmoB (12%)
(F9) NVIE_004540 (47%), NVIE_004550 (35%), AmoX (8%)
170
kDa
130
100
70
55
40
35
25
15
10
kDa
245
190
135
100
80
58
46
32
25
22
17
Silver Staining (Classic)
Silver Staining (Farmer’s Reducer)
SimplyBlue SafeStain
C
B
X
A
C
B
X
A
96%
4%
<0.05%
AmoC6/C4
AmoC1/C2
AmoC3
Composition of AmoC Peptides
(unique peptides identified)
Fig. 2
Tricine-SDS-PAGE gels of AMO cut-outs. Tricine-SDS-PAGE of AMO bands from BN-PAGE gels. Comparison of three different staining
methods for Tricine-SDS-PAGE gels with size markers on left side. Bands cut for analysis from a gel stained with SimplyBlue SafeStain and
digested using trypsin are indicated by brackets. Percentages represent the percentage of iBAQ normalized protein intensities for each
individual band. Band identifiers are indicated in parentheses. Green arrows marked A, B, C, and X represent expected heights of bands fo
AMO subunits AmoA, AmoB, AmoC, and AmoX, respectively. Orange arrows marked A, B, C, and X represent equivalent bands of AmoA
AmoB, AmoC, and AmoX, respectively, from silver-stained gels. A pie chart from the band with the highest amount of AmoC shows the
percentage of AmoC bands coming from distinguishable AmoC homologs. 0 Most Abundant Proteins (% iBAQ Intensity of Band) (F5) NVIE_024150 (56%) Fig. 2
Tricine-SDS-PAGE gels of AMO cut-outs. Tricine-SDS-PAGE of AMO bands from BN-PAGE gels. Comparison of three different staining
methods for Tricine-SDS-PAGE gels with size markers on left side. Bands cut for analysis from a gel stained with SimplyBlue SafeStain and
digested using trypsin are indicated by brackets. Percentages represent the percentage of iBAQ normalized protein intensities for each
individual band. Band identifiers are indicated in parentheses. RESULTS
C
l Complexome analysis of native membrane complexes displays
the AMO composition of Nitrososphaera viennensis
Nitrososphaera viennensis was grown in continuous culture for
several weeks under optimal growth conditions in order to obtain
enough biomass for biochemical analyses (Melcher et al. [45]). Between 800–2000 µg of membrane proteins were obtained from
450–550 mg of biomass per preparation, of which approximately
40–50 µg were loaded per lane on blue-native PAGE gels [39]. After optimization of conditions, 22 bands were cut out and
subjected to mass spectrometry (see Supplementary Materials and
Methods; Fig. S1A). AMO subunits (AmoA, AmoB, and AmoC) were
among the most abundant proteins (22% of iBAQ normalized
intensity) detected overall in these membrane fractions. The
relative intensity profiles of AmoA, AmoB, and AmoC showed
three distinct peaks corresponding to bands 4, 7, and 12, with the
most prominent peak occurring at band 7 (Fig. 1A). The subunits
AmoA,
AmoB,
and
AmoC
made
up
10%,
5%,
and
14%,
respectively, of the total protein found in band 7 based on iBAQ
normalized intensities. AmoX was also present in band 7
representing 10%. The most intense signals for the AmoC subunit
were represented by two of the six AmoC homologs, AmoC6 and
AmoC4. These two homologs could not be distinguished based on
the peptides identified in the BN-PAGE gel. In denaturing Tricine-
SDS-PAGE of cutouts from band 7, all known components of the
AMO complex were visualized and confirmed by proteomics
(Fig. 2). In addition, this allowed for the identification of unique
peptides of the AmoC6 subunit (see Supplementary Discussion). T
id
if
ddi i
l
i
h
i h
b
f
h Harvested biomass was concentrated in three centrifugation steps. First
with a continuous centrifuge (CEPA model LE) operating at maximum
speed. Biomass from the continuous centrifuge was then suspended in
400 mL volumes and concentrated using a Sorvall Lynx 4000 with an
F12–6 × 500 rotor for 30 min at 4 °C and 16,000 × g. Finally, biomass was
resuspended in small volumes and aliquoted to 1.5 mL Eppendorf tubes
and concentrated to a final pellet for 30 min at 4 °C and 16,000 × g using a
bench-top centrifuge. Pellets were frozen at −70 °C until further analysis. MATERIALS AND METHODS
Reactor growth Green arrows marked A, B, C, and X represent expected heights of bands for
AMO subunits AmoA, AmoB, AmoC, and AmoX, respectively. Orange arrows marked A, B, C, and X represent equivalent bands of AmoA,
AmoB, AmoC, and AmoX, respectively, from silver-stained gels. A pie chart from the band with the highest amount of AmoC shows the
percentage of AmoC bands coming from distinguishable AmoC homologs. PAGE of N. viennensis, BN-PAGE of N. cavascurensis, and cross-linked
samples, respectively. Relevant scripts for analysis can be found in the
GitHub repository https://github.com/hodgskiss/Archaeal_AMO. Nitrosocaldus cavascurensis was grown as a batch culture in the same
reactors, volume, and medium as described for N. viennensis, but at 68 °C
with 1 mM NH4Cl, 1 mM pyruvate, and pH 7.0. Carbonate was also supplied
by gassing, but with a mixture of air/ N2/ CO2 to achieve a 10% O2 and 2%
CO2 mixture. To increase the biomass, NH4Cl was added stepwise with
syringes via a septum to increase the final NO2
−concentration to
approximately 2.5 mM before harvesting the cultures. Sample and data processing Detailed
information
for
bioinformatic
analysis,
membrane
protein
extraction, BN-PAGE methods, Tricine-SDS-PAGE methods, mass spectro-
metry preparation, cross-linking, data analysis, and AlphaFold multimer
predictions can be found in Supplementary Materials and Methods. Briefly, cells were lysed and membrane fractions were isolated using
ultracentrifugation
(Beckman
Coulter
Ultracentrifuge;
SW
41
Ti
Swinging-Bucket Rotor, kmax = 124; 200,000 × g) for 90 min at 4 °C
using 13.2 mL thinwall polypropylene tubes with a level of decelera-
tion set to 7. Membrane proteins were extracted using n-dodecyl-β-D-
maltoside (DDM; Invitrogen BN2005) and loaded on a 3–12% pre-cast
BN-PAGE gel (Invitrogen BN1001). Selected bands were cut out and
analyzed via mass spectrometry for protein identification. Procedures
for protein extraction and running a BN-PAGE gel were based on
previous studies [39, 40] and the NativePAGE Novex Bis-Tris Gel
System manual from Life Technologies (MAN0000557). Study design
and analysis for membrane extraction and BN-PAGE was guided by
previous
studies
[41,
42]
Cross-linking
methods
were
based
on
protocols from Hevler et al. (2021) [43]. The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE [44] partner repository with
the dataset identifiers PXD035349, PXD034632, and PXD034475 for BN- To identify additional proteins that might be part of the
archaeal AMO complex, a correlation analysis was conducted to The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 591 Table 1. Correlations of proteins with occurrence of AmoA,B, and C in (A) N. viennensis and (B) N. cavascurensis BN-PAGE gels. Gene
Proteina
Conserved in Extant AOAb
Exclusive to AOAb
Correlationsc
AmoA
AmoB
AmoC6
A
AMO Correlation Results for N. viennensis
NVIE_016740
surface associated S-layer protein
X
X
X
X
nuoJ
Complex I
X
X
X
X
nuoM
Complex I
X
X
X
X
coxB
Complex IV
X
X
X
coxA1
Complex IV
X
NVIE_013530
protein of unknown function
X
X
X
X
NVIE_027260
conserved protein of unknown function
X
X
X
X
NVIE_017130
protein of unknown function DUF373
X
X
X
amoX
potential AMO subunit
X
X
X
X
X
NVIE_004540
hypothetical protein
X
X
X
X
X
B
AMO Correlation Results for N. Sample and data processing cavascurensis
coxA
Cytochrome c oxidase polypeptide 1
X
X
NCAV_1739
Uncharacterized protein
X
X
X
coxB
Putative heme-copper oxidase subunit II
X
X
X
NCAV_0011
ABC-1 domain-containing protein
X
X
amt
Ammonium transporter
X
X
X
petB
Putative cytochrome b/b6
X
NCAV_1587
Putative heme/copper-type cytochrome/
quinol oxidase, subunit
X
X
X
NCAV_0486
Uncharacterized protein (NVIE_004550
homolog)
X
X
X
X
X
NCAV_0488
Uncharacterized protein (NVIE_004540
homolog)
X
X
X
X
X
NCAV_0491
Uncharacterized protein (AmoX)
X
X
X
X
X
aProteins with at least one AmoA, AmoB, or AmoC protein correlation. bConservation and exclusiveness to AOA based on results from Abby et al. (2020) [46]. cCorrelation ≥0.7 and adjusted p value ≤0.001. able 1. Correlations of proteins with occurrence of AmoA,B, and C in (A) N. viennensis and (B) N. cavascurensis BN-PAGE gels. Nitrosocaldaceae and Nitrosopumilaceae were analyzed. Of these genes,
19 were conserved in AOA with five being found exclusively in AOA
(Supplementary Dataset 2). The five genes of interest included two
canonical amo genes (amoA and amoB) and the genes amoX,
NVIE_004540, and NVIE_004550. The absence of amoC in the genes
of interest is attributed to a truncated version existing within the
genome of “Candidatus Nitrosopumilus koreensis AR1” (likely due to
assembly issues) that precluded it from being identified as conserved in
all AOA. The amoX gene was previously identified in metagenomic
studies [15, 36] and NVIE_004540 was already a candidate identified
from the BN-PAGE correlation analysis. The additional conserved
protein, NVIE_004550, was newly identified and found to be located
directly upstream of NVIE_004540, indicating potential co-transcription. The two new candidates encode for polypeptides of 9.6 kDa and
12.8 kDa respectively, and – like the candidate subunit AmoX - their
predicted secondary structure is predominantly helical and their
subcellular localization transmembrane. The two new candidate amo
genes NVIE_004540 and NVIE_004550 have therefore been termed
amoY and amoZ, respectively. find candidates with a similar migration pattern as all three
primary AMO subunits AmoA, AmoB, and AmoC4/C6. Patterns of
the 50% most abundant proteins were compared to each other
using a Kendall correlation to determine the likelihood of
dependence between various proteins with a focus on proteins
correlated with known AMO subunits. Additional criteria were (i)
their presence in fully sequenced AOA, and (ii) their absence in
species that do not oxidize ammonia [46]. Sample and data processing The two proteins that
initially met these criteria were the putative AMO subunit AmoX
and a hypothetical protein, NVIE_004540 (Table 1). The migration
patterns for these proteins can be seen in Fig. 1A. While this
unbiased selection process produced additional AMO candidates,
further analysis was needed to verify the presence of these newly
identified and other potential subunits. Linkage analysis in AOA genomes supports proposed and
additional AMO subunits Earlier analyses of known subunits within the soil strains, or the family
Nitrososphaeraceae (as defined by the Genome Taxonomy Database
[47]; used throughout), has shown a general lack of spatial clustering of
all earlier known subunit genes. However, within the families
Nitrosopumilaceae and Nitrosocaldaceae, the genes for the canonical
AMO subunits, AmoABC, and the proposed subunit AmoX are syntenic
[36, 48, 49]. To investigate co-localization of potential additional subunit
genes, the syntenic status and conservation across AOA of the five
genes upstream and downstream of the amo gene cluster in A closer analysis in Nitrosocaldaceae, the earliest diverging
lineage in evolutionary reconstructions of AOA [46, 50], revealed
that the genes for the three candidate subunits for AMO (AmoX,
AmoY-homolog
of
NVIE_004540,
and
AmoZ-homolog
of
NVIE_004550) clustered spatially with the canonical subunits
(AmoABC) and were syntenic in Nitrosocaldus cavascurensis and
Ca. Nitrosocaldus islandicus. Spatial clustering of all six subunit The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 592 Cenarchaeum symbiosum A
Ca. Nitrosopelagicus brevis
NP-Theta
NP-Gamma
NT-Alpha
NP-Alpha
NP-Delta
NP-Eta
NP-Iota
NC-Alpha
NS-Gamma
NS-Zeta
164-166
15
1
2
11-12
12-123
9-10
627-733
151-175
3
203-236
4
1
3
2
263
Nitrosocaldus
Nitrosopelagicus
Nitrosoarchaeum
Nitrosomarinus
Nitrosopumilus
Nitrosospongia
Nitrosotenuis
Nitrosothermus
Nitrosocosmicus
Nitrososphaera
Nitrosotalea
Non-AOA Thermoproteota
NS-Alpha
0.1
amoA
amoX
amoC
amoB
amoY
amoZ
Fig. 3
Genomic comparison of AMO subunit synteny in AOA. Left: Phylogenetic tree of AOA based on 32 conserved ribosomal proteins,
ultrafast bootstrap values of 100% are indicated as blue circles. Taxonomic labels are colored according to GTDB family identity [47],
Nitrosocaldaceae-red, Nitrosopumilaceae-blue, Nitrososphaeraceae-orange. Clades/organisms in bold were included in syntenic analysis. Clades
are named according to Alves et al. (2018) [34]. Right: Representation of general syntenic patterns in different clades of AOA. Gaps between
genes on the same contig are marked by a zig-zag line A double forward slash indicates separate contigs. Numbers under the zig-zag lines
represent number of genes between amo subunit genes. A finer analysis and a full list of species can be found in Fig. S11 and Supplementary
Dataset 2, respectively. Non-AOA Thermoproteota amoB amoX amoA 203-236 Fig. 3
Genomic comparison of AMO subunit synteny in AOA. Left: Phylogenetic tree of AOA based on 32 conserved ribosomal proteins,
ultrafast bootstrap values of 100% are indicated as blue circles. Taxonomic labels are colored according to GTDB family identity [47],
Nitrosocaldaceae-red, Nitrosopumilaceae-blue, Nitrososphaeraceae-orange. Clades/organisms in bold were included in syntenic analysis. BN-PAGE protein gel indicates same AMO composition in the
thermophilic archaeon Nitrosocaldus cavascurensis BN-PAGE protein gel indicates same AMO composition in the
thermophilic archaeon Nitrosocaldus cavascurensis Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus Linkage analysis in AOA genomes supports proposed and
additional AMO subunits Clades
are named according to Alves et al. (2018) [34]. Right: Representation of general syntenic patterns in different clades of AOA. Gaps between
genes on the same contig are marked by a zig-zag line A double forward slash indicates separate contigs. Numbers under the zig-zag lines
represent number of genes between amo subunit genes. A finer analysis and a full list of species can be found in Fig. S11 and Supplementary
Dataset 2, respectively. genes is also found in recently obtained MAGs [51] within the
genus Nitrosocaldus. In the case of the newly proposed genus Ca. Nitrosothermus [51], amo genes were split on multiple contigs
and synteny could not be definitively determined (Fig. 3). Additionally, all six amo genes are inferred to have been newly
acquired by the last common ancestor of AOA [46]. was obtained (Fig. 1B) a correlation of the additional subunits was
also observed with AmoA, AmoB, and AmoC in this thermophilic
organism (Kendall correlation of proteins, as performed for N. viennensis). The three proteins AmoX, NCAV_0488 (AmoY), and
NCAV_0486 (AmoZ) all had migration patterns within the gel that
strongly
correlated
with
AmoABC
(Table
1). This
analysis
confirmed that the proposed subunits were translated in N. cavascurensis, and potentially had a physical connection within the
AMO complex. The emergence of Nitrosopumilaceae was accompanied by a
separation of this genomic region into a primary cluster contain-
ing amoABCX and a secondary cluster containing amoYZ (Fig. 3). Within Nitrosotalea sp., these clusters are 11–12 genes apart, while
the rest of Nitrosopumilaceae species have these clusters only 1–2
genes apart (with the exception of the sponge symbiont Ca. Cenarchaeum symbiosum). The emergence of the family Nitroso-
sphaeraceae led to a scattering of all subunit genes across the
genome with the exception of amoA and amoX, which are
typically linked. Chemical cross-linking supports physical interaction of
additional subunits To estimate the physical proximity of the proposed subunits to
known subunits and other proteins within the BN-PAGE gel, in-gel
cross-linking [43] was performed using the cross-linker disuccini-
midyl sulfoxide (DSSO) on an additional BN-PAGE cut-out from
band 7 (Fig. S1B). Mass spectrometry and cross-linking analysis
showed multiple cross-links among AmoA, AmoB, AmoC, and
AmoX as well as with the two newly proposed subunits AmoY and
AmoZ (Fig. 4C). Many cross-links were also connected to
NVIE_016740, a putative S-layer protein that likely represents a
highly abundant surface layer protein as known from other
archaea (SlaA) [52, 53]. As this protein presumably helps establish
the pseudo-periplasm in AOA, it is not surprising to find it heavily
cross-linked to membrane proteins. Although amoZ was identified in the genomic analysis, the
protein AmoZ (NVIE_004550) was not correlated with AmoABC in
the BN-PAGE gel of N. viennensis. When examining the relative
abundance profile for AmoZ, the general pattern of AMO peptide
peaks was followed. However, this remained undetected in the
correlation analysis due to a high relative abundance peak
occurring at the bottom of the gel peaking at the last band
taken at approximately 66 kDa based on the BN-PAGE ladder
(Fig. 1A). This is above the predicted mass of 12.8 kDa, but
suggests that AmoZ could also be part of the AMO but a weaker
association possibly lead to its dissociation from the complex and
migration to the bottom of the gel. p
AmoX also had individual cross-links to several other proteins
(Supplementary Dataset 1). As only single connections were found,
and these proteins did not appear in any other syntenic or
correlative analyses, they were not taken to represent a structural
role in the AMO complex. These cross-links can rather be attributed
to the high abundance of those proteins in the cell membrane. BN-PAGE protein gel indicates same AMO composition in the
thermophilic archaeon Nitrosocaldus cavascurensis
To test the composition of the AMO complex outside of the
context of N. viennensis, the BN-PAGE approach was applied to
membrane protein fractions of N. cavascurensis, a distantly related
thermophilic AOA species of the Nitrosocaldaceae family [48] that
was recently
obtained
in pure
culture
(Melcher
et al. in
preparation). Although a slightly different pattern of complexes Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus cavascurensis
AmoZ
(NVIE_004550)
1
123
AmoB
1
185
AmoA
1
216
AmoC
1
187
AmoX
1
102
SlaA (NVIE_016740)
1
200
400
600
800
1000
1202
AmoY
(NVIE_004540)
1
82
SASD allowed
SASD violating
Euclidean allowed
Euclidean violating
0
2
4
6
8
10
12
14
16
18
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-80
>80
Cross-links
Cα pair distance (Å)
Proportion of cross-links (%)
Cross-linked chain combinations
ABC
X(ABC)
Y(ABC)
Z(ABC)
XY
XZ
YZ
intra
0
5
10
15
20
25
SASD allowed
SASD violating
extracellular
cytoplasmic
The ISME Journal (2023) 17:588 – 599 C
D
AmoZ
(NVIE_004550)
1
123
AmoB
1
185
AmoA
1
216
AmoC
1
187
AmoX
1
102
SlaA (NVIE_016740)
1
200
400
600
800
1000
1202
AmoY
(NVIE_004540)
1
82 D C AmoZ
(NVIE_004550) AmoC
7 AmoY
(NVIE_004540) E
SASD allowed
SASD violating
Euclidean allowed
Euclidean violating
0
2
4
6
8
10
12
14
16
18
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-80
>80
Cross-links
Cα pair distance (Å) F
Proportion of cross-links (%)
Cross-linked chain combinations
ABC
X(ABC)
Y(ABC)
Z(ABC)
XY
XZ
YZ
intra
0
5
10
15
20
25
SASD allowed
SASD violating F E Cross-linked chain combinations identified for most amo genes, an obvious conserved promoter motif
for all six genes was not identified. identified for most amo genes, an obvious conserved promoter motif
for all six genes was not identified. identified for most amo genes, an obvious conserved promoter motif
for all six genes was not identified. A re-evaluation of these transcriptomic data (see Methods) also
revealed amoC6 as the primarily transcribed amoC homolog (Fig. 5),
thus confirming the identification of a unique AmoC6 peptide from a
Tricine-SDS-PAGE band digested with chymotrypsin (Supplementary
Discussion; Supplementary Dataset 1). Together this indicates that copper limitation in N. viennensis [54] confirmed that the genes amoA,
amoB, and amoC have some of the highest transcription levels in the
cell, as also shown in previous studies [55–57]. A clustering analysis of
the same dataset revealed that amoA, amoB, amoC, amoX, amoY, and
amoZ all appear to be co-regulated, and fell into the clusters
containing the most highly expressed genes. (Fig. S2; Supplementary
Dataset 2). While putative transcriptional promoter sequences can be g
A re-evaluation of these transcriptomic data (see Methods) also
revealed amoC6 as the primarily transcribed amoC homolog (Fig. Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus Available transcriptomic studies of AOA were inspected to explore
whether the expression patterns of the newly predicted subunits
would corroborate their involvement in the AMO. A recent study on The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 593 A
B
N. viennensis
N. cavascurensis
extracellular
cytoplasmic N. cavascurensis N. cavascurensis B A N. viennensis copper limitation in N. viennensis [54] confirmed that the genes amoA,
amoB, and amoC have some of the highest transcription levels in the
cell, as also shown in previous studies [55–57]. A clustering analysis of
he same dataset revealed that amoA, amoB, amoC, amoX, amoY, and
amoZ all appear to be co-regulated, and fell into the clusters
containing the most highly expressed genes. (Fig. S2; Supplementary
Dataset 2). While putative transcriptional promoter sequences can be
identified for most amo genes, an obvious conserved promoter motif
for all six genes was not identified. A re-evaluation of these transcriptomic data (see Methods) also
revealed amoC6 as the primarily transcribed amoC homolog (Fig. 5),
thus confirming the identification of a unique AmoC6 peptide from a
Tricine-SDS-PAGE band digested with chymotrypsin (Supplementary
Discussion; Supplementary Dataset 1). Together this indicates that
A
C
D
E
F
B
N. viennensis
N. Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus viennensis showing the location of amo genes and
average log2 transformed transcript per million (TPM) values from copper replete conditions in Reyes et al. (2020) [54]. Orange bars on
the genome indicate the locations of AMO subunit genes that are strongly expressed. Blue bars on the genome indicate amo genes that have
low transcriptional activity. Boxes show amo genes (in bold) and immediate neighbors colored based on average gene expression from
copper replete cultures. Red indicates a strong expression while blue represents a low or absent expression. All strongly expressed amo genes
were found in clusters of highly expressed genes across both limited and replete conditions (see Fig. S2). 0.0 Mb
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NVIE_2916
amoB
NVIE_028420
radA
…7 genes…
amoC6
NVIE_028550
NVIE_028520
amoC1
nirK
NVIE_000550
NVIE_000520
amoC2
NVIE_002340
NVIE_002320
NVIE_004530
amoY
(NVIE_004540)
amoZ
(NVIE_004550)
NVIE_0459
amoC3
gds2
NVIE_011550
amoA
amoX
NVIE_027290
NVIE_027260
NVIE_2798
amoC5
ypfJ
NVIE_024070
amoC4
NVIE_019090
NVIE_019070
Nitrososphaera
viennensis
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
7.5
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0 Fig. 5
Transcription of AMO subunit genes in N. viennensis. Genomic representation of N. viennensis showing the location of amo genes and
average log2 transformed transcript per million (TPM) values from copper replete conditions in Reyes et al. (2020) [54]. Orange bars on
the genome indicate the locations of AMO subunit genes that are strongly expressed. Blue bars on the genome indicate amo genes that have
low transcriptional activity. Boxes show amo genes (in bold) and immediate neighbors colored based on average gene expression from
copper replete cultures. Red indicates a strong expression while blue represents a low or absent expression. All strongly expressed amo genes
were found in clusters of highly expressed genes across both limited and replete conditions (see Fig. S2). discovered in the Thermoplasmata phylum [61]. A HMMER search
using the extended regions of the bacterial homologs against the
genomes of collected AOA did not reveal any significant
similarities. Therefore, a general structural search using Phobius
[62] was carried out with the N. Structural search for missing components in the archaeal AMO
complex p
As previously observed [60], comparisons of the amino acid
sequences of the three subunits AmoA, AmoB, and AmoC from
archaea
with
those
of
bacteria
indicate
that
the
primary
differences between the archaeal AMO subunits and the bacterial
AMO subunits are missing transmembrane helices, at least one in
AmoB and two in AmoC, and a missing C-terminal soluble portion
found in bacterial AmoB/PmoB (Figs. S3–S5). These observations
also hold true for the new clade of archaeal AMO recently Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus Blue bars on the genome indicate amo genes that have
low transcriptional activity. Boxes show amo genes (in bold) and immediate neighbors colored based on average gene expression from
copper replete cultures. Red indicates a strong expression while blue represents a low or absent expression. All strongly expressed amo genes
were found in clusters of highly expressed genes across both limited and replete conditions (see Fig. S2). 4 Fig. 4
Structural support of proposed AMO subunits based on BN-PAGE cross-linking and AlphaFold modeling. A, B Cartoon
representations of the AlphaFold structure models of the N. viennensis (A) and N. cavascurensis (B) hexamers, indicating their putative
membrane orientation based on sequence hydropathy analysis. Subunits are colored as follows: AmoA, light grey; AmoB, black; AmoC,
salmon; AmoX, lavender; AmoY, cyan; AmoZ, blue. Residues in the CuB and CuC copper sites are represented in red sticks. Disulfide bonds are
indicated in yellow. C Representation of identified cross-links among existing and proposed AMO subunits of an AMO band cut from a BN-
PAGE gel of N. viennensis. Green: suspected subunits based on comparative genomics. Blue: newly proposed subunits based on BN-PAGE
correlation and syntenic analysis. D Cross-links within the solvent accessible surface distance (SASD) threshold for DSSO, depicted in green,
mapped on the N. viennensis AlphaFold model. The single observed cross-link between the AmoZ and AmoB subunits is depicted in magenta,
as it violates SASD distance criteria (50.0 Å) but is within range of Euclidean distance (31.8 Å). E Distribution of SASD and Euclidean Cα–Cα
distances of unique DSSO cross-links identified with Annika and MeroX. Twenty-seven out of 67 unique cross-links satisfied distance criteria
(SASD < 35.0 Å). F Percentage of cross-linked subunit combinations. 0.0 Mb
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NVIE_2916
amoB
NVIE_028420
radA
…7 genes…
amoC6
NVIE_028550
NVIE_028520
amoC1
nirK
NVIE_000550
NVIE_000520
amoC2
NVIE_002340
NVIE_002320
NVIE_004530
amoY
(NVIE_004540)
amoZ
(NVIE_004550)
NVIE_0459
amoC3
gds2
NVIE_011550
amoA
amoX
NVIE_027290
NVIE_027260
NVIE_2798
amoC5
ypfJ
NVIE_024070
amoC4
NVIE_019090
NVIE_019070
Nitrososphaera
viennensis
log2 TPM
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
7.5
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Fig. 5
Transcription of AMO subunit genes in N. viennensis. Genomic representation of N. Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus viennensis genome to search for
genes that could encode a protein with the following criteria: (i)
1–3 transmembrane helices, (ii) conservation across all AOA [46],
and (iii) present in the top 100 transcribed genes [54] (similar
levels as the primary AMO subunits). This revealed six possible
candidates (Table 2). The only candidates to meet the structural
requirements while maintaining syntenic and similar patterns of
migration in BN-PAGE were amoX, amoY, and amoZ. AmoC6 is the primary structural AmoC homolog in the AMO complex
of N. viennensis, at least under the applied growth conditions. Transcriptomics of the marine strain, Nitrosopumilus maritimus,
also showed high expression of amoA, amoB, amoC, amoX, and
amoY (Nmar_1506). The gene amoZ (Nmar_1507), albeit syntenic
with amoY, exhibited lower expression levels [55]. The three newly proposed AMO subunits were also inspected in
proteomic datasets that were generated with methods allowing for
the improved recovery of membrane proteins. All six of the known
and proposed subunits were found in membrane fractions from N. viennensis from a previous study [15] as well as in the proteome of N. maritimus [55]. In other proteomic studies of AOA [58, 59], the three
new subunits were not always present, likely due to their small size
and limited number of trypsin cleavage sites. The addition of the three proposed subunits in archaea
increases the number of transmembrane helices from 10–11 to
approximately 14 per protomer making it comparable to the
number found in bacterial crystal structures of pMMO where each
protomer of the trimer (i.e., one unit of PmoABC), contains 14–15
transmembrane helices [23, 63]. Expression patterns of AMO subunits in Nitrososphaera
viennensis and Nitrosopumilus maritimus 5),
thus confirming the identification of a unique AmoC6 peptide from a
Tricine-SDS-PAGE band digested with chymotrypsin (Supplementary
Discussion; Supplementary Dataset 1). Together this indicates that The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 594 Fig. 4
Structural support of proposed AMO subunits based on BN-PAGE cross-linking and AlphaFold modeling. A, B Cartoon
representations of the AlphaFold structure models of the N. viennensis (A) and N. cavascurensis (B) hexamers, indicating their putative
membrane orientation based on sequence hydropathy analysis. Subunits are colored as follows: AmoA, light grey; AmoB, black; AmoC,
salmon; AmoX, lavender; AmoY, cyan; AmoZ, blue. Residues in the CuB and CuC copper sites are represented in red sticks. Disulfide bonds are
indicated in yellow. C Representation of identified cross-links among existing and proposed AMO subunits of an AMO band cut from a BN-
PAGE gel of N. viennensis. Green: suspected subunits based on comparative genomics. Blue: newly proposed subunits based on BN-PAGE
correlation and syntenic analysis. D Cross-links within the solvent accessible surface distance (SASD) threshold for DSSO, depicted in green,
mapped on the N. viennensis AlphaFold model. The single observed cross-link between the AmoZ and AmoB subunits is depicted in magenta,
as it violates SASD distance criteria (50.0 Å) but is within range of Euclidean distance (31.8 Å). E Distribution of SASD and Euclidean Cα–Cα
distances of unique DSSO cross-links identified with Annika and MeroX. Twenty-seven out of 67 unique cross-links satisfied distance criteria
(SASD < 35.0 Å). F Percentage of cross-linked subunit combinations. 0.0 Mb
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NVIE_2916
amoB
NVIE_028420
radA
…7 genes…
amoC6
NVIE_028550
NVIE_028520
amoC1
nirK
NVIE_000550
NVIE_000520
amoC2
NVIE_002340
NVIE_002320
NVIE_004530
amoY
(NVIE_004540)
amoZ
(NVIE_004550)
NVIE_0459
amoC3
gds2
NVIE_011550
amoA
amoX
NVIE_027290
NVIE_027260
NVIE_2798
amoC5
ypfJ
NVIE_024070
amoC4
NVIE_019090
NVIE_019070
Nitrososphaera
viennensis
log2 TPM
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
7.5
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Fig. 5
Transcription of AMO subunit genes in N. viennensis. Genomic representation of N. viennensis showing the location of amo genes and
average log2 transformed transcript per million (TPM) values from copper replete conditions in Reyes et al. (2020) [54]. Orange bars on
the genome indicate the locations of AMO subunit genes that are strongly expressed. DISCUSSION
Th
h
l The archaeal AMO complex is a key enzyme of AOA energy
metabolism that is highly expressed in all ammonia oxidizing
organisms investigated and has large implications for the
environment due to its overwhelming presence in many
ecosystems [8–14, 67]. The work here profits from the recent
improvements in the cultivation of AOA in continuous cultures
(Melcher et al. in preparation) and presents novel biochemical
and comparative genomic evidence on the composition of the
AMO complex in Nitrososphaera viennensis and other AOA that
contrasts with the proposed composition of this complex
within AOB. p
p
p
p
g
To compare the degree of conservation of the predicted
hexameric organization of the AMO complex, a structural model
of the AMO complex of N. cavascurensis was also obtained
(Fig. 4B). The resultant models were similar in their overall
arrangement to each other and to the N. viennensis model, with
high overall confidence scores (top model, pLDDT = 77.7 and
ptm score = 0.591). Differences between the N. viennensis and N. cavascurensis models include the localization of the transmem-
brane (TM) helix of AmoZ. In N. viennensis the TM helix is
predicted to interact mostly with the TM helix of AmoY, while in
N. cavascurensis it is predicted to interact with the TMs of AmoB
and AmoA (Figs. 4A, B, S7A, C). This would affect the relative
positioning of the N-terminal domain of AmoZ with respect to
the
AmoB
soluble
domain,
allowing
for
a
more
“open”
conformation. However, the extended loop connecting the
N-terminal pair of helices in AmoZ with the TM domain
theoretically allows for some flexibility (additional information
in Supplementary Discussion, Fig. S8). The present analysis has verified that AmoX, NVIE_004540,
and NVIE_004550 are all likely present within the archaeal AMO
complex
and
proposes
the
naming
of
NVIE_004540
and
NVIE_004550 as AmoY and AmoZ, respectively. This finding is
based on a host of independent analyses including proteomic,
genomic, transcriptomic, structural, and modeling approaches. The presence of six subunits rather than three is unique to the
archaeal domain and could represent a more complex regula-
tory strategy for the AMO complex in archaea. Differences in the
ammonia
oxidation
pathway
are
already
well
established
between the archaeal and bacterial domains (i.e., unresolved
second step in archaea [19, 21]; iron-based c cytochromes
[68, 69] and ubiquinone in bacteria [70, 71] vs. copper-
based plastocyanins [15, 16] and menaquinone in archaea
[72]). Predicted structure of the archaeal AMO complex supports the
integration of new subunits To gain insights into the structural context of the archaeal AMO
complex in the light of three additionally proposed subunits, a
structural model for the organization of the N. viennensis AMO
complex was obtained by employing the multimer-capable The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 595 Table 2. Structural search for missing AMO subunits in N. viennensis. Table 2. Structural search for missing AMO subunits in N. viennensis. Gene Expression & Structural Search
Previous Analyses
Protein
Gene
TPM log2a
TMb
AOA Conservationc
BN-PAGE Corr.d
AMO Syntenye
NVIE_013530
13.82
1
X
protein of unknown function
NVIE_004540
13.55
1
X
X
X
hypothetical protein
NVIE_004550
13.39
1
X
X
X
hypothetical protein
amoX
12.14
2
X
X
X
putative ammonia monooxygenase -
associated protein
coxB
11.87
1
X
putative heme-copper oxidase subunit II
NVIE_014370
11.75
1
putative Copper binding protein, plastocyanin/
azurin family
NVIE_010490
11.75
1
putative phosphoesterase, DHHA1
NVIE_027520
11.40
1
X
putative heme/copper-type cytochrome/
quinol oxidase, subunit
NVIE_027550
11.33
1
X
putative blue (Type 1) copper domain protein
atpK
11.30
2
archaeal A1A0-type ATP synthase, subunit K
NVIE_021780
10.90
1
exported protein of unknown function
NVIE_006540
10.90
1
exported protein of unknown function
NVIE_029580
10.20
1
X
blue (Type 1) copper domain-containing
protein
aBased on transcriptomic counts averaged from replete copper conditions from Reyes et al. (2020) [54]. bNumber of predicted transmembrane helices. cAs predicted from Abby et al. (2020) [46]. dRepresents proteins correlated with AmoA, AmoB, and AmoC6 in BN-PAGE bands from N. viennensis in this study. eGenes syntenic with amoA, amoB, and amoC based on the syntenic analysis from this study. tructural search for missing AMO subunits in N. viennensis Table 2. Structural search for missing AMO subunits in N. viennensis. Predicted structure of the archaeal AMO complex supports the
integration of new subunits Gene Expression & Structural Search
Previous Analyses
Protein
Gene
TPM log2a
TMb
AOA Conservationc
BN-PAGE Corr.d
AMO Syntenye
NVIE_013530
13.82
1
X
protein of unknown function
NVIE_004540
13.55
1
X
X
X
hypothetical protein
NVIE_004550
13.39
1
X
X
X
hypothetical protein
amoX
12.14
2
X
X
X
putative ammonia monooxygenase -
associated protein
coxB
11.87
1
X
putative heme-copper oxidase subunit II
NVIE_014370
11.75
1
putative Copper binding protein, plastocyanin/
azurin family
NVIE_010490
11.75
1
putative phosphoesterase, DHHA1
NVIE_027520
11.40
1
X
putative heme/copper-type cytochrome/
quinol oxidase, subunit
NVIE_027550
11.33
1
X
putative blue (Type 1) copper domain protein
atpK
11.30
2
archaeal A1A0-type ATP synthase, subunit K
NVIE_021780
10.90
1
exported protein of unknown function
NVIE_006540
10.90
1
exported protein of unknown function
NVIE_029580
10.20
1
X
blue (Type 1) copper domain-containing
protein
aBased on transcriptomic counts averaged from replete copper conditions from Reyes et al. (2020) [54]. bNumber of predicted transmembrane helices. cAs predicted from Abby et al. (2020) [46]. dRepresents proteins correlated with AmoA, AmoB, and AmoC6 in BN-PAGE bands from N. viennensis in this study. eGenes syntenic with amoA, amoB, and amoC based on the syntenic analysis from this study. Gene Expression & Structural Search involved all subunit combinations with the exception of AmoZ
(Fig. 4F). AmoZ only participated in cross-linking interactions
>35 Å, which supports a weaker association with the complex as
observed in the BN-PAGE migration patterns. version of AlphaFold 2.1 [64–66]. The resultant models were all
similar
and
represented
confident
predictions
(top
model,
pLDDT = 71.4 and ptm score = 0.668). All predicted transmem-
brane helices from AmoX, AmoY, and AmoZ play a role in
anchoring the complex in the membrane along with the
transmembrane helices from AmoA, AmoB, and AmoC (Fig. 4A). Additionally, the N-terminal domain of AmoZ was predicted to
contain two alpha helices that interact with the N-terminal domain
of AmoB, thereby possibly replacing the role of the missing
C-terminal soluble domain found in PmoB and offering the final
piece of the missing complex in archaea (additional information in
Supplementary Discussion). A disulfide bond was also predicted to
form within the soluble domain of AmoZ. The overall structure is
comparable to a protomer of the pMMO complex (Fig. S6). DISCUSSION
Th
h
l Although there is debate on which subunit harbors the primary
active site in CuMMO complexes, there is clear evidence that the
metal site(s) in PmoC plays a critical role in the complex of
methanotrophs [27, 28, 31, 32]. While the archaeal AmoC lacks a
substantial section found in all bacteria that corresponds to two
transmembrane helices (Fig. S5), the metal site observed in earlier
crystal structures as well as the newly proposed metal site
identified via cryo-EM [28], are conserved across all archaeal and
bacterial species (Fig. S5). The importance of this subunit is also
supported by site directed mutagenesis studies in the genetically
tractable Actinobacteria that contain the homologous hydrocar-
bon monooxygenase [32]. Regardless of species-specific differences in archaea, the
overall predicted archaeal structure, with the new subunits, is
reflective of the known bacterial protomer composition (Fig. S6). Definitive proof of the oligomerization and organization of these
subunits will not be possible until a definitive structure (i.e.,
crystal or cryo-EM) of archaeal AMO is realized. Putative
protomer interaction sites in the cryo-EM structure of Methylo-
coccus capsulatus str Bath (PDB structure 7S4H) [28] appear to be
in the section of PmoB that is missing in archaea (Fig. S4). However, the placement of AmoY and AmoZ on the outer
regions of the protomer could be facilitating these interactions
instead (Fig. S7). This could also explain the high amount of
SASD violating interactions between AmoY and AmoZ as the
analysis only takes into account a single protomer (Fig. 4F). It is
possible that these interactions may instead be between
subunits of AmoY and AmoZ in different protomers. Therefore,
the
predicted
protomer
models
of
N. viennensis
and
N. cavascurensis do not rule out the possibility of a trimeric
archaeal AMO complex. With respect to orientation, the present
modeling approach is not able to predict exactly how the
archaeal AMO sits in the membrane. However, it is likely that the
active site as well as the soluble AmoB and AmoZ domains are
situated toward the pseudo-periplasmic space. This is supported
by previous modeling efforts of nutrient transport in the S-layer
of AOA [91] as well as activity-based immunogold labeling of
CuMMO complexes in AOB and methanotrophs [92]. The soil model AOA, N. viennensis, like most other soil dwelling
AOA from
the
family
Nitrososphaeraceae,
encodes
multiple
homologs of the amoC gene while retaining only single copies
of amoA and amoB [15] (Supplementary Dataset 2). DISCUSSION
Th
h
l The varying characteristics within the AMO complex
observed in this work further underscore these differences and
add to a growing body of evidence that AOA and AOB
participate in nitrification under different environmental and/or
functional constraints. Data from cross-linking experiments in N. viennensis were
mapped to the predicted model and strongly supported the
predicted interactions (Fig. 4D) with some exceptions. Out of 67
unique observed cross-links, 27 (40%) satisfied a maximum solvent
accessible surface distance (SASD) threshold of ≤35 Å (Fig. 4E) and The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 596 In blue native PAGE protein gels, the AMO complex in both N. viennensis and Nitrosocaldus cavascurensis migrated well above the
predicted height of a trimeric protomer complex, even when
considering the additional subunits (predicted molecular weight
of a homotrimeric complex with six subunits per protomer:
296.9 kDa N. viennensis; 305.1 kDa N. cavascurensis). The archaeal
AMO bands are also observed at a higher molecular weight in the
gel when compared to the homologous bacterial PMO complex
from a Methylomirabilis species that was also extracted using n-
dodecyl-β-D-maltoside (DDM) [73]. This could be explained by
differences in membrane composition of the strains or potential
differences in oligomerization of the protomer. AOA contain
unique ether-linked lipids (i.e., crenarchaeols) [37, 74–78] and rely
on a proteinaceous S-layer rather than an outer membrane to
create a pseudo-periplasmic space [52, 53]. The observation of
three distinct peaks of AMO can most likely be explained by the
co-migration with other proteins or complexes that it could be
physically interacting with, in particular with the S-layer protein. Nitrosocaldus yellowstonensis [86]. This structure confirmed the
lack of the C-terminal cupredoxin domain and revealed an
extended amino acid region not found in bacteria made up of
two helices and two loops. It was proposed that this additional
region could help stabilize the existing cupredoxin domain as
supportive interactions are lacking due to the absence of the
C-terminal domain. However, this amino acid extension is only
found within the proposed genus of Nitrosocaldus (Fig. S4). It is
more likely that the soluble domain of AmoZ, which is conserved
across all AOA, is conferring this stabilizing role. DISCUSSION
Th
h
l ,
g
g
In the absence of additional structure-function analyses, it is
unclear if the additional subunits in the archaeal complex simply
reflect the vast evolutionary distance to all other known protein
complexes of the CuMMO family [34], or if this difference in
structure also has relevant functional implications. For instance,
the bacterial AMO complexes are promiscuous enzymes able to
oxidize methane and other compounds [87–90]. Such investiga-
tions on alternative substrates have not yet been performed with
the archaeal complex but would be important for evaluating the
functional role of archaea (and possibly the new subunits) in the
environment. Additionally, differences in the AMO complex
between AOA species have been identified that have the potential
to affect the function of the AMO complex. This includes the extra
AmoB loop found within Nitrosocaldus, but is also clearly
demonstrated by the newly proposed subunit AmoZ. The genus
Nitrososphaera and the family Nitrosocaldaceae (both investigated
in this study) are predicted to form a disulfide bond linking the
two alpha helices making up the soluble domain of AmoZ (Figs. S7B, D, S10C) via two cysteines that are not conserved in other
AOA. Additionally, the genus Nitrosocosmicus is predicted to
contain an additional zinc ribbon domain represented by four
cysteine residues at the C-terminal end, presumably residing
within the cytoplasm (Fig. S10C). The observations of a disulfide
bond and zinc ribbon domain within certain AOA lineages could
be linked to sensitivity to reactive oxygen species and unique
regulation strategies, respectively, and may reflect unique patterns
of evolution that complement yet unknown aspects of the
metabolism of these specific groups. y
y
y
Previous work on bacteria that rely on CuMMOs have identified
other putative proteins involved with the complex. Monocistronic
transcripts containing amoABC from the AOB Nitrosococcus oceani
ATCC 19707 contained two additional genes assigned as amoR
and amoD [79]. The amoR gene was found to be only present in
Nitrosococcus and was therefore not thought to be a conserved
part of bacterial AMO. A recent study indicated that AmoD/PmoD
(and likely the homologous AmoE) play crucial roles in copper
homeostasis, but they are not suspected to be a structural part of
any CuMMO complex [80]. Rather, crystal and cryo-EM structures
of bacterial PMO have consistently confirmed a trimeric protomer
structure with one subunit of PmoA, PmoB, and PmoC making up
each protomer [22–28]. DISCUSSION
Th
h
l Considering the wide
distribution of AOA in virtually all ecosystems [8–14, 34] and
their ecological relevance, developing genetic tools for AOA and
improving their biomass production will be needed to enable
structure-function analysis and to elucidate the full pathway of
ammonia oxidation in these archaea. would therefore consist of six subunits instead of three as in
other complexes of the CuMMO family. The addition of the new
subunits would make the number of transmembrane helices
comparable to CuMMO complexes found within bacteria. As the
anchoring of pMMO in the membrane has previously been
shown to be critical for its activity [26, 28], it seems plausible
that the newly identified subunits play an important role for the
structural and functional integrity of the archaeal AMO complex. The presence of a soluble domain within AmoZ that could
replace the stabilizing function of the missing soluble domain in
AmoB also fulfills a potentially crucial missing piece of the AMO
complex. Since AmoXYZ appear to have important structural
roles, it will be imperative to include all subunits in future
expression
and
structural
studies
of
this
environmentally
relevant protein complex in archaea. Considering the wide
distribution of AOA in virtually all ecosystems [8–14, 34] and
their ecological relevance, developing genetic tools for AOA and
improving their biomass production will be needed to enable
structure-function analysis and to elucidate the full pathway of
ammonia oxidation in these archaea. adaptations
of
archaeal
ammonia
oxidizers. Proc
Natl
Acad
Sci
USA. 2016;113:E7937–46. 16. Walker CB, De La Torre JR, Klotz MG, Urakawa H, Pinel N, Arp DJ, et al. Nitroso-
pumilus maritimus genome reveals unique mechanisms for nitrification and
autotrophy in globally distributed marine crenarchaea. Proc Natl Acad Sci USA. 2010;107:8818–23. 17. Berg IA, Kockelkorn D, Buckel W, Fuchs G. A 3-hydroxypropionate 4-hydro-
xybutyrate autotrophic carbon dioxide assimilation pathway in archaea. Science. 2007;218:1782–6. 18. Könneke M, Schubert DM, Brown PC, Hügler M, Standfest S, Schwander T, et al. Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for
CO2 fixation. Proc Natl Acad Sci USA. 2014;111:8239–44. 19. Lancaster KM, Caranto JD, Majer SH, Smith MA. Alternative bioenergy: updates to
and challenges in nitrification metalloenzymology. Joule. 2018;2:1–21. 20. Simon J, Klotz MG. Diversity and evolution of bioenergetic systems involved in
microbial nitrogen compound transformations. Biochim Biophys Acta - Bioenerg. 2013;1827:114–35. 21. Kozlowski JA, Stieglmeier M, Schleper C, Klotz MG, Stein LY. REFERENCES 1. Wong-Chong GM, Loehr RC. The kinetics of microbial nitrification. Water Res. 1975;9:1099–106. 27. Chang WH, Lin HH, Tsai IK, Huang SH, Chung SC, Tu IP, et al. Copper centers in the
cryo-EM structure of particulate methane monooxygenase reveal the catalytic
machinery of methane oxidation. J Am Chem Soc. 2021;143:9922–32. 2. Hyman MR, Wood PM. Suicidal inactivation and labelling of ammonia mono-
oxygenase by acetylene. Biochem J. 1985;227:719–25. 2. Hyman MR, Wood PM. Suicidal inactivation and labelling of ammonia mono-
oxygenase by acetylene. Biochem J. 1985;227:719–25. 28. Koo CW, Tucci FJ, He Y, Rosenzweig AC. Recovery of particulate methane mono
oxygenase structure and activity in a lipid bilayer. Science. 2022;375:1287–91. 3. Hollocher TC, Tate ME, Nicholas DJ. Oxidation of ammonia by Nitrosomonas
europaea. Definitive 18O-tracer evidence that hydroxylamine formation involves a
monooxygenase. J Biol Chem. 1981;256:10834–6. 29. Balasubramanian R, Smith SM, Rawat S, Yatsunyk LA, Stemmler TL, Rosenzweig
AC. Oxidation
of
methane
by
a
biological
dicopper
centre. Nature. 2010;465:115–9. 4. Winogradsky S. Recherches sur les organismes de la nitrification. Ann Inst Pateur. 1890;4:213–31. 30. Op den Camp HJM, Islam T, Stott MB, Harhangi HR, Hynes A, Schouten S, et al. Environmental, genomic and taxonomic perspectives on methanotrophic Ver-
rucomicrobia. Environ Microbiol Rep. 2009;1:293–306. 5. Könneke M, Bernhard AE, De La Torre JR, Walker CB, Waterbury JB, Stahl DA. Isolation of an autotrophic
ammonia-oxidizing
marine archaeon. Nature. 2005;437:543–6. 31. Ross MO, MacMillan F, Wang J, Nisthal A, Lawton TJ, Olafson BD, et al. Particulate
methane monooxygenase contains only mononuclear copper centers. Science. 2019;364:566–70. 6. Treusch AH, Leininger S, Kietzin A, Schuster SC, Klenk HP, Schleper C. Novel genes
for nitrite reductase and Amo-related proteins indicate a role of uncultivated
mesophilic crenarchaeota in nitrogen cycling. Environ Microbiol. 2005;7:1985–95. 32. Liew EF, Tong D, Coleman NV, Holmes AJ. Mutagenesis of the hydrocarbon
monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is
essential for copper-containing membrane monooxygenase activity. Micro-
biology. 2014;160:1267–77. 7. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science. 2004;304:66–74. 7. Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA, et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science. 2004;304:66–74. 8. Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, et al. Archaea pre-
dominate
among
ammonia-oxidizing
prokaryotes
in
soils. Nature. 2006;442:806–9. 33. DISCUSSION
Th
h
l Pathways and key
intermediates required for obligate aerobic ammonia-dependent chemolitho-
trophy in bacteria and Thaumarchaeota. ISME J. 2016;10:1836–45. 22. Lieberman RL, Rosenzweig AC. Crystal structure of a membrane-bound metalloen-
zyme that catalyses the biological oxidation of methane. Nature. 2005;434:177–82. 23. Hakemian AS, Kondapalli KC, Telser J, Hoffman BM, Stemmler TL, Rosenzweig AC. The metal centres of particulate methane monooxygenase from Methylosinus
trichosporium OB3b. Biochemistry. 2008;47:6793–801. DATA AVAILABILITY 24. Smith SM, Rawat S, Telser J, Hoffman BM, Stemmler TL, Rosenzweig AC. Crystal
structure and characterization of particulate methane monooxygenase from
Methylocystis species strain M. Biochemistry. 2011;50:10231–40. All proteomic data was deposited to the ProteomeXchange Consortium via PRIDE
[44] partner repository with identifiers PXD035349, PXD034632, and PXD034475 for
BN-Page of N. viennensis, BN-PAGE of N. cavascurensis, and cross-linked samples,
respectively. Relevant scripts and code for data analysis can be found at the GitHub
repository https://github.com/hodgskiss/Archaeal_AMO. 25. Sirajuddin S, Barupala D, Helling S, Marcus K, Stemmler TL, Rosenzweig AC. Effects
of zinc on particulate methane monooxygenase activity and structure. J Biol
Chem. 2014;289:21782–94. 26. Ro SY, Ross MO, Deng YW, Batelu S, Lawton TJ, Hurley JD, et al. From micelles to
bicelles: effect of the membrane on particulate methane monooxygenase
activity. J Biol Chem. 2018;293:10457–65. DISCUSSION
Th
h
l Additional
copies of amoC that are spatially disconnected from the AMO
operon are encoded by some terrestrial AOB and were implicated
in stress response based on transcriptional studies [81, 82]. Within
Nitrososphaeraceae, no conserved AMO operons exist (Fig. 3). Duplications of the amoC gene (spatially distant from the other
AMO genes) also occur in some species of the AOA marine
associated family (Nitrosopumilaceae) and in two MAGs from AOA
thermophiles (Nitrosocaldaceae), all discovered in sediments
[51, 83, 84]. An amoC duplication is also found in an AOA sponge
symbiont and copies of archaeal amoC are even found in marine
viruses [85]. These findings together may indicate the metabolic
importance of the AmoC subunit for ecophysiological adaptations
in ammonia oxidation. While this work identified one particular
AmoC (AmoC6; NVIE_028540) to be the primary homolog within
the complex of N. viennensis, it is possible that (some of) the other
AmoC subunits, which arose by gene duplications at the genus
level (Fig. S9), might be incorporated under certain environmental
conditions and provide different activity profiles to the enzyme. In conclusion, this study provides evidence through genomic,
proteomic, and transcriptomic data for the presence of AmoX
and the inclusion of AmoY and AmoZ as subunits within the
archaeal AMO complex. A single protomer of the archaeal AMO Beyond comparative genomics, the only confirmed structural
information for archaea stems from the crystal structure of a
heterologously expressed AmoB originating from Candidatus The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 597 would therefore consist of six subunits instead of three as in
other complexes of the CuMMO family. The addition of the new
subunits would make the number of transmembrane helices
comparable to CuMMO complexes found within bacteria. As the
anchoring of pMMO in the membrane has previously been
shown to be critical for its activity [26, 28], it seems plausible
that the newly identified subunits play an important role for the
structural and functional integrity of the archaeal AMO complex. The presence of a soluble domain within AmoZ that could
replace the stabilizing function of the missing soluble domain in
AmoB also fulfills a potentially crucial missing piece of the AMO
complex. Since AmoXYZ appear to have important structural
roles, it will be imperative to include all subunits in future
expression
and
structural
studies
of
this
environmentally
relevant protein complex in archaea. REFERENCES Can-
didatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermo-
philic archaeon with a highly mobile genome. Front Microbiol. 2018;9:28. 75. Villanueva L, Damsté JSS, Schouten S. A re-evaluation of the archaeal membrane
lipid biosynthetic pathway. Nat Rev Microbiol. 2014;12:438–48. 49. Nicol GW, Schleper C. Ammonia-oxidising Crenarchaeota: important players in
the nitrogen cycle? Trends Microbiol. 2006;14:207–12. 50. Sheridan PO, Raguideau S, Quince C, Holden J, Zhang L, Gaze WH, et al. Gene
duplication drives genome expansion in a major lineage of Thaumarchaeota. Nat
Commun. 2020;11:5494. 76. Sinninghe Damsté JS, Schouten S, Hopmans EC, Van Duin ACT, Geenevasen JAJ. Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether mem-
brane lipid of cosmopolitan pelagic crenarchaeota. J Lipid Res. 2002;43:1641–51. 51. Luo Z-H, Narsing Rao MP, Chen H, Hua Z-S, Li Q, Hedlund BP, et al. Genomic
insights of “Candidatus Nitrosocaldaceae” based on nine new metagenome-
assembled
genomes,
including
“Candidatus
Nitrosothermus”
gen
nov. and
two
new
species
of
“Candidatus
Nitrosocaldus”. Front
Microbiol. 2021;11:608832. 77. Sinninghe Damsté JS, Rijpstra WIC, Hopmans EC, Jung MY, Kim JG, Rhee SK, et al. Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids of group I.1a 77. Sinninghe Damsté JS, Rijpstra WIC, Hopmans EC, Jung MY, Kim JG, Rhee SK, et al. Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids of group I.1a
and I.1b Thaumarchaeota in soil. Appl Environ Microbiol. 2012;78:6866–74. 78. Elling FJ, Könneke M, Mußmann M, Greve A, Hinrichs KU. Influence of tempera-
ture, pH, and salinity on membrane lipid composition and TEX86 of marine
planktonic thaumarchaeal isolates. Geochim Cosmochim Acta. 2015;171:238–55. 52. Stieglmeier M, Klingl A, Alves RJE, Rittmann SKMR, Melcher M, Leisch N, et al. Nitrososphaera viennensis gen. nov., sp. nov., an aerobic and mesophilic,
ammonia-oxidizing archaeon from soil and a member of the archaeal phylum
Thaumarchaeota. Int J Syst Evol Microbiol. 2014;64:2738–52. 79. El Sheikh AF, Poret-Peterson AT, Klotz MG. Characterization of two new genes,
amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitroso-
coccus oceani ATCC 19707. Appl Environ Microbiol. 2008;74:312–8. Thaumarchaeota. Int J Syst Evol Microbiol. 2014;64:2738–52. 80. Fisher OS, Kenney GE, Ross MO, Ro SY, Lemma BE, Batelu S, et al. Characterization
of a long overlooked copper protein from methane- and ammonia-oxidizing
bacteria. Nat Commun. 2018;9:4276. 53. Albers SV, Meyer BH. The archaeal cell envelope. Nat Rev Microbiol. 2011;9:414–26. 54. Reyes C, Hodgskiss LH, Kerou M, Pribasnig T, Abby SS, Bayer B, et al. REFERENCES 598 42. Berger S, Cabrera-orefice A, Jetten MSM, Brandt U, Welte CU. Investigation of
central energy metabolism-related protein complexes of ANME-2d methano-
trophic archaea by complexome profiling. BBA - Bioenerg. 2021;1862:148308. 67. Klotz MG, Stein LY. Nitrifier genomics and evolution of the nitrogen cycle. FEMS
Microbiol Lett. 2008;278:146–56. 68. Yamanaka T, Shinra M. Cytochrome c-552 and cytochrome c-554 derived from
Nitrosomonas europaea. J Biochem. 1974;75:1265–73. 43. Hevler JF, Lukassen MV, Cabrera‐Orefice A, Arnold S, Pronker MF, Franc V, et al. Selective cross‐linking of coinciding protein assemblies by in‐gel cross‐linking
mass spectrometry. EMBO J. 2021;40:e106174. 69. Arciero DM, Hooper AB, Balny C. Spectroscopic and rapid kinetic studies of
reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitroso-
monas europaea. Biochemistry. 1991;30:11466–72. 44. Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ,
et al. The PRIDE database and related tools and resources in 2019: improving
support for quantification data. Nucleic Acids Res. 2019;47:D442–50. 70. Hooper AB, Erickson RH, Terry KR. Electron transport systems of Nitrosomonas:
isolation of a membrane-envelope fraction. J Bacteriol. 1972;110:430–8. 71. Whittaker M, Bergmann D, Arciero D, Hooper AB. Electron transfer during the
oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas euro-
paea. Biochim Biophys Acta - Bioenerg. 2000;1459:346–55. 45. Melcher M, Hodgskiss LH, Mardini MA, Schleper C, Rittmann SMKR. Analysis of
biomass productivity and physiology of Nitrososphaera viennensis grown in
continuous culture. Frontiers in Microbiology. (in preparation) 14:206. 72. Elling FJ, Becker KW, Könneke M, Schröder JM, Kellermann MY, Thomm M, et al. Respiratory quinones in Archaea: phylogenetic distribution and application as
biomarkers in the marine environment. Environ Microbiol. 2016;18:692–707. 46. Abby SS, Kerou M, Schleper C. Ancestral reconstructions decipher major adap-
tations of ammonia-oxidizing archaea upon radiation into moderate terrestrial
and marine environments. MBio. 2020;11:e02371–20. 73. Versantvoort W, Guerrero-Castillo S, Wessels HJCT, Van Niftrik L, Jetten MSM, Brandt
U, et al. Complexome analysis of the nitrite-dependent methanotroph Methylo-
mirabilis lanthanidiphila. Biochim Biophys Acta - Bioenerg. 2019;1860:734–44. 47. Rinke C, Chuvochina M, Mussig AJ, Chaumeil PA, Davín AA, Waite DW, et al. A
standardized archaeal taxonomy for the Genome Taxonomy Database. Nat
Microbiol. 2021;6:946–59. 74. Pitcher A, Rychlik N, Hopmans EC, Spieck E, Rijpstra WIC, Ossebaar J, et al. Cre-
narchaeol dominates the membrane lipids of Candidatus Nitrososphaera gar-
gensis, a thermophilic Group I.1b Archaeon. ISME J. 2010;4:542–52. 48. Abby SS, Melcher M, Kerou M, Krupovic M, Stieglmeier M, Rossel C, et al. REFERENCES Musiani F, Broll V, Evangelisti E, Ciurli S. The model structure of the copper-
dependent ammonia monooxygenase. JBIC J Biol Inorg Chem. 2020;25:995–1007. 34. Alves RJE, Minh BQ, Urich T, Von Haeseler A, Schleper C. Unifying the global
phylogeny and environmental distribution of ammonia-oxidising archaea based
on amoA genes. Nat Commun. 2018;9:1517. 9. Nicol GW, Leininger S, Schleper C, Prosser JI. The influence of soil pH on the
diversity, abundance and transcriptional activity of ammonia oxidizing archaea
and bacteria. Environ Microbiol. 2008;10:2966–78. 35. Khadka R, Clothier L, Wang L, Lim CK, Klotz MG, Dunfield PF. Evolutionary history
of copper membrane monooxygenases. Front Microbiol. 2018;9:2493. 10. Adair KL, Schwartz E. Evidence that ammonia-oxidizing archaea are more
abundant than ammonia-oxidizing bacteria in semiarid soils of northern Arizona,
USA. Micro Ecol. 2008;56:420–6. 36. Bartossek R, Spang A, Weidler G, Lanzen A, Schleper C. Metagenomic analysis of
ammonia-oxidizing archaea affiliated with the soil group. Front Microbiol. 2012;3:208. 11. Karner MB, Delong EF, Karl DM. Archaeal dominance in the mesopelagic zone of
the Pacific Ocean. Nature. 2001;409:507–10. 37. De La Torre JR, Walker CB, Ingalls AE, Könneke M, Stahl DA. Cultivation of a
thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ
Microbiol. 2008;10:810–8. 12. Shi Y, Tyson GW, Eppley JM, Delong EF. Integrated metatranscriptomic and
metagenomic analyses of stratified microbial assemblages in the open ocean. ISME J. 2011;5:999–1013. 38. Tourna M, Stieglmeier M, Spang A, Könneke M, Schintlmeister A, Urich T. Nitro-
sosphaera viennensis, an ammonia oxidizing archaeon from soil. Proc Natl Acad
Sci USA. 2011;108:8420–5. 13. Baker BJ, Lesniewski RA, Dick GJ. Genome-enabled transcriptomics reveals
archaeal populations that drive nitrification in a deep-sea hydrothermal plume. ISME J. 2012;6:2269–79. 39. Wittig I, Braun H-P, Schägger H. Blue native PAGE. Nat Protoc. 2006;1:418–28. 14. Hollibaugh JT, Gifford S, Sharma S, Bano N, Moran MA. Metatranscriptomic
analysis of ammonia-oxidizing organisms in an estuarine bacterioplankton
assemblage. ISME J. 2011;5:866–78. 40. Reisinger V, Eichacker LA. Solubilization of membrane protein complexes for blue
native PAGE. J Proteom. 2008;71:277–83. 41. De Almeida NM, Wessels HJCT, De Graaf RM, Ferousi C, Jetten MSM, Keltjens JT,
et al. Membrane-bound electron transport systems of an anammox bacterium: a
complexome analysis. Biochim Biophys Acta - Bioenerg. 2016;1857:1694–704. 15. Kerou M, Offre P, Valledor L, Abby SS, Melcher M, Nagler M, et al. Proteomics and
comparative genomics of Nitrososphaera viennensis reveal the core genome and The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. REFERENCES Genome wide transcriptomic analysis of the soil ammonia oxidizing archaeon
Nitrososphaera
viennensis
upon
exposure
to
copper
limitation. ISME
J. 2020;14:2659–74. 81. Berube PM, Samudrala R, Stahl DA. Transcription of all amoC copies is associated
with recovery of Nitrosomonas europaea from ammonia starvation. J Bacteriol. 2007;189:3935–44. 55. Qin W, Amin SA, Lundeen RA, Heal KR, Martens-Habbena W, Turkarslan S, et al. Stress response of a marine ammonia-oxidizing archaeon informs physiological
status of environmental populations. ISME J. 2017;12:508–19. 82. Berube PM, Stahl DA. The divergent AmoC3 subunit of ammonia mono-
oxygenase functions as part of a stress response system in Nitrosomonas euro-
paea. J Bacteriol. 2012;194:3448–56. 56. Carini P, Dupont CL, Santoro AE. Patterns of thaumarchaeal gene expression in
culture and diverse marine environments. Environ Microbiol. 2018;20:2112–24. 83. Lebedeva EV, Hatzenpichler R, Pelletier E, Schuster N, Hauzmayer S, Bulaev A,
et al. Enrichment and genome sequence of the group I.1a ammonia-oxidizing
archaeon ‘Ca. Nitrosotenuis uzonensis’ representing a clade globally distributed
in thermal habitats. PLoS One. 2013;8:e80835. 57. Stewart FJ, Ulloa O, Delong EF. Microbial metatranscriptomics in a permanent
marine oxygen minimum zone. Environ Microbiol. 2012;14:23–40. 58. Bayer B, Pelikan C, Bittner MJ, Reinthaler T, Könneke M, Herndl GJ, et al. Proteomic
response of three marine ammonia-oxidizing archaea to hydrogen peroxide and
their metabolic interactions with a heterotrophic alphaproteobacterium. mSystems. 2019;4:e00181–19. 84. Qin W, Heal KR, Ramdasi R, Kobelt JN, Martens-Habbena W, Bertagnolli AD, et al. Nitrosopumilus maritimus gen. nov., sp. nov., Nitrosopumilus cobalaminigenes sp. nov., Nitrosopumilus oxyclinae sp. nov., and Nitrosopumilus ureiphilus sp. nov., four
marine ammonia-oxidizing archaea of the phylum Thaumarchaeota. Int J Syst
Evol Microbiol. 2017;67:5067–79. 59. Santoro AE, Dupont CL, Richter RA, Craig MT, Carini P, McIlvin MR, et al. Genomic
and proteomic characterization of ‘Candidatus Nitrosopelagicus brevis’: an
ammonia-oxidizing archaeon from the open ocean. Proc Natl Acad Sci USA. 2015;112:1173–8. 85. Ahlgren NA, Fuchsman CA, Rocap G, Fuhrman JA. Discovery of several novel,
widespread, and ecologically distinct marine Thaumarchaeota viruses that
encode amoC nitrification genes. ISME J. 2019;13:618–31. 60. Tolar BB, Herrmann J, Bargar JR, van den Bedem H, Wakatsuki S, Francis CA. Integrated structural biology and molecular ecology of N-cycling enzymes from
ammonia-oxidizing archaea. Environ Microbiol Rep. 2017;9:484–91. 86. Lawton TJ, Ham J, Sun T, Rosenzweig AC. Structural conservation of the B subunit
in the ammonia monooxygenase/particulate methane monooxygenase super-
family. Proteins. 2014;82:2263–7. 61. AUTHOR CONTRIBUTIONS LHH, MK, SW, MH, and CS conceptualized the research project. Investigation was
carried out by LHH, MK, WC, SNS, and RIPT. Biomass production was performed by
MM. LHH, MK, and CS wrote the paper with edits and contributions from MM, WC,
RIPT, SNS, SW, and MH. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly
from
the
copyright
holder. To
view
a
copy
of
this
license,
visit
http://
creativecommons.org/licenses/by/4.0/. REFERENCES Diamond S, Lavy A, Crits-Christoph A, Matheus Carnevali PB, Sharrar A, Williams
KH, et al. Soils and sediments host Thermoplasmata archaea encoding novel
copper membrane monooxygenases (CuMMOs). ISME J. 2022;16:1348–62. 87. Hyman MR, Wood PM. Methane oxidation by Nitrosomonas europaea. Biochem J. 1983;212:31–37. 62. Käll L, Krogh A, Sonnhammer ELL. A combined transmembrane topology and
signal peptide prediction method. J Mol Biol. 2004;338:1027–36. 88. Hyman MR, Wood PM. Ethylene oxidation by Nitrosomonas europaea. Arch
Microbiol. 1984;137:155–8. 89. Hyman MR, Murton IB, Arp DJ. Interaction of ammonia monooxygenase from
Nitrosomonas europaea with alkanes, alkenes, and alkynes. Appl Environ Micro-
biol. 1988;54:3187–90. 63. Hakemian AS, Rosenzweig AC. The biochemistry of methane oxidation. Annu Rev
Biochem. 2007;76:223–41. 64. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly
accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9. 90. Jones RD, Morita RY. Methane oxidation by Nitrosococcus oceanus and Nitroso-
monas europaea. Appl Environ Microbiol. 1983;45:401–10. 65. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. AlphaFold Protein Structure Database: massively expanding the structural cov-
erage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50:D439–D444. 91. Li P, Herrmann J, Tolar BB, Poitevin F, Ramdasi R, Bargar JR, et al. Nutrient
transport suggests an evolutionary basis for charged archaeal surface layer
proteins. ISME J. 2018;12:2389–402. 92. Sakoula D, Smith GJ, Frank J, Mesman RJ, Kop LFM, Blom P, et al. Universal
activity-based labeling method for ammonia- and alkane-oxidizing bacteria. ISME
J. 2022;16:958–71. 66. Evans R, O’Neill M, Pritzel A, Antropova N, Senior A, Green T, et al. Protein
complex prediction with AlphaFold-Multimer. bioRxiv. 2022. https://doi.org/
10.1101/2021.10.04.463034. The ISME Journal (2023) 17:588 – 599 L.H. Hodgskiss et al. 599 Correspondence and requests for materials should be addressed to Christa Schleper. Correspondence and requests for materials should be addressed to Christa Schleper. Reprints and permission information is available at http://www.nature.com/
reprints Reprints and permission information is available at http://www.nature.com/
reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. The ISME Journal (2023) 17:588 – 599 ACKNOWLEDGEMENTS We thank Anas Mohammed Mardini for excellent technical assistance in the
cultivation of N. viennensis and Wolfram Weckwerth for valuable input in the initial
discussions of the project. We also thank Florian Sikora and Dr. Boris Görke for
technical assistance and usage of the OneShot machine for cell lysis and Dr. Stephanie Eichorst for assistance and usage of the ultracentrifuge. We are also
appreciative of Dr. Thomas Rattei, Florian Goldenberg, and Johann Dorn of the
Division of Computational Systems Biology (CUBE) for providing maintenance and
access to the Life Science Computer Cluster (LiSC) at the University of Vienna. Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41396-023-01367-3. FUNDING This project was supported by Doktoratskolleg (DK) plus: Microbial nitrogen cycling—
from single cells to ecosystems (Austrian Science Fund W1257), ERC Advanced Grant
TACKLE (No. 695192), and the European Union’s Horizon 2021–2027 research and
innovation programme under grant agreement No 101079299. COMPETING INTERESTS COMPETING INTERESTS COMPETING INTERESTS
The authors declare no competing interests. The authors declare no competing interests. © The Author(s) 2023, corrected publication 2023 The ISME Journal (2023) 17:588 – 599
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To cite this version: Parisa Saadati, Jose Abdelnour-Nocera, Torkil Clemmensen. Proposed System for a Socio-Technical
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teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Keywords:sociotechnical, human work interaction design, automation, augmentation,
Industry 4.0. Proposed System for a Socio-technical Design
Framework for Improved User Collaborations with
Automation Technologies Parisa Saadati1 [0000-0002-0525-4654], José Abdelnour-Nocera1,2 [0000-0001-7935-7368] andTorkil
Clemmensen3 [0000-0002-0934-2336] Parisa Saadati1 [0000-0002-0525-4654], José Abdelnour-Nocera1,2 [0000-0001-7935-7368] andTorkil
Clemmensen3 [0000-0002-0934-2336] 1University of West London, UK
parisa.saadati@uwl.ac.uk 1University of West London, UK
parisa.saadati@uwl.ac.uk 2 ITI/Larsys Portugal
abdejos@uwl.ac.uk 1
Introduction During the life cycle of any organisation, a variety of environmental stimuli will in-
fluence its operations and decision-making processes. These external factors are de-
pendent on economic and social factors, political and legislative changes, and devel-
opments in technology and human knowledge. The internal environment may also
influence various processes and elements of an organisation such as the staff, infor-
mation and monitoring systems or management policies [1]. Complex organisational
systems inevitably rely now on large-scale software-intensive systems. In this paper,
we hint at a possible sociotechnical HCI framework with customized value proposi-
tions and a case presentation for a future investigation of three different scenarios
with different levels of automation. Socio-Technical System Design (STSD) developments have identified and ad-
dressed several problems in understanding and developing complex systems. Despite
many positive outcomes, these methods have not materially changed industrial soft-
ware engineering practices. One of the main reasons behind this is involving users
only in the testing stage of any new system development instead of the design process
[2]. Currently, ‘automation’ is one of the main means for supporting operators using
systems that feature high complexity. Automation allows designers to transfer the
burden from operators to machine by re-allocating the system tasks that were previ-
ously performed by human [3].Researchers have studied different aspects of imple-
mentation of advanced interactive technologies employing automation in different
platforms [1, 3-7]. Organisations can now improve operations and decision making by implementing
cyber-physical systems (CPS) and internet of things (IoT), and potentially linking
them to blockchain technology in the future. Rising integration of Internet of Every-
thing (IoE) into the industrial value chain is the foundation of “Industry 4.0” technol-
ogies [8]. These technologies can improve the end-users’ experience via increasing
the self-service options, optimising operations and security processes, and enhancing
ground asset management and connectivity. An important point to consider is that implementing new technologies in a complex
service-driven work environment (e.g. an airport terminal) does not necessarily and
automatically guarantee a positive response from workers and customers [9]. Hence,
developments towards future ‘smart workplaces’ need to be carefully designed in
order to achieve expected service quality goals for both end-users and employees. The
main purpose of this study is to identify all humanistic/social and technological ele-
ments in the design of newly automated systems applicable to Industry 4.0 that are
affecting the human and machine collaborations. 3Copenhagen Business School
tc.digi@cbs.dk 3Copenhagen Business School
tc.digi@cbs.dk Abstract.To improve human performance, interactive technologies are going towards more
automated systems that involve computers, robots and cyber-physical systems into the deci-
sion-making process. While automation can lead to increased performance and reduced impact
of human errors, interactive technologies without optimal design can have a negative impact on
the experience of operators and end-users, leading to suboptimal performance of the automated
systems. In this research, we aim to evaluate and refineHuman Work Interaction Design
(HWID) framework to be applicable in various highly-automated settings including Industry
4.0 environments. This will be performed via a thorough literature review as the first step. The
list of identified factors playing a potential role in various interactive systems will then be eval-
uated and optimised in three case studies. We will try to understand how to maximise collabo-
rations between the users and the machine in interactive systems. A practical approach for
evaluating both employees’ and end-users' perspectives in three scenarios with different levels
of automation will be assessed. We will evaluate the outputs in multiple levels of organisations,
employees and end-users. The ultimate output of the study will be a framework or model that
will help in designing future research studies for various automation scenarios, especially semi-
autonomous systems that involve high levels of interaction between users and the machine. We
will provide guidelines for implementation of the proposed framework in different scenari-
os. We expect that the framework output of this research will provide a comprehensive guide-
line applicable to many Industry 4.0 technologies. Keywords:sociotechnical, human work interaction design, automation, augmentation,
Industry 4.0. 2 2 2
Review Automating a process that is embedded into people’s everyday lives will surely im-
pact their experience. Automation replaces or rearranges people’s practices and habits
that may have been developed over long periods. Therefore, using automation in in-
teractive systems requires consideration of potential changes on human activity and
the new coordination demands on the human operators. These experiences highly
depend on the type and level of automation [7] and to what extent the developer has
allowed the machine to make decisions. 1
Introduction This paper is organised as follows. Section 2 introduces the findings of the literature review on different factors affecting
the human and machine collaborations and categorising them into three main catego-
ries. Section 3 proposes the future research outcome by investigating into these fac-
tors from three case studies; university library, research platform and an airport. 3 3 2.1
Technological elements of interactive systems Around 1970s and after a series of technological advances labelled as the third indus-
trial revolution (also called “the digital revolution”), the transition towards the fourth
industrial revolution (Industry 4.0) is now undergoing that will transform the design,
manufacturing, and operation of various products and systems [7]. The increasing
integration of the Internet of Everything (IoE) into the industrial value chain has built
the foundation for this revolution [8]. The increased connectivity and interaction
among systems, humans and machines support the integration of various automated or
semi-automated systems, and hence, increasing flexibility and productivity [10]. These automated systems will lead to interconnected manufacturing systems and sup-
ply chains with their own challenges. To achieve sufficient autonomous awareness in a system, efficient integration of
smart sensors and mobile devices is required alongside industrial communication
protocols and standards. Economic impact of this industrial revolution is supposed to
be huge [10], as it promises substantial increase in operational effectiveness as well as
the development of new business models, services, products and organisational struc-
tures and culture [10-12]. Three key components of Industry 4.0 are Internet of Things (IoT), Cyber-Physical
Systems (CPS), and smart workplaces. The main objects commonly used in the Indus-
try 4.0 are RFID (radio-frequency identifiers), sensors, actuators, and mobile phones
that interacts with each other and cooperate with their neighbouring smart compo-
nents to reach the common goal. For all these smart objects and people who are going
to collaborate with them, there is a need for setting technical standards to enable them
to work. Industry 4.0 advancements [7] are categorised into 4 main principles in general: 1. Technical assistance, 2. Interconnections, 3. Decentralised decisions, and 4. Information transparency. The main focus of this research will be on the “Collaborations” sub-principle of the
“Interconnections” principle (which includes Collaborations, Standards and Security). 4 4 Three type of collaborations are considered in the context of Industry 4.0: human-
human, human-machine and machine-machine collaborations. As a result of recent
advances in smart interactive systems, employees’ experience and access to technolo-
gy have increased substantially. Recent development of using smart technologies in
new domains such as health, education, finance and the impact of Industry 4.0 tech-
nologies in manufacturing and logistics have raised new challenges for Human Com-
puter Interaction (HCI) researchers and practitioners. Figure 1. The HWID framework [5] Figure 1. The HWID framework [5] For applying HWID models to specific workplaces we need to consider several in-
dependent and entangled factors[5]. Considering numerous theories, concepts, tech-
niques and methods developed for other work environments is the first step. Envi-
ronmental contexts such as national, cultural, geographic, social and organisational
factors will have an important role in designing optimal HWID models, as they im-
pact interaction between users (i.e. both operators and employees) and smart systems
in their work and life. There are more work-related factors including the users’
knowledge/skills, application domain, work contents and goals, as well as the nature
of tasks or newly introduced technologies to be considered in the interaction perfor-
mance. Developing HWID models requires establishing design goals, evaluation of
usability and user experience, engagement of all stakeholders, and provision of trans-
parent design processes. 2.3
Smart Workplace “Smart Workplace” is a vision where the organisation is fully connected with all
stakeholders via proactive adaptation to the real-time needs of the organisation includ-
ing operational necessities and customer requests. As an example, security concerns
in airports necessitate more investigations prior to the boarding, which results in long
queues and waiting times for passengers. Hence, airports need to be more innovative
in operations and handling of stakeholders (passengers and workers) and their needs
in real time. 2.2
Human Work Interaction Design Human Work Interaction Design (HWID) is a comprehensive framework that aims to
establish relationships between extensive empirical work-domain studies and HCI
designs. It builds on the foundation of Cognitive Work Analysis (CWA) [5]. HWID is
currently positioned as a modern lightweight version of CWA. HWID studies how to understand, conceptualise, and design for the complex and
emergent contexts in which information and communication technologies (ICT) and
work are entangled [1]. HWID models are based on the characteristics of humans and
work domain contents and the interactions during their tasks and decision making
activities (Figure 1). HWID focuses on the integration of work analysis (i.e., CWA
methods) and interaction design methods (e.g. goal-oriented design and HCI usabil-
ity) for smart workplaces. The ultimate goal of HWID is to empower users by design-
ing smarter workplaces in various work domains. 5 5 2.4
Humanistic elements of interactive systems To address human element in designing complex interactive systems, design fiction
and design ethnography should be linked[13]. This is in line with considering the
impact of anthropology on the design’s future-orientedness by understanding the cul-
tural meanings and sensitivity to values and context[14]. Analysis of the allocation of
functions is necessary to identify the optimal distribution of both functions and tasks
between a partly-autonomous system and the user[3]. Physical support of human workers by robots or machines is an important aspect of
new technologies. This is due to involvement of users in conducting a range of tasks
that are unpleasant, too exhausting or unsafe [15,16]. For an effective, successful, and
safe support of users in physical tasks, it is necessary that robots or machines interact
smoothly and intuitively with their human counterparts [15], and that humans are
properly trained for this kind of human-machine collaboration[8]. The value of information. In collaborations between human and machine, the value
of information is now more recognised given high power of the machine in decision-
making in highly-automated systems. For instance, informing users about the sensor’s
reading power of Tesla’s automated car can significantly increase their trust [6]. 6 However, other studies show that the number of information items or tasks users re-
ceive in an automated process should be personalised and up to the point of their de-
sire/tolerability. Not enough functions allocated to a user will lead to underload and
boredom and thus decreased performance. [17] Too many allocated functions will
lead to cognitive, perceptive or motoric overload and increase negative emotions (e.g. stress, anxiety) [18] and user’s error. [17, 3] Meanwhile, users can cope with emo-
tions after spending some time with the autonomous technology and developing some
routines. Providing an abundance of information and transparency is an important hypothe-
sis in interactive technologies. Trust, transparency and acceptance of losing control
(i.e. shared authority between the user and system [8]) can improve the interaction of
the user by revealing the ambiguous feelings toward the automation. Other psycho-
logical factors under study include worries about practical challenges and security of
the technology (e.g. hacking a system) and reliability of the process itself (e.g. flat
mobile phone battery for systems that rely on applications). 2.4
Humanistic elements of interactive systems Users may lose their trust
in decision-making of an automated system when other humans who will not follow
the same process are involved and can impact on the outcome (e.g. if fishermen not
using a specific application access to more fish than those using that application). An important situation is when responsibilities are shared between users and the
system. Ability to identify responsible party related to a bad outcome (i.e. user error
versus system failure) can impact the performance of users [9]. Controllable designed
interface and environment of work, as well as feeling safe while using new technolo-
gies, are among other factors that can increase the performance of the users. Involving users in the design process. The design process should determine the
content and format of information to be shared with users in order to create an experi-
ence of certainty and trust. Feedback from the users plays a major role for designing
such systems. However, the amount and format of the feedback must be well chosen,
otherwise it might question the main advantage of automation itself. Research needs to bridge the gap between the micro-perspective of technology
specifications and the macro-perspective of how life will and should change through
implementation of that technology. Enacting future systems “in the wild”, as a partic-
ular form of prototyping, is certainly an important element of this bridge. Motivating the users to engage with the new technologies is still a challenge due to
lack of understanding of the end-users’ individual experience and interaction with
such technologies. Users can have different roles or backgrounds that can affect their
discovery, collaboration and learning of the interactive system[11].Researchers have
tried to recruit users for testing their interaction via use of flyers or instructions ex-
plaining the technology (a process known as augmentation) [19]. Furthermore, engaging users in designing the automated or augmented product will
change their interaction time. The development teams need to familiarise themselves
with space and environment of practices, build trust with the employees and improve
design ideas. Studies suggest the relations between modes of discovery, design im-
provements, interaction and socio-spatial aspects. These relations can be developed 7 more as an analytical and design tool to redefine the borders of opportunities for so-
cial interaction in daily automated spaces. 2.5
Summary of the review We believe that there are unmet needs for evaluation and identification of both tech-
nical and humanistic factors involved in partly-autonomous systems[7, 3]. Unlike the
extensive technical literature on automation, there is a small research base examining
the human capabilities involved in work with automated systems[7]. Several factors such as sociological and psychological exchanges, ergonomic, cul-
tural relativity, technology availability and acceptance, etc.,have been proposed to be
involved in human-machine collaboration in various settings, especially in higher
levels of automation. However, the main problem is that there is no comprehensive
list of these factors, and no previous study has tried to develop a model based on these
factors. Such a model will be helpful to system designers for developing any new
interactive highly-automated system. We therefore see HWID framework as a funnel for socio-technical design, automa-
tion technologies, and information system (Figure 2). Figure 2. Main scopes of proposed HWID research on user collaboration with au-
tomation in complex settings Figure 2. Main scopes of proposed HWID research on user collaboration with au-
tomation in complex settings 3
Proposed further research In the
final scenario, current shortcomings and future opportunities will be evaluated by
using an HWID model for future smart workplaces using Industry 4.0 framework. Given the variety of environments and different levels of automation, we will poten-
tially achieve different lists of factors that affect the performance of both operators
and systems. This will help us to update the list for different environments. In the
final scenario, current shortcomings and future opportunities will be evaluated by
using an HWID model for future smart workplaces using Industry 4.0 framework. 3
Proposed further research For investigating independent and entangled factors related to human and machine
collaborations in automated systems, we propose a practical approach for evaluating
both end-users’ and employees' (or operators’) perspectives in an automatous envi-
ronment. First step (current stage) in this research is to produce a list of relevant factors from
different sources including: review of the relevant literature, contact and interview
with experts in this domain, and observation of some smart workplaces. This compre-
hensive list will then be evaluated and optimised in two scenarios (scenario 1, Univer- 8 8 sity of West London Library, and scenario 2, Indian Research Platform). These sce-
narios were selected carefully based on potentially important factors including socio-
behavioural (e.g., work pattern), psychological (e.g., trust in system), demographical
(e.g., wealth and ethnicity), and geographical characteristics of their user populations. We will analyse previously available (via literature review and expert opinions)
and newly-gathered data (via questionnaires and interviews) to produce a model to be
validated onscenario 3 settings (i.e.,London based airport). By several iterations in
this highly automated environment we will refine and provide the final output of the
study, which will be a tool/guideline for designing HWID models for various interac-
tive technologies. Figure 3 below depicts the proposed process of research in this
study. Figure 3. Research procedure
Given the variety of environments and different levels of automation, we will poten-
tially achieve different lists of factors that affect the performance of both operators
and systems. This will help us to update the list for different environments. In the
final scenario, current shortcomings and future opportunities will be evaluated by
using an HWID model for future smart workplaces using Industry 4.0 framework. Literature
Review&
ex-
pertopinio
n
Research Plat-
form– Applica-
tion data &
interview find-
ings
Refined
list
of Sociotech-
nical factors
AirportKi-
osks’ data
-
Interview
findings
Proposed
HWID
model
UWLLS
Kiosks’ data-
interview
Findings Figure 3. Research procedure
Literature
Review&
ex-
pertopinio
n
Research Plat-
form– Applica-
tion data &
interview find-
ings
Refined
list
of Sociotech-
nical factors
AirportKi-
osks’ data
-
Interview
findings
Proposed
HWID
model
UWLLS
Kiosks’ data-
interview
Findings Figure 3. Research procedure Figure 3. Research procedure Given the variety of environments and different levels of automation, we will poten-
tially achieve different lists of factors that affect the performance of both operators
and systems. This will help us to update the list for different environments. Conclusion In summary, the overall objective of this paper was to present a review of the possible
theoretical background for a to-be-developed sociotechnical HCI framework, includ-
ing customized value propositions for the work domain of choice, and, finally, to
present three scenarios to be considered in future research. One of the outcomes that
the current stage is a comprehensive list category in main principle and number of
sub-principles of the factors impact the machine and human counterpart collaboration
from sociotechnical perspective. This is what we hoped to illustrate with this paper as
start of a series of papers in different scenarios with various automation level. 9 9 References 1. Barricelli, B.R., Roto, V., Clemmensen, T., Campos, P., Lopes, A., Goncalves, F., Ab-
delnour-Nocera, J. (2018) Human Work Interaction Design 2018. Designing Engaging Au-
tomation. IFIP AICT 554, pp.215-228.http://doi.org/10.1007/978-3-030-05297-3_15 2. Baxter, G. and Sommerville, I. (2011) 'Socio-technical systems: From design methods to
systems engineering', Interacting with Computers, 23(1), pp.4-17. https://doi:
10.1016/j.intcom.2010.07.003 3. Barricelli, B.R., Roto, V., Clemmensen, T., Campos, P., Lopes, A., Goncalves, F., Ab-
delnour-Nocera, J. (2018) Human Work Interaction Design 2018. Designing Engaging Au-
tomation. IFIP AICT 554, pp.36-56. http://doi.org/10.1007/978-3-030-05297-3_3 4. Abdelnour-Nocera J, Oussena S, Burns C (2015). Human Work Interaction Design of the
Smart University. 4th IFIP 13.6 Working Conference on Human Work Interaction Design,
AICT-468: pp.127-140 5. Clemmensen T (2011) A Human Work Interaction Design (HWID) Case Study in E-
Government and Public Information Systems. International Journal of Public Information
Systems 3: pp.105-113 6. Dikmen, M., Burns, C.(2017) Trust in Autonomous Vehicles. 2017 IEEE International
Conference
on
Systems,
Man,
and
Cybernetics:
pp.1093–1098. https://doi.org/10.1109/SMC.2017.8122757 7. Parasuraman, R., Sheridan, T.,Wickens, C. (2000) A model for types and level of human
interaction with automation. IEEE Transactions on Systems, Man, and Cybernetics - Part
A:System and Humans, 30(3), pp.286-297 8. Hermann, M., Pentek, T. and Otto, B. (2016) Design Principles for Industrie 4.0 Scenarios. IEEE, pp. 3928-3937. http://doi.10.1109/HICSS.2016.48 9. World
Economic
Forum. (2018)
The
fourth
Industrial
Revolution,
https://www.weforum.org/pages/the-fourth-industrial-revolution-by-klaus-schwab, last ac-
cessed 22nd August, 2018 10. Davies, R. (2015) Industry 4.0: Digitalisation for productivity and growth. European Par-
liamentary Research Service 11. Lesser, E., Prusak, L. (1999) Communities of Practice, Social Capital and Organizational
Knowledge. Information
Systems
Review
1,
pp.3–10. [Online]
Available
from:http://providersedge.com/docs/km_articles/Cop_-_Social_Capital_-_Org_K.pdf, last
accessed 25th Feb, 2019 12. International
Airport
reviews,
[Online]
Available
from:
https://www.internationalairportreview.com/article/25929/personalisation-smart-airport/,
last accessed 18th Feb, 2019 13. Lindley, J., Sharma, D., and Potts, R. (2014) Anticipatory Ethnography: Design Fiction as
an Input to Design Ethnography. Ethnographic Praxis in Industry Conference Proceedings
2014, pp.237–253. https://doi.org/10.1111/1559-8918.01030 14. Otto, T., Smith, RC., Gunn, W.(2013) Design Anthropology: A Distinct Style of Knowing. Bloomsbury, London, New York, pp.1–29. 15. Awais, M., Henrich, D.(2013) “Human- Robot Interaction in an Unknown Human Inten-
tion Scenario”, 11th International Conference on Frontiers of Information Technology,
2013, pp. 89-94.https://doi.org/10.1109/FIT.2013.24 16. Kiesler, S. Hinds, P (2004) “Human-Robot Interaction”, Human-Computer Interaction,
Volume 19, 2004 17. Yerkes, RM., Dodson, J.D. (1908) The relation of strength of stimulus to rapidly of habit-
formation. Journal of Comparative Neurology and psychology, 18(5), pp.459-482. 10 18. Kirk, A. Brown,D.F. g
( ) pp
19. Wouters, N., Downs, J., Harrop, M., Cox, T., Oliveira, E., Webber, S., Vetere, F., Vande-
moere, A. (2016). Uncovering the Honeypot Effect: How Audiences Engage with Public
Interactive Systems. Proceedings of the 2016 ACM Conference on Designing Interactive
Systems, ACM, pp.5–16. http://doi.org/10.1145/2901790.2901796 References (2003) Employee assistance programs: A review of the management
of stress and wellbeing through workplace counselling and consulting, Australian Psy-
chologist 38(2), pp.138–143. g
( ) pp
19. Wouters, N., Downs, J., Harrop, M., Cox, T., Oliveira, E., Webber, S., Vetere, F., Vande-
moere, A. (2016). Uncovering the Honeypot Effect: How Audiences Engage with Public
Interactive Systems. Proceedings of the 2016 ACM Conference on Designing Interactive
Systems, ACM, pp.5–16. http://doi.org/10.1145/2901790.2901796
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Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry
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Stem cell research & therapy
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Zurich Open Repository and
Archive Zurich Open Repository and
Archive
University of Zurich
University Library
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CH-8057 Zurich
www.zora.uzh.ch University of Zurich
University Library
Strickhofstrasse 39
CH-8057 Zurich
www.zora.uzh.ch Year: 2021 Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential
in human adipose-derived mesenchymal stromal cells by mass cytometry RESEARCH Open Access Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential
in human adipose-derived mesenchymal stromal cells by mass cytometry Canepa, Daisy D ; Casanova, Elisa A ; Arvaniti, Eirini ; Tosevski, Vinko ; Märsmann, Sonja ; Eggerschwiler,
Benjamin ; Halvachizadeh, Sascha ; Buschmann, Johanna ; Barth, André A ; Plock, Jan A ; Claassen, Manfred ;
Pape, Hans-Christoph ; Cinelli, Paolo DOI: https://doi.org/10.1186/s13287-020-02044-4 Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-195855
Journal Article
Published Version
The following work is licensed under a Creative Commons: Attribution 4.0 International ( Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-195855
Journal Article
Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at:
Canepa, Daisy D; Casanova, Elisa A; Arvaniti, Eirini; Tosevski, Vinko; Märsmann, Sonja; Eggerschwiler, Ben-
jamin; Halvachizadeh, Sascha; Buschmann, Johanna; Barth, André A; Plock, Jan A; Claassen, Manfred; Pape,
Hans-Christoph; Cinelli, Paolo (2021). Identification of ALP+/CD73+ defining markers for enhanced osteogenic
potential in human adipose-derived mesenchymal stromal cells by mass cytometry. Stem Cell Research Therapy,
12:7.t DOI: https://doi.org/10.1186/s13287-020-02044-4 (2021) 12:7 Canepa et al. Stem Cell Research & Therapy
https://doi.org/10.1186/s13287-020-02044-4 Identification of ALP+/CD73+ defining
markers for enhanced osteogenic potential
in human adipose-derived mesenchymal
stromal cells by mass cytometry Daisy D. Canepa1,2†, Elisa A. Casanova1†, Eirini Arvaniti3, Vinko Tosevski4, Sonja Märsmann1,
Benjamin Eggerschwiler1,2, Sascha Halvachizadeh1, Johanna Buschmann5, André A. Barth5, Jan A. Plock5,
Manfred Claassen3, Hans-Christoph Pape1 and Paolo Cinelli1* Manfred Claassen3, Hans-Christoph Pape1 and Paolo Cinelli1* Abstract Background: The impressive progress in the field of stem cell research in the past decades has provided the
ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD-
MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and
variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for
their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it
would be essential to analyze AD-MSCs at single-cell level. Method: Recent advances in single-cell technologies have opened the way for high-dimensional, high-throughput,
and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF)
technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic
commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches. Result: This study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon
isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic
differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced
osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation
exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We
finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular
fractions (SVFs) and that could ultimately be used for cell therapies. (Continued on next page) * Correspondence: paolo.cinelli@usz.ch
†Daisy D. Canepa and Elisa A. Casanova contributed equally to this work. 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091
Zurich, Switzerland
Full list of author information is available at the end of the article * Correspondence: paolo.cinelli@usz.ch
†Daisy D. Canepa and Elisa A. Casanova contributed equally to this work. 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091
Zurich, Switzerland
Full list of author information is available at the end of the article * Correspondence: paolo.cinelli@usz.ch
†Daisy D. Canepa and Elisa A. Casanova contributed equally to this work.
1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091
Zurich, Switzerland
Full list of author information is available at the end of the article Introduction surface markers that could be used to purify cells from
tissues to homogeneity. Surgical interventions for bone repair are required for
numerous reasons, such as trauma-resulting non-union
fractures, or diseases including osteoporosis and osteo-
necrosis. Currently, autologous bone grafting is the most
commonly used approach, but has a number of short-
comings such as the limited amount of harvested spon-
giosa and donor site pain [1]. Alternative approaches,
including the use of synthetic bone substitutes, are not
optimal because they lack the osteoinductive properties
which are extremely important for healing large bone
defects [2]. Cell therapies based on ex vivo expanded
mesenchymal stromal stem cells (MSCs) in combination
with appropriate scaffolds may be valuable alternatives
to autologous bone grafting [3]. MSCs hold the ability to
differentiate into osteoblasts and are available from a
wide variety of tissue sources [4]. In particular, human
fat tissue has been demonstrated to be a valuable source
of MSCs—the so-called adipose-derived stromal cells
(AD-MSCs) [3]. An additional advantage of using fat tis-
sue is the relatively simple isolation procedure compared
to autologous bone isolation [5]. We and others have
shown that the combination of AD-MSCs in association
with synthetic calcium phosphate bone substitutes may
be a good alternative to autologous bone grafting [6–10]. Nevertheless, there are drawbacks linked to the use of
MSCs for clinical therapy in humans. In contrast to
other stem cell types (e.g., embryonic stem cells), the
mechanisms that regulate self-renewal and lineage speci-
fication in MSCs are largely unexplored. In particular,
MSC heterogeneity exists among donors, tissue sources,
and within cell populations [11–14]. The knowledge re-
garding how different functional and differentiation
attributes of MSCs are specified at the population level
is insufficient. This poses significant obstacles in efforts
to develop clinical manufacturing protocols that reprodu-
cibly generate functionally equivalent MSC populations
[15, 16]. Currently, MSCs are defined by cell surface phe-
notypes, as well as their functional ability to differentiate
into multiple cell lineages including osteoclast, chondro-
cyte, adipocyte, or skeletal myocyte lineages [17–19]. With
respect to the clinical application of MSCs, much effort
has been directed toward the identification of unique cell In 2006, the International Society for Cell Therapy
(ISCT) published the minimal criteria for defining MSCs
[20]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Page 2 of 16 (Continued from previous page) (Continued from previous page)
Conclusion: The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and
highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/
CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but
also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose
tissue for therapeutic applications. Keywords: Adipose-derived mesenchymal stromal cells, Stromal vascular fraction, Osteogenic potential, CyTOF,
Multidimensional analysis, Cell subpopulation (Continued from previous page)
Conclusion: The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and
highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/
CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but
also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose
tissue for therapeutic applications. Keywords: Adipose-derived mesenchymal stromal cells, Stromal vascular fraction, Osteogenic potential, CyTOF,
Multidimensional analysis, Cell subpopulation Classification of osteogenic differentiation ability of 17
human AD-MSCs We have isolated 17 AD-MSCs from the stromal vascular
fraction (SVF) of human fat tissue following standard pro-
tocols [34]. We further assessed the trilineage potential of
the established cell lines by inducing differentiation toward
osteogenic, chondrogenic, and adipogenic fate. Expression
of
lineage-specific
markers
during
the
differentiation
process was monitored by RTQ-PCR (data not shown) and
by classical staining assays (Alizarin Red, Alcian Blue, and
Oil Red: Figs. 1 and S1A) at days 14, 17, and 21. Staining in-
tensity was quantified using a highly standardized, auto-
mated digital image quantification approach [35]. This
approach takes into consideration not only the amount of
deposited dye in the whole cell culture dish but also the
time needed for differentiation [35]. Shortly, for each cell
line, the calculated pixels for each differentiation day (days
14, 17, and 21) were summed up to obtain one single value
per line (Figs. 1a, b and S1A). Next, the lines were catego-
rized into “good,” “intermediate,” and “bad” differentiating
cells based on the interquartile range distribution. We cate-
gorized lines in the 1st quartile as “bad,” lines in the 2nd
and 3rd quartile as “intermediate,” and lines in the 4th
quartile as “good” (Figs. 1a, b and S1A). Cells from different
donors clearly showed variable differentiation abilities
(Figs. 1b, c and S1A). For example, in “good” osteogenic dif-
ferentiating lines, calcium deposition was already detected
at day 14 whereas “bad” lines did not show differentiation
at day 21 but needed in average at least 30 days to fully dif-
ferentiate (Fig. 1c). The “intermediate” AD-MSC lines
showed Alizarin Red staining around day 17 and classified
therefore between the “good” and the “bad” lines (Fig. 1c). A similar trend was also observed for chondrogenic and
adipogenic differentiation (Figure S1A). Of interest, “good”
lines for one lineage were not necessarily “good” for the
other two lineages and the same was true for “bad” lines
(Figure S1B). These data suggest either an impairment of
the cells to differentiate or the existence of different sub-
populations with varying differentiation potential. p
( g
)
Visualization of the distribution of the 31 markers in
the AD-MSC lines with the dimensionality reduction
method Uniform Manifold Approximation and Projec-
tion (UMAP) [32] highlighted the intra- and inter-donor
heterogeneity (Figs. 2b, c and S2A). Classification of osteogenic differentiation ability of 17
human AD-MSCs Interestingly, all 17
AD-MSC lines formed one compact cloud showing high
degree of similarity among cells not only within the cell
lines but also among donors (Figs. 2b, c and S2A). Des-
pite the high degree of similarity among cells, the ex-
pression profiles of the investigated markers were not
homogeneously distributed over the cloud but showed a
gradient-like distribution all over the 17 AD-MSC lines. Interestingly, this was also the case for the widely ac-
cepted MSC markers CD73, CD105, and CD90 [20]. The
expression of these key markers mostly co-localized in
the same region of the cloud and was overlapping with
the expression of other markers described in the litera-
ture to be critical for MSCs, such as EGFRα and PDGF
Rα (Figs. 2b and S2A). In agreement with the minimal
criteria definition [20], the negative markers were indeed
not expressed in the AD-MSC lines (Figs. 2b and S2A). Other markers such as CD146, NG2, CD271, and
STRO-1 were expressed only by a relatively low number
of cells and were heterogeneously distributed over the
cloud (Figure S2A). We next generated UMAPs for each
individual AD-MSC donor for all 31 markers. Although
very small, each marker showed inter-donor variation re-
garding not only the amount of positive cells but also
the expression intensity of the markers (Fig. 2c). These
data clearly highlight in an unprecedented, multiparametric,
and multidimensional way the heterogeneous composition
of AD-MSC from several donors at single-cell level, sug-
gesting the presence of specific subpopulations. Introduction These criteria comprise, besides plastic adherence
and trilineage differentiation potential (osteogenic, chon-
drogenic, and adipogenic), the expression of CD105, CD73,
and CD90, coincident with the lack of the hematopoietic
markers CD45, CD34, CD14, CD19b, CD79a, and HLA-DR
[20]. Additional markers have been identified over the years
and are widely accepted for characterizing MSCs [21–31]. Even though all these markers were identified through
functional experiments, in the sense that they correlate with
the trilineage potential of the cells, it is not clear how their
distribution and expression correlate with the observed dif-
ferentiation capacity. Furthermore, questions remain open
regarding whether MSCs express any unique surface epi-
topes, and more importantly, it is unknown whether the
epitopes described to date have value in predicting MSC
function. In recent years, it was attempted to identify subpopu-
lations of MSCs that show enhanced bone regenerative
capability. Of note, most of these studies used a limited
number of markers alone or in combinations, thus making
comparison and reproducibility of the data difficult. It
would be therefore essential to be able to analyze the ex-
pression of the identified markers in toto and at single-cell
level in order to fully understand which subpopulations
are undergoing osteogenic lineage commitment. Recent advances in single-cell technologies have allowed
multidimensional, high-throughput, and high-resolution
measurements of biological systems. In this study, we ap-
plied cytometry by time-of-flight (CyTOF) to explore the
cellular composition of 17 human AD-MSCs, interrogating
31 markers at single-cell level. The goal of this study was to
investigate the subcellular composition of AD-MSCs in
their naïve state as well as during osteogenic commitment
via unsupervised dimensionality reduction [32], as well as
by supervised representation learning approaches [33]. The
data obtained reveal for the first time, in an unbiased way
and at single-cell level, the heterogeneity of AD-MSCs from
several donors and highlight the presence of subpopulations
of cells with osteogenic lineage commitment properties. Page 3 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Page 3 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy This information is of paramount importance considering
the emerging need of MSCs for biomedical applications. This information is of paramount importance considering
the emerging need of MSCs for biomedical applications. Introduction unravel cellular heterogeneity in the context of cancer,
immune diseases, or cellular differentiation [38–40] as
well as for identifying subcellular markers for diseases
[41]. However, this technology was never employed to
characterize human AD-MSCs. We coupled the high di-
mensionality of mass cytometry with advanced cellular
barcoding to simultaneously investigate 31 markers in
17 primary human AD-MSC lines to dissect at single-
cell level their cellular composition (Fig. 2a). Single-cell, multidimensional analyses reveal high cellular
heterogeneity in AD-MSCs We further wondered whether the variable differenti-
ation ability of the “good” and “bad” AD-MSCs is due to
the presence of specific subpopulations. Since it was pre-
viously shown that lineage specification occurs during
the first 4 days of differentiation [42], we cultured all 17
AD-MSC lines under osteogenic condition and investi-
gated at the single-cell level with CyTOF the population
dynamics. Shortly, at five different time points, cells for In order to dissect the differences between the AD-
MSCs obtained from different donors, we firstly per-
formed single-cell analyses with mass cytometry at their
naïve/undifferentiated state. CyTOF allows the simultan-
eous analysis at single-cell level of up to 50 different
parameters using antibodies conjugated with metal iso-
topes [36, 37]. This technique combines flow cytometry
and mass spectrometry and has already been used to Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Page 4 of 16 (2021) 12:7 A A Interquartile Osteogenic
Distribution
Osteogenic Quantification
«Good»
«Interm.»
«Bad»
F18
F28
F04
F22
F14
F11
F16
F17
F05
F29
F27
F19
F15
F10
F30
F32
F31
0
1×104
2×104
3×104
4×104
5×104
2×106
4×106
6×106
8×106
1×107
5×107
1×108
1.5×108
2×108
d14
d17
d21
Alizarin Red [a.u.]
Alizarin Red [a.u.]
0
5×103
1×104
1.5×104
2×104
1×107
2×107
3×107
4×107
5×107
1×108
1.5×108
2×108
2.5×108
3×108
B
C
Fig. 1 Classification of in vitro osteogenic differentiation potential of 17 AD-MSC lines. a Strategy used for the quantification of differentiation and
AD-MSC classification: (1) Cells were differentiated in vitro into osteogenic lineage, and at three time points (days 14, 17, and 21), they were
stained with Alizarin Red staining. (2) For each cell line, images of the whole well were acquired and pixels were counted and summed for the
three time points (days 14, 17, and 21). (3) Interquartile distribution was applied, and it was decided that the 4th quartile was representing
“good,” the 3rd and the 2nd quartile represented the “intermediate,” and the 1st quartile represented the “bad” differentiating lines. b Sum of the
pixels acquired at the three time points (days 14, 17, and 21) for osteogenic differentiation of all 17 AD-MSC lines and interquartile categorization
into “good,” “intermediate,” and “bad” AD-MSCs. c In vitro differentiation of one representing “good,” one “intermediate,” and one “bad” AD-MSC
after 14, 17, and 21 days under osteogenic condition assessed by Alizarin Red staining. Single-cell, multidimensional analyses reveal high cellular
heterogeneity in AD-MSCs Depicted are triplicates of undifferentiated cells (control)
and cells cultured under differentiation conditions C Osteogenic Quantification
«Good»
«Interm.»
«Bad»
F18
F28
F04
F22
F14
F11
F16
F17
F05
F29
F27
F19
F15
F10
F30
F32
F31
0
1×104
2×104
3×104
4×104
5×104
2×106
4×106
6×106
8×106
1×107
5×107
1×108
1.5×108
2×108
d14
d17
d21
Alizarin Red [a.u.]
B C C B Osteogenic Quantification Interquartile Osteogenic
Distribution
Alizarin Red [a.u.]
0
5×103
1×104
1.5×104
2×104
1×107
2×107
3×107
4×107
5×107
1×108
1.5×108
2×108
2.5×108
3×108 Interquartile Osteogenic
Distribution Interquartile Osteogenic
Distribution Fig. 1 Classification of in vitro osteogenic differentiation potential of 17 AD-MSC lines. a Strategy used for the quantification of differentiation and
AD-MSC classification: (1) Cells were differentiated in vitro into osteogenic lineage, and at three time points (days 14, 17, and 21), they were
stained with Alizarin Red staining. (2) For each cell line, images of the whole well were acquired and pixels were counted and summed for the
three time points (days 14, 17, and 21). (3) Interquartile distribution was applied, and it was decided that the 4th quartile was representing
“good,” the 3rd and the 2nd quartile represented the “intermediate,” and the 1st quartile represented the “bad” differentiating lines. b Sum of the
pixels acquired at the three time points (days 14, 17, and 21) for osteogenic differentiation of all 17 AD-MSC lines and interquartile categorization
into “good,” “intermediate,” and “bad” AD-MSCs. c In vitro differentiation of one representing “good,” one “intermediate,” and one “bad” AD-MSC
after 14, 17, and 21 days under osteogenic condition assessed by Alizarin Red staining. Depicted are triplicates of undifferentiated cells (control)
and cells cultured under differentiation conditions each of the 17 AD-MSC lines were collected (day 0: un-
differentiated cells, day 1–4: differentiation) and stra-
tegically barcoded (Table S2). At day 4, all samples were
simultaneously stained and processed for CyTOF acqui-
sition (Fig. 3a and Table S1). Cell density plots on the UMAPs of the 17 AD-MSC
lines during the initial 4 days of differentiation highlighted
an emerging subpopulation, which was very small at day 0
and increased over the differentiation period (Fig. 3b). This subpopulation was clearly visible already at day 0 in Page 5 of 16 Canepa et al. Single-cell, multidimensional analyses reveal high cellular
heterogeneity in AD-MSCs Stem Cell Research & Therapy (2 (2021) 12:7 A
Human fat tissue
isolation
Isolation
of AD-MSCs
Ab Panel
development
Identification of osteogenic
subpopulations
tSNE1
tSNE2
31 Ab staining
CyTOF
BC-strategy
C
B
Fig. 2 Mass cytometry analyses of human AD-MSCs reveal high heterogeneity. a Scheme of mass cytometry analysis on 17 human AD-MSCs
from AD-MSC collection to the identification of osteogenic subpopulations. b UMAPs of selected markers in all 17 analyzed AD-MSC lines. c
UMAPs of three selected markers (CD73, CD105, PDGFR) in 4 AD-MSC donors. Each dot represents one cell. Blue denotes minimal, green
intermediate, and red high expression A
Human fat tissue
isolation
Isolation
of AD-MSCs
Ab Panel
development
Identification of osteogenic
subpopulations
tSNE1
tSNE2
31 Ab staining
CyTOF
BC-strategy A Human fat tissue
isolation BC-strategy Identification of osteogenic
subpopulations
tSNE1
tSNE2
31 Ab staining
CyTOF Identification of osteogenic
subpopulations 31 Ab staining C
B B C Fig. 2 Mass cytometry analyses of human AD-MSCs reveal high heterogeneity. a Scheme of mass cytometry analysis on 17 human AD-MSCs
from AD-MSC collection to the identification of osteogenic subpopulations. b UMAPs of selected markers in all 17 analyzed AD-MSC lines. c
UMAPs of three selected markers (CD73, CD105, PDGFR) in 4 AD-MSC donors. Each dot represents one cell. Blue denotes minimal, green
intermediate, and red high expression absent in the “bad” cell lines and was confirmed and vali-
dated on all later days of the differentiation process
(Fig. 3d). Analysis of the percentages of cells positive for
ALP and CD73 in each category always confirmed signifi-
cant high frequency of ALP+ cells in the “good” lines,
moderate frequency in the “intermediate” lines, and very
low frequency of ALP+ cells in the “bad” lines over the
four osteogenic days (Figure S3A). The percentage of
CD73-positive cells was constant during the 4 differenti-
ation days in the three categories, but significantly in-
creased in the bad lines at day 2 and day 4 compared to
“good” lines (Figure S3A). the “good” lines whereas in the “bad” lines it was barely
present even at day 4 (Fig. 3b). To further investigate
whether it was possible to discriminate the differentiation
potential of AD-MSC lines at their undifferentiated state,
we applied the CellCNN algorithm [33] to the mass cy-
tometry data obtained at day 0 (undifferentiated state). Single-cell, multidimensional analyses reveal high cellular
heterogeneity in AD-MSCs Presented with the task of comparing “good” versus “bad”
cell lines, CellCNN detected a subpopulation character-
ized by high alkaline phosphatase (ALP+) expression and
low expression of the MSC marker CD73 (CD73low)
(Fig. 3c). This subpopulation was highly frequent in “good,
” moderately present in the “intermediate,” and almost Page 6 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy Population
Frequency (%)
Day 0
(Training)
Day 1
(Validation)
Day 2
(Validation)
Day 3
(Validation)
Day 4
(Validation)
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
A
C
B
All Cells
Selected Population
D
Fig. 3 Identification of AD-MSC osteogenic subpopulation. a Sample collection and CyTOF approach scheme during 4 days of osteogenic
differentiation (d0 = undifferentiated state, d1–d4 = differentiation). b Cell density plots on the UMAPs of the five analyzed days (d0, d1, d2, d3,
d4) during osteogenic differentiation. Once the pool of all 17 AD-MSC lines is represented, once only the “good,” the “intermediate” (interm.), and
the “bad” AD-MSC lines. Highlighted is the emerging population during osteogenic differentiation. Bright color indicates lower density, and dark
color indicates higher cellular density. c Empirical distribution densities of all analyzed 31 marker abundances for the entire cell population (blue)
and the cell subset selected by CellCNN (red). The identified subpopulation is characterized by alkaline phosphatase-positive (ALP+) and CD73low
expressing cells. d Boxplots indicating the frequencies of the ALP+/CD73low subpopulation selected by CellCNN in all “good,” “intermediate”
(interm.), and “bad” osteogenic differentiating lines during the five analyzed days. Error bars represent the mean of the percentage of positive
cells present in “good” (n = 6), “intermediate” (n = 4), and “bad” (n = 7) AD-MSCs A A B B Population
Frequency (%)
Day 0
(Training)
Day 1
(Validation)
Day 2
(Validation)
Day 3
(Validation)
Day 4
(Validation)
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
C
B
All Cells
Selected Population
D
Fig. 3 Identification of AD-MSC osteogenic subpopulation. a Sample collection and CyTOF approach scheme during 4 days of ost
differentiation (d0 = undifferentiated state, d1–d4 = differentiation). b Cell density plots on the UMAPs of the five analyzed days (d
d4) during osteogenic differentiation. Once the pool of all 17 AD-MSC lines is represented, once only the “good,” the “intermediat
the “bad” AD-MSC lines. Single-cell, multidimensional analyses reveal high cellular
heterogeneity in AD-MSCs Highlighted is the emerging population during osteogenic differentiation. Bright color indicates lower de
color indicates higher cellular density. c Empirical distribution densities of all analyzed 31 marker abundances for the entire cell po
and the cell subset selected by CellCNN (red). The identified subpopulation is characterized by alkaline phosphatase-positive (ALP+
expressing cells. d Boxplots indicating the frequencies of the ALP+/CD73low subpopulation selected by CellCNN in all “good,” “int
(interm.), and “bad” osteogenic differentiating lines during the five analyzed days. Error bars represent the mean of the percentage
cells present in “good” (n = 6), “intermediate” (n = 4), and “bad” (n = 7) AD-MSCs Population
Frequency (%)
Day 0
(Training)
Day 1
(Validation)
Day 2
(Validation)
Day 3
(Validation)
Day 4
(Validation)
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
Bad
Interm
Good
C C Day 3
(Validation) Day 4
(Validation) All Cells
Selected Population
D D Fig. 3 Identification of AD-MSC osteogenic subpopulation. a Sample collection and CyTOF approach scheme during 4 days of osteogenic
differentiation (d0 = undifferentiated state, d1–d4 = differentiation). b Cell density plots on the UMAPs of the five analyzed days (d0, d1, d2, d3,
d4) during osteogenic differentiation. Once the pool of all 17 AD-MSC lines is represented, once only the “good,” the “intermediate” (interm.), and
the “bad” AD-MSC lines. Highlighted is the emerging population during osteogenic differentiation. Bright color indicates lower density, and dark
color indicates higher cellular density. c Empirical distribution densities of all analyzed 31 marker abundances for the entire cell population (blue)
and the cell subset selected by CellCNN (red). The identified subpopulation is characterized by alkaline phosphatase-positive (ALP+) and CD73low
expressing cells. d Boxplots indicating the frequencies of the ALP+/CD73low subpopulation selected by CellCNN in all “good,” “intermediate”
(interm.), and “bad” osteogenic differentiating lines during the five analyzed days. Error bars represent the mean of the percentage of positive
cells present in “good” (n = 6), “intermediate” (n = 4), and “bad” (n = 7) AD-MSCs Page 7 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 We further investigated the correlation between ALP+
frequency (measured by CyTOF during the 5 days) and
the ability to differentiate into osteocytes (based on the
quantification of the staining at days 14, 17, and 21). ALP+/CD73+ cells are present in the SVF of human fat
tissue To ultimately prove the clinical utility of the identified ALP
and CD73 marker combination, we investigated whether
ALP+/CD73+ cells were also present in freshly isolated hu-
man adipose stromal vascular fractions (SVFs), and if, upon
isolation, they displayed similar properties as the ALP+/
CD73+ cells present in AD-MSC lines. For this purpose, hu-
man adipose tissues were collected from 3 healthy donors,
and the presence of ALP+/CD73+ cells was investigated. Immunohistochemical staining revealed the presence of
ALP+/CD73+ located in fat tissue capillaries (Figs. 5a and
S5A). SVFs from the same donors were further processed by
FACS sorting, and the fractions (control: unstained cells
sorted through FACS; CD45−/ALP+/CD73+, CD45−/ALP
−/CD73low,
CD45−/ALP−/CD73high)
were
plated
for
osteogenic differentiation (Figs. 5b and S5B). Quantification
of osteogenic differentiation at d14, d17, and d21 confirmed
higher osteogenic differentiation in the ALP+/CD73+ sorted
cells compared to the other ones (Figs. 5b and S5C). ALP+CD73+ marker combination can be used for
monitoring the osteogenic potential of undifferentiated
AD-MSC populations after expansion in vitro p p
p
A major problem during in vitro expansion of MSCs
(and also AD-MSCs) is that they show signs of aging
and changes in the subcellular composition, which fi-
nally lead to a decrease of the differentiation potential
over the passages [14]. To follow the dynamic of the cell
composition over prolonged cell culture, we analyzed
with our CyTOF antibody panel 3 “good” and 1 “inter-
mediate” AD-MSC lines (F28, F14, F22, and F05) from
passage 3 (p3) to passage 20 (p20) (Table S4). We could
confirm that the median intensity of expression of CD73
was increasing whereas ALP was rapidly diminishing
after prolonged culture, mirroring the situation observed
in all “bad” lines at p10 (Figs. 4e and S4E). ALP+/CD73+ cells possess enhanced osteogenic
differentiation ability In order to further characterize the identified subpopula-
tion, we selected four AD-MSC lines (F28, F14, F04, and
F22) and sorted three distinct cell subpopulations by
FACS:
ALP+/CD73+,
ALP−/CD73low,
and
ALP
−/CD73high. Although CellCNN analysis on CyTOF
data revealed the presence of an ALP+/CD73low popula-
tion, this phenotype was not clearly definable by FACS
sorting. We could select ALP−/CD73high and ALP
−/CD73low, but it was not possible to unambiguously
distinguish
between
ALP+/CD73low
and
ALP+/
CD73high cells. For this reason, we selected the double
positive ALP+ and CD73+ (ALP+/CD73+) population
for further experiments (Figure S4A). As a control, we
used for each AD-MSC line unstained cells processed
through the FACS. After sorting, the different subpopu-
lations were directly plated for differentiation into the
three lineages followed by lineage-specific staining at
days 14, 17, and 21 and quantification according to
Eggerschwiler et al. [35]. The sorted ALP+/CD73+ frac-
tion showed enhanced osteogenic differentiation when
compared to the other sorted populations (Figs. 4a, b
and S4B). We next differentiated the cells at p5, p9, and p20, and
we observed a decrease in the differentiation capacity
over the passages and these changes correlate with the
expression of ALP and CD73 (Figs. 4e, f and S4F). In
conclusion, we confirmed that ALP+/CD73+ expressing
cells possess higher osteogenic differentiation potential
and the marker combination of ALP and CD73 can be
used to predict the osteogenic differentiation potential
of cultured AD-MSCs. Single-cell, multidimensional analyses reveal high cellular
heterogeneity in AD-MSCs As
expected ALP+ always correlated with the osteogenic
differentiation ability (Figure S3B) confirming once more
that ALP+ expression correlates with osteogenic lineage
commitment. In conclusion, our approach allowed the
identification of an osteogenic subpopulation character-
ized by the markers ALP+/CD73low that hallmarked ex-
clusively the “good” differentiating lines. interquartile categorization of these lines confirmed two
predicted “good” lines, one predicted “intermediate” line,
and two predicted “bad” lines (Figs. 4c, d and S4C-D). Thus, we could confirm that the presence of the marker
combination ALP+/CD73+ is sufficient to predict the
osteogenic differentiation ability of a donor AD-MSC line
in its undifferentiated state. ALP+CD73+ marker combination is predictive for
osteogenic potential in undifferentiated AD-MSC
populations We next assessed whether the marker ALP+ (which is also
CD73+, see Figure S4A) could be used as a predictor for
osteogenic differentiation potential of AD-MSCs in their
undifferentiated state. For this purpose, we selected five
new AD-MSC lines, which had never been characterized
or used in previous experiments. Undifferentiated cells
from the new lines, together with 9 already characterized
lines (as reference cells), were subjected to CyTOF (Table
S3). Quantification of the presence of ALP+ cells in the
new lines allowed a predicted categorization based on the
9 AD-MSCs, into “good,” “intermediate,” and “bad” lines
(Fig. 4c). We further compared the outcome from the
CyTOF data with the differentiation ability observed
in vitro (Fig. 4d). Alizarin Red quantification and the Page 8 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy A
B
Alizarin Red [a.u.]
control
sorted
ALP+
CD73+
ALP-
CD73 low
ALP-
CD73 high
d14
d17
d21 A
C
B
Alizarin Red [a.u.]
F22
F14
F04
Alizarin Red [a.u.]
control
sorted
ALP+
CD73+
ALP-
CD73 low
ALP-
CD73 high
control
sorted
ALP+
CD73+
ALP-
CD73 low
ALP-
CD73 high
d14
d17
d21
d14
d17
d21
d14
d17
d21
d14
d17
d21
D
Alizarin Red [a.u.]
ALP frequency (%)
d14
d17
d21
0
2×103
4×103
6×103
8×103
1×104
2×105
4×105
6×105
8×105
2×107
4×107
6×107
8× 07
p5
p9
p20
E
F
0
5
10
15
<Good>
<Bad>
<Interm.>
Fig. 4 ALP+/CD73+ markers possess higher and predictive osteogenic potential. a Alizarin Red staining and quantification of F28 AD-MSC line
sorted subpopulations (ALP+/CD73+, ALP−/CD73low, ALP−/CD73high) after 14, 17, and 21 days of osteogenic differentiation. Controls sorted are
unstained cells, which were run through the FACS machine. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells
cultured under osteogenic differentiation conditions (differentiation). b Quantification of Alizarin Red staining for F04, F14, and F22 AD-MSC lines
for the same sorted subpopulations after 14, 17, and 21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and
are presented as mean ± s.d. c Predicted categorization based on alkaline phosphatase (ALP) frequency in five new AD-MSC lines (green) and
nine already characterized AD-MSC lines (reference) measured by CyTOF. d Alizarin Red staining at day 21 of the five new AD-MSC lines. ALP+CD73+ marker combination is predictive for
osteogenic potential in undifferentiated AD-MSC
populations Depicted
are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). e Histogram for the
median intensity of expression of CD73 and ALP of F22 “good” AD-MSC line from passage 3 (p3) till passage 20 (p20). Black is the lowest
intensity, and white represents the highest intensity. f Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and
21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the
one-way ANOVA Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within
h
d
d
f B F22
F14
F04
Alizarin Red [a.u.]
control
sorted
ALP+
CD73+
ALP-
CD73 low
ALP-
CD73 high
d14
d17
d21
d14
d17
d21
d14
d17
d21 Alizarin Red [a.u.]
d14
d17
d21
0
2×103
4×103
6×103
8×103
1×104
2×105
4×105
6×105
8×105
2×107
4×107
6×107
8× 07
p5
p9
p20
E
F C
ALP frequency (%)
0
5
10
15
<Good>
<Bad>
<Interm.> Alizarin Red [a.u.]
d14
d17
d21
0
2×103
4×103
6×103
8×103
1×104
2×105
4×105
6×105
8×105
2×107
4×107
6×107
8× 07
p5
p9
p20
F C F F E D D Fig. 4 ALP+/CD73+ markers possess higher and predictive osteogenic potential. a Alizarin Red staining and quantification of F28 AD-MSC line
sorted subpopulations (ALP+/CD73+, ALP−/CD73low, ALP−/CD73high) after 14, 17, and 21 days of osteogenic differentiation. Controls sorted are
unstained cells, which were run through the FACS machine. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells
cultured under osteogenic differentiation conditions (differentiation). b Quantification of Alizarin Red staining for F04, F14, and F22 AD-MSC lines
for the same sorted subpopulations after 14, 17, and 21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and
are presented as mean ± s.d. c Predicted categorization based on alkaline phosphatase (ALP) frequency in five new AD-MSC lines (green) and
nine already characterized AD-MSC lines (reference) measured by CyTOF. d Alizarin Red staining at day 21 of the five new AD-MSC lines. Depicted
are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). e Histogram for the
median intensity of expression of CD73 and ALP of F22 “good” AD-MSC line from passage 3 (p3) till passage 20 (p20). ALP+CD73+ marker combination is predictive for
osteogenic potential in undifferentiated AD-MSC
populations Black is the lowest
intensity, and white represents the highest intensity. f Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and
21 days of osteogenic differentiation. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the
one-way ANOVA Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within
the same day: *p ≤0.05, **p ≤0.01, ***p ≤0.001, and ****p ≤0.0001. ns, not significant Page 9 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 A A Th
d
d
h
ALP /CD 3
ll
Di
i
A
B
control sorted
ALP+CD73+
ALP-CD73low
ALP-CD73high
Fig. 5 ALP+/CD73+ cells are present in human fat tissue and possess enhanced osteogenic potential. a Immunofluorescence of ALP and CD73 in
human fat tissue. Scale 100 μm. BF, bright field. b Alizarin Red staining and quantification of 3 healthy donors’ SVF after 21 days of osteogenic
differentiation. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells cultured under osteogenic differentiation
conditions. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA
Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within the same day: *p ≤
0.05, **p ≤0.01, and ***p ≤0.001. ns, not significant B B control sorted
ALP+CD73+
ALP-CD73low
ALP-CD73high Fig. 5 ALP+/CD73+ cells are present in human fat tissue and possess enhanced osteogenic potential. a Immunofluorescence of ALP and CD73 in
human fat tissue. Scale 100 μm. BF, bright field. b Alizarin Red staining and quantification of 3 healthy donors’ SVF after 21 days of osteogenic
differentiation. Depicted is one triplicate of undifferentiated cells (control) and triplicates of cells cultured under osteogenic differentiation
conditions. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA
Dunnett’s multiple comparisons test was used to compare the ALP+/CD73+ population with the other sorted fractions within the same day: *p ≤
0.05, **p ≤0.01, and ***p ≤0.001. ns, not significant Discussion However, variability can also be observed
when BM-MSCs were autologously isolated over different
periods of time or even when isolated bilaterally from the
same donor [15], indicating that the cellular composition of
MSCs plays an important role and is highly heterogeneous. A possibility to explain this heterogeneity is the variable
composition of the tissues used for the establishment of
MSC lines (e.g., amount of blood vessels). Dissecting this
heterogeneity at single-cell level and identifying subpopula-
tions of cells with specific differentiation attributes are ur-
gently
needed
for
developing
clinical
manufacturing
protocols that reproducibly generate functionally equivalent
MSC populations. In this study, we have aimed at identifying specific
AD-MSC subpopulations of cells with higher osteogenic
differentiation potential. The novel approach used herein
enabled the simultaneous visualization of 31 selected
markers in 17 primary AD-MSC lines, thereby offering
unprecedented observational dimensionality in a large
sample set. This approach allowed circumvention of the
classical bulk assays most frequently used for character-
izing MSCs and their differentiation potential, which
pool signals across entire cell populations, masking cell-
to-cell variation. Unexpectedly, the dimensionality re-
duction algorithm UMAP revealed a high degree of cel-
lular similarity, as observed from the compact clouds
that all AD-MSC lines generated. This is in contrast to
hematopoietic cells, for example, where UMAPs clearly
separate the different cell subpopulations (Bendall et al. [37]). Nevertheless, despite the high degree of similarity
among cells, the distribution of the markers within the
clouds was highly heterogeneous, forming in some cases
gradients (such as CD73, EGFR, PDGFR, SOX9) or small
islands (ALP, CD166, STRO-1) (Figs. 2b and S2A). Fur-
thermore, each marker showed inter-donor variation re-
garding not only the amount of positive cells but also
the expression intensity of the markers (Fig. 2c). In vitro selection after prolonged culture represents a
major concern for the use of MSCs for therapeutic ap-
plications. Expansion on hard tissue culture surfaces
may promote cellular divergence and/or reduction in po-
tency [57, 58]. Additionally, the culture conditions used
are very permissive when compared with the ones
employed by other stem cell types, e.g., embryonic stem
cells or induced pluripotent stem cells, where specific
factors are necessary to maintain the self-renewal cap-
acity of the stem cells [59–61]. Discussion These data demonstrate that ALP+/CD73+ cells are
present in freshly isolated human fat tissue and possess
enhanced osteogenic potential, representing therefore in-
teresting cells for therapeutic applications. Even with the most effective protocols, different MSC prep-
arations show strong variation in their differentiation per-
formance. One possible explanation for this phenomenon Page 10 of 16 Page 10 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 it is an accepted osteoblast marker. CD73 and ALP are
GPI (glycophosphatidylinositol)-anchored ectoenzymes
with 5′-nucleotidase activity; thus, they share similar
functions. CD73 and ALP regulate the extracellular
breakdown of ATP to adenosine [51]. Released ATP
serves as an autocrine or paracrine regulator of both
osteoblast and osteoclast functions [52, 53], and hydroly-
zation of pyrophosphate provides inorganic phosphate to
promote mineralization. The extracellular nucleotide
ATP can be one of the key mediators in bone metabol-
ism, not only as a phosphate source, but also as a signal-
ing molecule via P2 receptors. In fact, osteoblasts have
been reported to release ATP into the extracellular en-
vironment constitutively followed by engagement of P2
receptors [54]. Most importantly, ALP+/CD73+ cells are
also present and even more abundant in freshly isolated
SVFs. The origin of these cells has to be better charac-
terized, but it is reasonable to assume that these cells
could be of pericytic origin. ALP is a known pericytic
marker which was previously described as a marker for
the prospective isolation of pericytes from different tis-
sues [55, 56]. This is in agreement with our observation
that ALP+/CD73+ cells are localized in the capillaries of
fat tissue. In this sense, the difference observed between
“good” and “bad” AD-MSC lines could be explained with
differences in the amount of blood vessels in the isolated
fat tissue. Our data suggest the existence of a balanced
regulation of ALP and CD73 in human AD-MSCs,
which is crucial for the determination of osteogenic
lineage commitment. is the high heterogenic cellular composition of MSCs, con-
sisting of different cells harboring diverse lineage commit-
ment ability [43]. The high donor-to-donor variability
observed when MSCs are derived from the same tissue of
origin may be due to different factors including donor
health [11, 44], age, MSC availability, and/or self-renewal
capacity [45–47]. Discussion Our data indicate that
progressive loss of ALP+/CD73low cells during passa-
ging precludes osteogenic differentiation and constantly
monitoring ALP+/CD73low can be used as a quality
control procedure to monitor AD-MSC expansion for
bone regeneration purposes. Although ALP and CD73 have never been associated
together with osteogenic potential, singularly they were
previously correlated with osteogenic differentiation. CD73 was shown to regulate bone formation and re-
modeling in intramembranous bone repair [48]. In our
study, we demonstrated that CD73 expression levels in-
versely correlate with the osteogenic differentiation abil-
ity
of
17
human
AD-MSC
primary
preparations
(Figs. 4e, f and S4E-F). Tissue nonspecific ALP has been
found in several tissues and cell types, such as activated
B cells or pluripotent embryonic stem cells [49, 50], and In conclusion, our study highlights that single-cell and
multiparametric analysis identifies gradient expression
and co-localization of markers which have not been pre-
viously observed. The combination of ALP+/CD73low
markers can not only (1) discriminate between “good”
and “bad” differentiating lines but can also be used for
(2) prospective isolation of selected cells from SVF for
bone tissue engineering and (3) to assess the differenti-
ation potential of AD-MSC preparation in culture. Page 11 of 16 Page 11 of 16 Page 11 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Osteogenesis Kit or StemPro® Adipogenesis Kit (Gibco/
Life Technologies). For chondrogenic differentiation,
cells were cultured at a density of 5 × 103 cells/cm2 in a
Nunc™24-well plate (Thermo Fisher Scientific) and dif-
ferentiation was induced at the 4th day of culture using
the StemPro® Chondrogenesis Kit (Gibco/Life Technolo-
gies). All media were changed every 4 days. Osteogenesis Kit or StemPro® Adipogenesis Kit (Gibco/
Life Technologies). For chondrogenic differentiation,
cells were cultured at a density of 5 × 103 cells/cm2 in a
Nunc™24-well plate (Thermo Fisher Scientific) and dif-
ferentiation was induced at the 4th day of culture using
the StemPro® Chondrogenesis Kit (Gibco/Life Technolo-
gies). All media were changed every 4 days. The use of MSCs in clinical medicine will likely con-
tinue to grow rapidly, yet it still is unclear how clinical
manufacturing affects MSC biology, particularly regard-
ing lineage specification. The development of assays
allowing for the monitoring of the production process
and assessment of cellular function are urgently needed. Assessment and classification of trilineage differentiation
potential Differentiation assessment via specific staining was per-
formed for all three differentiation lineages after 14, 17,
and 21 days of differentiation. For Alizarin Red S (Sigma)
staining, cells were washed with PBS and fixed with 4%
(v/v) formaldehyde (Sigma) for 30 min at RT. Upon
washing twice with ddH20, Alizarin Red S solution (0.7 g
Alizarin Red S diluted in 50 ml ddH2O at pH = 4.2) was
added for 20 min at RT. Afterwards, cells were washed
four times with ddH2O, dried, and stored in the dark
until image acquisition. For Oil Red O (Sigma) staining,
cells were washed once with PBS and fixed with 10% (v/
v) formaldehyde (Roth) for 1 h at RT. Afterwards, cells
were washed twice with ddH2O, rinsed twice with 60%
(v/v) 2-propanole (Sigma) in ddH20, and dried. Oil Red
O working solution (0.15 g Oil Red O in 50 ml 60% (v/v)
2-propanole in ddH2O) was added for 10 min at RT. After four ddH2O washing steps, cells were dried and
images were directly taken. For Alcian Blue 8GX (Sigma)
staining, cells were washed with PBS and then fixed with
4% (v/v) formaldehyde (Sigma) for 20 min at RT. After-
wards, cells were washed twice with ddH2O and incu-
bated for 3 min with 3% (v/v) acetic acid (Merck
Millipore) in ddH20. Alcian Blue solution (0.1 g Alcian
Blue 8GX in 100 ml of 3% acetic acid in ddH20 at pH =
2.5) was given for 1 h at RT. Cells were washed four
times with ddH2O, dried, and stored in the dark until
image acquisition. Images of the entire wells at days 14,
17, and 21 of differentiation were acquired with Cytation
5 imaging reader (BioTek). Quantification of differenti-
ation was performed according to [35], and subsequent
classification of AD-MSC into “good,” “bad,” and “inter-
mediate” differentiating lines was performed applying
the interquartile range distribution. We defined cell lines
present in the 4th quartile as “good,” lines present in the
2nd and 3rd as “intermediate,” and lines in the 1st quar-
tile as “bad.” Discussion The approach chosen in this work might provide a basis
for better understanding how different functional attri-
butes of MSCs are specified at the population level, and
can be used in the development of clinical manufactur-
ing protocols that reproducibly generate functionally
equivalent MSC populations. Cells and cell culture Twenty-two human adipose tissue samples (100–600 g)
were
obtained
from
lipectomies
and
liposuctions
(healthy donors, no diabetic donors) [62]. AD-MSCs
were isolated from fat tissue, with the consent of the do-
nors according to Swiss (KEK-ZH: StV 7-2009) and
international ethical guidelines (ClinicalTrials.gov; Iden-
tifier: NCT01218945) [62]. The extraction procedure
was performed according to [34]. AD-MSCs were cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM)
(PAN Biotech) supplemented with 10% of fetal bovine
serum (FBS) (Biowest), 1% of antibiotics (100× penicillin,
100× streptomycin) (Biowest), and 1% L-glutamine 200
mM (Sigma) (called AD-MSC medium). Medium was
changed every 3 days, and cells were passaged with 1×
Trypsin-EDTA (Life Technologies) for 5 min at 37 °C
when cells were about 80% confluent. Cells were incu-
bated at 37 °C in an atmosphere with 95% humidity and
5% CO2. Material and methods
Ethics statement Adipose-derived stromal cells (AD-MSCs) were obtained
from lipectomies and liposuctions (healthy donors, no dia-
betic donors) upon written informed consent of the do-
nors, following the guidelines approved by the Kantonale
Ethik Kommission (KEK) Zurich Swiss (KEK-ZH: StV 7-
2009) and international ethical guidelines (ClinicalTrials. gov; Identifier: NCT01218945). The stromal vascular frac-
tion (SVF) isolated from human fat tissue was obtained
with the consent of the patient according to Swiss ethics
(BASEC-Nr.: 2019-01504). Isolation of the stromal vascular fraction For osteogenic differentiation, AD-MSCs were seeded at
a density of 1.6 × 104 cells/cm2 in Nunc™24-well plates
(Thermo Fisher Scientific) or at a density of 1 × 104 in
96-well plates (TPP). For adipogenic differentiation, cells
were cultured at a density of 1.6 × 104 cells/cm2 in
Nunc™24-well plates (Thermo Fisher Scientific). Differ-
entiation was started 24 h after seeding with StemPro® Stromal vascular fraction (SVF) was isolated from hu-
man fat tissue with the consent of the patient according
to Swiss ethics (BASEC-Nr.: 2019-01504) and according
to [34]. Briefly, lipectomies were cut in small pieces and
extensively washed with PBS. Enzymatic digestion was
performed with 0.075% collagenase I (Gibco) at 37 °C for Page 12 of 16 Page 12 of 16 Page 12 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Mass cytometry antibody panel and staining procedures
The antibody panel consisted of 31 monoclonal anti-
human metal-conjugated antibodies, which included cell
surface, cytoplasmic, and transcription targets (Table S1). When possible, already metal-conjugated antibodies were
purchased from Fluidigm; otherwise, antibodies were con-
jugated in-house with isotopically pure lanthanide metals
according to the commercially available MaxPar Antibody
Labelling Kit (Fluidigm). Labeled antibodies were stored at
4 °C in antibody stabilizer solution (Candor Bioscience). Titration of each antibody was performed on a one-to-one
mix of cells consisting of PBMCs (peripheral blood mono-
nuclear cells), HEK (human embryonic kidney cells 293),
Hela (cervical cancer cells), Jurkat (human T lymphocyte
cells), Saos2 (sarcoma cells), Nalm6 (B cell precursor
leukemia cells), SHSY5S (neuroblastoma cells), and hu-
man AD-MSCs. These different cell lines, which we called
MIX, were chosen in order to have for each marker a
positive and a negative control cell type. Sample staining
was performed as described in the MaxPar Cell Surface,
MaxPar Cytoplasmic/Secreted Antigen, and MaxPar Nu-
clear Target protocols (Fluidigm) with minor changes. Briefly, cells were first subjected to cell surface antibody
staining, followed by cytoplasm staining, and nuclear
staining. For the cytoplasmic and intranuclear staining,
cell fixation steps were shortened to 10 min. Cells were
then resuspended in 4% paraformaldehyde (Electron
Microscopy Sciences) and stored at 4 °C until acqui-
sition. Fluorescence activating cell sorting (FACS) g
g (
)
AD-MSC lines were washed with PBS and stained with
ALP-APC (R&D) (1/50) and CD73-FITC (Biolegend) (1/
160) for 25 min at 4 °C. Upon washing, the cell fractions
(controls sorted, ALP+/CD73+, ALP−/CD73high, ALP
−/CD73low) were sorted with a FACS BD Aria III 5L
and seeded in Nunc™96-well plates (TPP) at a density of
1.2 × 104 cells/cm2 for osteogenic differentiation. Con-
trols sorted were unstained cells processed through the
FACS and collected without sorting specific subpopula-
tions. Differentiation was induced 24 h after seeding. Freshly isolated SVFs were washed with PBS and stained
with ALP-APC (R&D) (1/50), CD73-FITC (Biolegend)
(1/160), and CD45-PE (Biolegend) (1/160) for 25 min at
4 °C. SVF fractions (controls sorted, CD45−/ALP+/CD73+,
CD45−/ALP−/CD73high,
CD45−/ALP−/CD73low)
were
sorted with FACS BD Aria III 5L and plated in vitro at a
density of 1 × 104 in 96-well plates (TPP) for osteogenic dif-
ferentiation. All media were changed every 4 days. Immunohistochemistry and immunofluorescence Paraffin-embedded samples of human fat tissue were se-
lected for immunohistochemical and immunofluores-
cence analysis. Samples were deparaffinized with xylene
and rehydrated by an increasing ethanol gradient for
hematoxylin and eosin (H&E) staining. Target retrieval
was performed using the PT Link (DAKO) at pH solu-
tion 9.0 (DAKO). Immunohistochemistry staining was
performed using a Dako Autostainer Link 48. Primary
antibodies used were as follows: rabbit monoclonal ALP
(Abcam, 1/200), mouse monoclonal CD73 (Abcam, 1/
200), mouse monoclonal CD31 (DAKO, 1/200), and the
appropriate EnVision HRP secondary antibody (EnVi-
sion HRP rabbit or mouse, DAKO, 1/500) according to
the manufacturer’s instruction. Immunofluorescence was
performed using a Dako Autostainer Link 48 with the
following antibodies: rabbit monoclonal ALP (Abcam, 1/
200), mouse monoclonal CD73 (Abcam, 1/200), Alexa
Fluor 488 goat anti-rabbit IgG (Thermo Fisher, 1/200),
and Alexa Fluor 546 goat anti-mouse IgG (Thermo
Fisher, 1/200) according to the manufacturer’s instruc-
tion. Sections were visualized with LEICA DM6600 with
a × 20 magnifying objective lens. Isolation of the stromal vascular fraction In the day of CyTOF acquisition, cells were
washed with MaxPar Fix and Perm Buffer (Flui-
digm) containing Cell-ID Intercalator-IR (Fluidigm)
and incubated at RT for 1 h. Cells were washed
with ddH2O and then diluted in ddH2O with 10%
EQ Calibration Beads (Fluidigm) at 1 million cells/
ml
before
acquisition
with
CyTOF
2
mass
cyt-
ometer (Fluidigm). 45 min in a rotating disk. The reaction was neutralized
with AD-MSC medium and centrifuged at 850g for 10
min. For lysis of the red blood cells, the pellet was incu-
bated for 10 min at RT in 160 mM NH4Cl and then ex-
tensively washed with PBS. The SVF was then filtered
through a 100-μm filter nylon mesh and was either dir-
ectly processed for FACS sorting followed by osteogenic
differentiation, or frozen in AD-MSC medium supple-
mented with 10% DMSO (Sigma). Barcoding strategies for the osteogenic differentiation
experiments Barcoding strategies for the osteogenic differentiation
experiments Mass-tag cellular barcoding g
g
For all CyTOF experiments, the Cell-ID 20-Plex Pd Bar-
coding Kit (Fluidigm) was used following the manufac-
tural instructions. In short, 1 million cells per condition
and per line were washed with PBS and then incubated
with Cell-ID Cisplatin (Fluidigm) for 10 min at RT. Afterwards, cells were fixed with MaxPar Fix Buffer
(Fluidigm) for 10 min at RT, washed with MaxPar
Barcode Perm Buffer (Fluidigm), and incubated with the
appropriate barcode for 30 min at RT. Finally, cells were
washed with Cell Staining Buffer (Fluidigm) and com-
bined depending on the CyTOF experiment in one or
more tubes before antibody staining. Depending on the
planned CyTOF experiment, a specific barcoding strat-
egy was developed in order to minimize technical bias
and highlight biological differences. Page 13 of 16 Page 13 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Barcoding strategy for prediction of differentiation
potential in five new AD-MSC lines Mass cytometry measurements were transformed using
the inverse hyperbolic sine (arcsinh) function with a co-
factor of 5 and subsequently median-centered on a per-
marker basis. Five not yet characterized AD-MSC lines (new AD-
MSCs) together with 9 already characterized AD-MSC
lines (reference) were collected in their undifferentiated
state (day 0). Next, together with one MIX, they were all
barcoded according to the barcode plan (Table S3) and
pooled into one single tube for antibody staining and
CyTOF acquisition as described above. Defining the selected cell subpopulation g
p p
The default CellCNN filter interpretation analysis was
performed to define and characterize the selected cell
subpopulation. Initially, learned filters were clustered
and a single representative filter was retained from each
cluster. As a second step, a score was derived for each
representative filter, measuring how well this filter alone
can classify the validation samples. Only one representa-
tive filter achieved a positive score, and this filter was used
to define the selected cell subpopulation (i.e., cells with
positive score with respect to that filter) in individual mass
cytometry samples at d0, d1, d2, d3, and d4. Statistical analyses y
Quantification of the staining of the triplicates of undif-
ferentiated cells (control) and cells cultured with differ-
entiation
medium
(differentiation)
is
presented
as
mean ± s.d. Quantification of the triplicates of the stain-
ing of the FACS sorted subpopulations is presented as
mean ± s.d. For statistical analyses, the one-way ANOVA
Dunnett’s multiple comparisons test was used to com-
pare the ALP+/CD73+ population with the other sorted
fractions within the same day as well as for comparing
the percentage of ALP+, CD73+, and CD271+ cells in
the “good” category for each day with the same day of
the “intermediate” and “bad” ones. *p ≤0.05, **p ≤0.01,
***p ≤0.001, and **** p ≤0.0001. Pearson’s correlation
was used to determine the correlation between the ALP
frequency measured by CyTOF at days 0, 1, 2, 3, and 4
with the staining intensity measured at days 14, 17, and
21 for the osteogenic differentiation lineage. For this differentiation experiment, we had a total of 102
samples. Thus, having only 20 different barcodes avail-
able, we distributed the barcoded samples into 6 tubes
(Tables S2). In each tube when possible, there was one
“good,” one “bad,” and one “intermediate” line for all the
collected time points. The 17 AD-MSC lines cultured
under osteogenic condition were collected during the
first 5 days (day 0, day 1, day 2, day 3, day 4) of differen-
tiation. At each day, the samples were barcoded, pooled
into the appropriate tube, and stored at 4 °C until day 4. At day 4, a unique antibody master mix was prepared
and distributed into the six tubes. In order to monitor
tube-to-tube variations, we added to each of the six
tubes twice the MIX (PBMCs, HEK, Hela, Jurkat, Saos2,
Nalm6m, SHSY5S, AD-MSCs) for a total of 102 samples
(Tables S2). Stability of the barcoded samples stored at
4 °C during the four collection days was extensively
proved in preliminary tests (data not shown). Barcoding strategy for the passage experiment Barcoding strategy for the passage experiment
AD-MSCs F28, F22, F5, and F14 were cultured in AD-
MSC medium in Nunc™6-cm plates (Thermo Fisher
Scientific) and passaged when 90% confluence was
reached. This was repeated from passage 3 (p3) to pas-
sage 20 (p20). At each passage, part of the cells was fro-
zen
in
AD-MSC
medium
supplemented
with
10%
DMSO (Sigma). All AD-MSC lines from p3 to p20 were
thawed the same day and barcoded according to the bar-
code plan (Table S4). All barcoded passages from the
same cell line were pooled into one tube. Each tube con-
tained also twice a MIX as a control. Cells were stained
with the antibody panel following the protocols men-
tioned above and then processed in CyTOF2 (Fluidigm). Model training
ll CellCNN was trained with the objective to classify
“good” versus “bad” AD-MSC lines from their corre-
sponding mass cytometry measurements at day 0 (undif-
ferentiated state). Training examples (multi-cell inputs)
comprised 2000 cells, sampled uniformly at random
from the original mass cytometry samples. In total, we
sampled 1000 training examples per class (“good” or
“bad” cell lines). For the top-k pooling layer, we consid-
ered values of k such that the ratio of k over the multi-
cell input size would be one of [0.5%, 1%, 3%, 5%]. The
remaining CellCNN parameters were set to their default
values. Data availability through the FACS sorting machine. Depicted are triplicates of undifferen-
tiated cells (control) and cells cultured with osteogenic differentiation
medium (differentiation). Error bars indicate the triplicates of the staining
and are presented as mean ± s.d. Table S1. Mass cytometry antibody
panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the
passage experiment. through the FACS sorting machine. Depicted are triplicates of undifferen-
tiated cells (control) and cells cultured with osteogenic differentiation
medium (differentiation). Error bars indicate the triplicates of the staining
and are presented as mean ± s.d. Table S1. Mass cytometry antibody
panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the
passage experiment. through the FACS sorting machine. Depicted are triplicates of undifferen-
tiated cells (control) and cells cultured with osteogenic differentiation
medium (differentiation). Error bars indicate the triplicates of the staining
and are presented as mean ± s.d. Table S1. Mass cytometry antibody
panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the
passage experiment. Mass cytometry data that support the findings of this
study are available on request from the corresponding
author [P.C.]. Supplementary Information
h
l
l The online version contains supplementary material available at https://doi. org/10.1186/s13287-020-02044-4. The online version contains supplementary material available at https://doi. org/10.1186/s13287-020-02044-4. Mass cytometry data analysis Mass cytometry data.fcs files collected from each set of
samples were normalized using the executable MATLAB
version of the Normalizer tool [63] and concatenated
using the .fcs concatenation tool from Cytobank. Indi-
vidual samples were debarcoded using the executable
MATLAB version of the single-cell debarcoder tool [64]. Page 14 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Authors’ contributions D.D.C., E.A.C., H.C.P., and P.C. conceived the study. D.D.C. and E.A.C. performed all experiments with help from S.M. and B.E. E.A. and M.C. performed the CellCNN analysis. D.D.C., E.A.C., P.C., and V.T. conceived the
antibody panel. V.T. assisted with the CyTOF experimental design. J.B., A.A.B.,
J.A.P., and S.H. provided the adipose tissue samples. D.D.C., E.A.C., and P.C. wrote the manuscript with input from all authors. All authors read and
approved the final manuscript. Blue denotes minimal, green intermediate, and red high expression. Fig
ure S3. Analyses of the osteogenic subpopulation. A) Means of the per-
centage of alkaline phosphatase (ALP) positive cells and CD73 positive
cells in the three AD-MSC categories during the five analyzed days of
osteogenic differentiation (d0, d1, d2, d3, d4). Error bars represent the
mean ± s.d. of the percentage of positive cells present in «good» (n = 6),
«intermediate» (n = 4),and «bad» (n = 7) AD-MSCs. B) Pearson correlations
of the ALP frequency measured by CyTOF at day 0, 1, 2, 3, 4 with the
staining intensities measured at day 14, 17, and 21 for osteogenic differ-
entiation. Red dots represent «good», green «intermediate» (interm.), and
black «bad» differentiating lines. Error bars indicate the triplicates of the
staining and are presented as mean ± s.d. For statistical analyses, the
one-way ANOVA Dunnett’s multiple comparisons test was used to com-
pare each day of the “good” AD-MSCs with the same day of “intermedi-
ate” and “bad” categories: * p≤0.05, ** p≤0.01, *** p ≤0.001, and **** p Acknowledgements The authors thank Yvonne Neldner for technical assistance, and Ines Kleiber-
Schaaf and Andrea Garcete-Bärtschi for paraffin embedding and staining. We
acknowledge assistance provided by the Cytometry Facility of the University
of Zurich, especially Tess Brodie, Paulina Kulig, and Christoph Schwärzler, and
thank Justin Douglas Walter for critically reading the manuscript. This work
was supported by the Gottfried and Julia Bangerter Foundation (to P.C.) and
the Olga Mayenfisch Foundation (to P.C.). Additional file 1: Figure S1. In vitro chondrogenic and adipogenic
categorization of 17 AD-MSCs A) Sum of the pixels acquired at the three
time points (day 14, 17, 21) for chondrogenic (left) and adipogenic (right)
differentiation of all 17 AD-MSC lines and interquartile categorization into
«good», «intermediate», and «bad» AD-MSCs. C) Summary of the
categorization of all 17 AD-MSCs for the three differentiation lineages
(osteogenic, chondrogenic, and adipogenic). interm. = intermediate. Fig-
ure S2. UMAP analyses in the 17 human AD-MSC lines. A) UMAP projec-
tions of all 31 markers in 17 AD-MSC lines. Each dot represents one cell. Blue denotes minimal, green intermediate, and red high expression. Fig-
ure S3. Analyses of the osteogenic subpopulation. A) Means of the per-
centage of alkaline phosphatase (ALP) positive cells and CD73 positive
cells in the three AD-MSC categories during the five analyzed days of
osteogenic differentiation (d0, d1, d2, d3, d4). Error bars represent the
mean ± s.d. of the percentage of positive cells present in «good» (n = 6),
«intermediate» (n = 4),and «bad» (n = 7) AD-MSCs. B) Pearson correlations
of the ALP frequency measured by CyTOF at day 0, 1, 2, 3, 4 with the
staining intensities measured at day 14, 17, and 21 for osteogenic differ-
entiation. Red dots represent «good», green «intermediate» (interm.), and
black «bad» differentiating lines. Error bars indicate the triplicates of the
staining and are presented as mean ± s.d. For statistical analyses, the
one-way ANOVA Dunnett’s multiple comparisons test was used to com-
pare each day of the “good” AD-MSCs with the same day of “intermedi-
ate” and “bad” categories: * p≤0.05, ** p≤0.01, *** p ≤0.001, and **** p
≤0.0001. ns=not significant. Figure S4. ALP+/CD73+ Sorting analysis
and prediction of osteogenic differentiation potential. A) Gating strategy
for FACS sorting for the following subpopulations: ALP+/CD73+, ALP-/
CD73low, and ALP-/CD73high. Acknowledgements B) Alizarin Red staining and quantification
of the sorted subpopulations in four AD- MSC lines (F04, F14, F22, F28)
after 14, 17, and 21 days. Control sorted are unstained cells, which were
run through the FACS sorting machine. Depicted are triplicates of undif-
ferentiated cells (control) and cells cultured with the differentiation
medium (differentiation). Error bars indicate the triplicates of the staining
and are presented as mean ± s.d. C) Categorization of the new AD-MSC
lines (depicted in green) together with all the 17 already analyzed lines,
based on Alizarin Red quantification after 14, 17, and 21 days of osteo-
genic differentiation and interquartile distribution of the five new AD-
MSCs (depicted in violet). D) Alizarin Red staining and quantification of
five new AD-MSCs: two «good» (F08, F26), one «intermediate» (F23), and
two «bad» (F20, F24). Depicted are triplicates of undifferentiated cells
(control) and cells cultured under osteogenic differentiation conditions
(differentiation). Error bars indicate triplicates of the staining and are pre-
sented as mean ± s.d. E) Histograms of median intensities of expression
of selected markers (CD73 and ALP) in F05, F14, F22 and F28 AD-MSC
lines from passage 3 (p3) till passage 20 (p20). Black is the lowest inten-
sity and white represents the highest intensity. F) Alizarin Red staining
and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21
days of osteogenic differentiation. Depicted are triplicates of undifferenti-
ated cells (control) and cells cultured under osteogenic differentiation
medium (differentiation). Error bars indicates the triplicates of the staining
and are presented as mean ± s.d. Figure S5. ALP+/CD73+ cells are
present in the human fat tissue and stromal vascular fraction A)
Hematoxylin/Eosin (H&E) and immunohistochemistry staining of human
fat tissue for ALP, CD73, and CD31. Scale 100 μm. B) Gating strategy for
sorting the selected subpopulations (CD45- /ALP+/CD73+, CD45-/ALP-/
CD73low, CD45-/ALP-/CD73high) in the SVFs. C) Alizarin Red staining and
pixel quantification of sorted SVF fractions (CD45-/ALP+/CD73+, CD45-/
ALP /CD73low CD45 /ALP /CD73high) after 21 days of osteogenic differ Author details
1 B) Gating strategy for
sorting the selected subpopulations (CD45- /ALP+/CD73+, CD45-/ALP-/
CD73low, CD45-/ALP-/CD73high) in the SVFs. C) Alizarin Red staining and
pixel quantification of sorted SVF fractions (CD45-/ALP+/CD73+, CD45-/
ALP-/CD73low, CD45-/ALP- /CD73high) after 21 days of osteogenic differ-
entiation in vitro. Control sorted are unstained SVFs, which were run Received: 7 September 2020 Accepted: 23 November 2020 Author details
1 1Department of Trauma, University Hospital Zurich, Rämistrasse 100, 8091
Zurich, Switzerland. 2Life Science Zurich Graduate School, University of
Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. 3Department of
Biology, Institute of Molecular Systems Biology, ETH Zurich, Otto-Stern-Weg
3, 8093 Zurich, Switzerland. 4Mass Cytometry Facility, University of Zurich,
Winterthurerstrasse 190, 8057 Zurich, Switzerland. 5Department of Plastic and
Hand Surgery, University Hospital Zurich, Rämistrasse 100, 8091 Zurich,
Switzerland. ate and bad categories:
p≤0.05,
p≤0.01,
p ≤0.001, and
p
≤0.0001. ns=not significant. Figure S4. ALP+/CD73+ Sorting analysis
and prediction of osteogenic differentiation potential. A) Gating strategy
for FACS sorting for the following subpopulations: ALP+/CD73+, ALP-/
CD73low, and ALP-/CD73high. B) Alizarin Red staining and quantification
of the sorted subpopulations in four AD- MSC lines (F04, F14, F22, F28)
after 14, 17, and 21 days. Control sorted are unstained cells, which were
run through the FACS sorting machine. Depicted are triplicates of undif-
ferentiated cells (control) and cells cultured with the differentiation
medium (differentiation). Error bars indicate the triplicates of the staining
and are presented as mean ± s.d. C) Categorization of the new AD-MSC
lines (depicted in green) together with all the 17 already analyzed lines,
based on Alizarin Red quantification after 14, 17, and 21 days of osteo-
genic differentiation and interquartile distribution of the five new AD-
MSCs (depicted in violet). D) Alizarin Red staining and quantification of
five new AD-MSCs: two «good» (F08, F26), one «intermediate» (F23), and
two «bad» (F20, F24). Depicted are triplicates of undifferentiated cells
(control) and cells cultured under osteogenic differentiation conditions
(differentiation). Error bars indicate triplicates of the staining and are pre-
sented as mean ± s.d. E) Histograms of median intensities of expression
of selected markers (CD73 and ALP) in F05, F14, F22 and F28 AD-MSC
lines from passage 3 (p3) till passage 20 (p20). Black is the lowest inten-
sity and white represents the highest intensity. F) Alizarin Red staining
and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21
days of osteogenic differentiation. Depicted are triplicates of undifferenti-
ated cells (control) and cells cultured under osteogenic differentiation
medium (differentiation). Error bars indicates the triplicates of the staining
and are presented as mean ± s.d. Figure S5. ALP+/CD73+ cells are
present in the human fat tissue and stromal vascular fraction A)
Hematoxylin/Eosin (H&E) and immunohistochemistry staining of human
fat tissue for ALP, CD73, and CD31. Scale 100 μm. References
h van de Peppel J, et al. Identification of three early phases of cell-fate
determination during osteogenic and adipogenic differentiation by
transcription factor dynamics. Stem Cell Reports. 2017;8:947–60. https://doi. org/10.1016/j.stemcr.2017.02.018. 19. Kobolak J, Dinnyes A, Memic A, Khademhosseini A, Mobasheri A. Mesenchymal stem cells: identification, phenotypic characterization,
biological properties and potential for regenerative medicine through
biomaterial micro-engineering of their niche. Methods. 2016;99:62–8. https://doi.org/10.1016/j.ymeth.2015.09.016. 43. Huang AH, Farrell MJ, Mauck RL. Mechanics and mechanobiology of
mesenchymal stem cell-based engineered cartilage. J Biomech. 2010;43:
128–36. https://doi.org/10.1016/j.jbiomech.2009.09.018. 20. Dominici M, et al. Minimal criteria for defining multipotent mesenchymal
stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–7. https://doi.org/10.1080/14653240600855905. 44. Wang J, Liao L, Wang S, Tan J. Cell therapy with autologous mesenchymal
stem cells-how the disease process impacts clinical considerations. Cytotherapy. 2013;15:893–904. https://doi.org/10.1016/j.jcyt.2013.01.218. 21. Dicker A, et al. Functional studies of mesenchymal stem cells derived from
adult human adipose tissue. Exp Cell Res. 2005;308:283–90. https://doi.org/
10.1016/j.yexcr.2005.04.029. 45. D'Ippolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-related
osteogenic potential of mesenchymal stromal stem cells from human
vertebral bone marrow. J Bone Miner Res. 1999;14:1115–22. https://doi.org/
10.1359/jbmr.1999.14.7.1115. 22. Festy F, et al. Surface protein expression between human adipose tissue-
derived stromal cells and mature adipocytes. Histochem Cell Biol. 2005;124:
113–21. https://doi.org/10.1007/s00418-005-0014-z. 46. Katsara O, et al. Effects of donor age, gender, and in vitro cellular aging on
the phenotypic, functional, and molecular characteristics of mouse bone
marrow-derived mesenchymal stem cells. Stem Cells Dev. 2011;20:1549–61. https://doi.org/10.1089/scd.2010.0280. 23. Gronthos S, et al. Surface protein characterization of human adipose tissue-
derived stromal cells. J Cell Physiol. 2001;189:54–63. https://doi.org/10.1002/
jcp.1138. 47. Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated with
decreased maximal life span and accelerated senescence of bone marrow
stromal cells. Bone. 2003;33:919–26. 24. Haniffa MA, et al. Adult human fibroblasts are potent immunoregulatory
cells and functionally equivalent to mesenchymal stem cells. J Immunol. 2007;179:1595–604. https://doi.org/10.4049/jimmunol.179.3.1595. 25. Mitchell JB, et al. Immunophenotype of human adipose-derived cells:
temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006;24:376–85. https://doi.org/10.1634/stemcells.2005-0234. 48. Bradaschia-Correa V, et al. Ecto-5′-nucleotidase (CD73) regulates bone
formation and remodeling during intramembranous bone repair in aging
mice. Tissue Cell. 2017;49:545–51. https://doi.org/10.1016/j.tice.2017.07.001. 26. Varma MJ, et al. Phenotypical and functional characterization of freshly
isolated adipose tissue-derived stem cells. Stem Cells Dev. 2007;16:91–104. https://doi.org/10.1089/scd.2006.0026. 49. Feldbush TL, Lafrenz D. References
h Multilineage cells from human adipose tissue: implications for
cell-based therapies. Tissue Eng. 2001;7:211–28. https://doi.org/10.1089/
107632701300062859. 11. Kuznetsov SA, et al. Single-colony derived strains of human marrow stromal
fibroblasts form bone after transplantation in vivo. J Bone Miner Res. 1997;
12:1335–47. https://doi.org/10.1359/jbmr.1997.12.9.1335. 35. Eggerschwiler B, Canepa DD, Pape HC, Casanova EA, Cinelli P. Automated
digital image quantification of histological staining for the analysis of the
trilineage differentiation potential of mesenchymal stem cells. Stem Cell Res
Ther. 2019;10:69. https://doi.org/10.1186/s13287-019-1170-8. 12. McLeod CM, Mauck RL. On the origin and impact of mesenchymal stem
cell heterogeneity: new insights and emerging tools for single cell analysis. Eur Cell Mater. 2017;34:217–31. https://doi.org/10.22203/eCM.v034a14. Ther. 2019;10:69. https://doi.org/10.1186/s13287-019-1170-8. 36. Nassar AF, Ogura H, Wisnewski AV. Impact of recent innovations in the use
of mass cytometry in support of drug development. Drug Discov Today. 2015;20:1169–75. https://doi.org/10.1016/j.drudis.2015.06.001. 13. Post S, Abdallah BM, Bentzon JF, Kassem M. Demonstration of the presence
of independent pre-osteoblastic and pre-adipocytic cell populations in
bone marrow-derived mesenchymal stem cells. Bone. 2008;43:32–9. https://
doi.org/10.1016/j.bone.2008.03.011. 37. Bendall SC, et al. Single-cell mass cytometry of differential immune and
drug responses across a human hematopoietic continuum. Science. 2011;
332:687–96. https://doi.org/10.1126/science.1198704. 14. Selich A, et al. Massive clonal selection and transiently contributing clones
during expansion of mesenchymal stem cell cultures revealed by lentiviral
RGB-barcode technology. Stem Cells Transl Med. 2016;5:591–601. https://doi. org/10.5966/sctm.2015-0176. 38. Amir el AD, et al. viSNE enables visualization of high dimensional single-cell
data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol. 2013;31:545–52. https://doi.org/10.1038/nbt.2594. 15. Phinney DG, et al. Donor variation in the growth properties and osteogenic
potential of human marrow stromal cells. J Cell Biochem. 1999;75:424–36. 39. Ferrell PB Jr, et al. High-dimensional analysis of acute myeloid leukemia
reveals phenotypic changes in persistent cells during induction therapy. PLoS One. 2016;11:e0153207. https://doi.org/10.1371/journal.pone.0153207. 16. Phinney DG. Functional heterogeneity of mesenchymal stem cells:
implications for cell therapy. J Cell Biochem. 2012;113:2806–12. https://doi. org/10.1002/jcb.24166. 40. Hamers AAJ, et al. Human monocyte heterogeneity as revealed by high-
dimensional mass cytometry. Arterioscler Thromb Vasc Biol. 2019;39:25–36. https://doi.org/10.1161/ATVBAHA.118.311022. 17. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast
colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970;3:393–403. 41. Galli E, et al. GM-CSF and CXCR4 define a T helper cell signature in multiple
sclerosis. Nat Med. 2019;25:1290–300. https://doi.org/10.1038/s41591-019-0521-4. 18. Pittenger MF, et al. Multilineage potential of adult human mesenchymal
stem cells. Science. 1999;284:143–7. 42. References
h 1. Bigham AS, Dehghani SN, Shafiei Z, Torabi Nezhad S. Xenogenic
demineralized bone matrix and fresh autogenous cortical bone effects on
experimental bone healing: radiological, histopathological and
biomechanical evaluation. J Orthop Traumatol. 2008;9:73–80. https://doi.org/
10.1007/s10195-008-0006-6. 2. Ikada Y. Challenges in tissue engineering. J R Soc Interface. 2006;3:589–601. https://doi.org/10.1098/rsif.2006.0124. 3. Dai R, Wang Z, Samanipour R, Koo KI, Kim K. Adipose-derived stem cells for
tissue engineering and regenerative medicine applications. Stem Cells Int. 2016;2016:6737345. https://doi.org/10.1155/2016/6737345. 4. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells
reside in virtually all post-natal organs and tissues. J Cell Sci. 2006;119:2204–
13. https://doi.org/10.1242/jcs.02932. 5. Ntege EH, Sunami H, Shimizu Y. Advances in regenerative therapy: a review
of the literature and future directions. Regen Ther. 2020;14:136–53. https://
doi.org/10.1016/j.reth.2020.01.004. 6. Gao S, et al. Proliferation of ASC-derived endothelial cells in a 3D
electrospun mesh: impact of bone-biomimetic nanocomposite and co-
culture with ASC-derived osteoblasts. Injury. 2014;45:974–80. https://doi.org/
10.1016/j.injury.2014.02.035. 7. Konig MA, et al. Direct transplantation of native pericytes from adipose
tissue: a new perspective to stimulate healing in critical size bone defects. Cytotherapy. 2016;18:41–52. https://doi.org/10.1016/j.jcyt.2015.10.002. 7. Konig MA, et al. Direct transplantation of native pericytes from adipose
tissue: a new perspective to stimulate healing in critical size bone defects. Cytotherapy. 2016;18:41–52. https://doi.org/10.1016/j.jcyt.2015.10.002. 8. Groninger O, et al. Directing stem cell commitment by amorphous calcium
phosphate nanoparticles incorporated in PLGA: relevance of the free
calcium ion concentration. Int J Mol Sci. 2020;21. https://doi.org/10.3390/
ijms21072627. Page 15 of 16 Page 15 of 16 Page 15 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 (2021) 12:7 9. Sordi MB, Cabral da Cruz AC, Aragones A, Rodriguez Cordeiro MM, de Souza
Magini R. PLGA+HA/betaTCP scaffold incorporating simvastatin: a promising
biomaterial for bone tissue engineering. J Oral Implantol. 2020. https://doi. org/10.1563/aaid-joi-D-19-00148. 31. Zhu Y, et al. Adipose-derived stem cell: a better stem cell than BMSC. Cell
Biochem Funct. 2008;26:664–75. https://doi.org/10.1002/cbf.1488. 32. Becht E, et al. Dimensionality reduction for visualizing single-cell data using
UMAP. Nat Biotechnol. 2018. https://doi.org/10.1038/nbt.4314. 10. De Luca A, et al. Improvement of osteogenic differentiation of human
mesenchymal stem cells on composite poly l-lactic acid/nano-
hydroxyapatite scaffolds for bone defect repair. J Biosci Bioeng. 2020;129:
250–7. https://doi.org/10.1016/j.jbiosc.2019.08.001. 33. Arvaniti E, Claassen M. Sensitive detection of rare disease-associated cell
subsets via representation learning. Nat Commun. 2017;8:14825. https://doi. org/10.1038/ncomms14825. 34. Zuk PA, et al. References
h MicroRNA-21 preserves the fibrotic mechanical memory of
mesenchymal stem cells. Nat Mater. 2017;16:379–89. https://doi.org/10.1038/
nmat4780. 58. Li CX, et al. MicroRNA-21 preserves the fibrotic mechanical memory of
mesenchymal stem cells. Nat Mater. 2017;16:379–89. https://doi.org/10.1038/
nmat4780. 59. Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity
which inhibits the differentiation of murine embryonal carcinoma and
embryonic stem cells. Dev Biol. 1987;121:1–9. https://doi.org/10.1016/0012-
1606(87)90132-1. 59. Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity
which inhibits the differentiation of murine embryonal carcinoma and
embryonic stem cells. Dev Biol. 1987;121:1–9. https://doi.org/10.1016/0012-
1606(87)90132-1. 60. Williams RL, et al. Myeloid leukaemia inhibitory factor maintains the
developmental potential of embryonic stem cells. Nature. 1988;336:684–7. https://doi.org/10.1038/336684a0. 61. Ying QL, et al. The ground state of embryonic stem cell self-renewal. Nature. 2008;453:519–23. https://doi.org/10.1038/nature06968. 61. Ying QL, et al. The ground state of embryonic stem cell self-renewal. Nature. 2008;453:519–23. https://doi.org/10.1038/nature06968. 62. Buschmann J, et al. Yield and proliferation rate of adipose-derived stromal
cells as a function of age, body mass index and harvest site-increasing the
yield by use of adherent and supernatant fractions? Cytotherapy. 2013;15:
1098–105. https://doi.org/10.1016/j.jcyt.2013.04.009. 63. Finck R, et al. Normalization of mass cytometry data with bead standards. Cytometry A. 2013;83:483–94. https://doi.org/10.1002/cyto.a.22271. 63. Finck R, et al. Normalization of mass cytometry data with bead standards. Cytometry A. 2013;83:483–94. https://doi.org/10.1002/cyto.a.22271. 64. Zunder ER, et al. Palladium-based mass tag cell barcoding with a doublet-
filtering scheme and single-cell deconvolution algorithm. Nat Protoc. 2015;
10:316–33. https://doi.org/10.1038/nprot.2015.020. References
h Alkaline phosphatase on activated B cells
characterization of the expression of alkaline phosphatase on activated B
cells. Kinetics and membrane anchor. J Immunol. 1991;147:3690–5. 50. Rohwedel J, Sehlmeyer U, Shan J, Meister A, Wobus AM. Primordial germ cell-
derived mouse embryonic germ (EG) cells in vitro resemble undifferentiated
stem cells with respect to differentiation capacity and cell cycle distribution. Cell Biol Int. 1996;20:579–87. https://doi.org/10.1006/cbir.1996.0076. 27. Wagner W, et al. Comparative characteristics of mesenchymal stem cells
from human bone marrow, adipose tissue, and umbilical cord blood. Exp
Hematol. 2005;33:1402–16. https://doi.org/10.1016/j.exphem.2005.07.003. 28. Yoshimura K, et al. Characterization of freshly isolated and cultured cells
derived from the fatty and fluid portions of liposuction aspirates. J Cell
Physiol. 2006;208:64–76. https://doi.org/10.1002/jcp.20636. 51. Yegutkin GG. Nucleotide- and nucleoside-converting ectoenzymes:
important modulators of purinergic signalling cascade. Biochim Biophys
Acta. 2008;1783:673–94. https://doi.org/10.1016/j.bbamcr.2008.01.024. 29. Yanez R, et al. Adipose tissue-derived mesenchymal stem cells have in vivo
immunosuppressive properties applicable for the control of the graft-
versus-host disease. Stem Cells. 2006;24:2582–91. https://doi.org/10.1634/
stemcells.2006-0228. 52. Grol MW, Panupinthu N, Korcok J, Sims SM, Dixon SJ. Expression, signaling,
and function of P2X7 receptors in bone. Purinergic Signal. 2009;5:205–21. https://doi.org/10.1007/s11302-009-9139-1. 53. Orriss IR, Burnstock G, Arnett TR. Purinergic signalling and bone
remodelling. Curr Opin Pharmacol. 2010;10:322–30. https://doi.org/10.1016/j. coph.2010.01.003. 30. Tapp H, Hanley EN Jr, Patt JC, Gruber HE. Adipose-derived stem cells:
characterization and current application in orthopaedic tissue repair. Exp
Biol Med (Maywood). 2009;234:1–9. https://doi.org/10.3181/0805/MR-170. Page 16 of 16 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 Canepa et al. Stem Cell Research & Therapy (2021) 12:7 54. Buckley KA, Golding SL, Rice JM, Dillon JP, Gallagher JA. Release and
interconversion of P2 receptor agonists by human osteoblast-like cells. FASEB J. 2003;17:1401–10. https://doi.org/10.1096/fj.02-0940com. 55. Dellavalle A, et al. Pericytes of human skeletal muscle are myogenic
precursors distinct from satellite cells. Nat Cell Biol. 2007;9:255–67. https://
doi.org/10.1038/ncb1542. 56. Gerlach JC, et al. Perivascular mesenchymal progenitors in human fetal and
adult liver. Stem Cells Dev. 2012;21:3258–69. https://doi.org/10.1089/scd. 2012.0296. 56. Gerlach JC, et al. Perivascular mesenchymal progenitors in human fetal and
adult liver. Stem Cells Dev. 2012;21:3258–69. https://doi.org/10.1089/scd. 2012.0296. 57. Yang C, Tibbitt MW, Basta L, Anseth KS. Mechanical memory and dosing
influence stem cell fate. Nat Mater. 2014;13:645–52. https://doi.org/10.1038/
nmat3889. 57. Yang C, Tibbitt MW, Basta L, Anseth KS. Mechanical memory and dosing
influence stem cell fate. Nat Mater. 2014;13:645–52. https://doi.org/10.1038/
nmat3889. 58. Li CX, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
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English
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The Intensity of the Plasmon–exciton of Three Spherical Metal Nanoparticles On the Semiconductor Quantum Dot Having Three External Fields
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Plasmonics
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Abstractl The influence of the plasmon of three spherical metal nanoparticles (MNPs) on the semiconductor quantum dot (SQD) having
three external fields is analyzed. The density matrix equations are modified for the description of the optical properties of the
SQD-MNPs nanosystem. We study theoretically the role of the plasmon–exciton dipole coupling in the SQD-MNPs nanosys-
tem. We investigate the dependence of the plasmon–exciton dipole coupling of the SQD-MNPs nanosystem on the position
of three spherical MNPs with respect to SQD as well as on the material parameters of the hybrid nanosystem. The direction
and detunings of the three external fields play an important role in the characterization of the SQD-MNPs nanosystem. Keywords Quantum dot · Plasmon · Dipole–dipole interactions https://doi.org/10.1007/s11468-022-01649-0
Plasmonics (2022) 17:1633–1644 https://doi.org/10.1007/s11468-022-01649-0
Plasmonics (2022) 17:1633–1644 The Intensity of the Plasmon–exciton of Three Spherical Metal
Nanoparticles On the Semiconductor Quantum Dot Having Three
External Fields Hagar M. Ali1 · Somia Abd‑Elnabi1 · Kariman I. Osman1 Received: 10 January 2022 / Accepted: 25 April 2022
© The Author(s) 2022
/ Published online: 27 May 2022 Introduction [17]. Investigating the spatial properties of coherent plas-
monic (CP) field and demonstrating how it depends on the
collective molecular states of the SQD-MNP system (bright
and dark states) are shown in [18, 19] that when the coherent
SQD-MNP molecule is in the dark state, i.e., the SQD does
not emit light, the CP field is spatially confined around the
MNP. It is studied that there were the states of polarization
of coherent plasmonic fields of a SQD-MNP system in the
environment surrounding the MNP and investigated how
the dynamics of these states were evolved with time when
this system was interacting with a time-dependent laser field
[20–22]. Plasmon-assisted two-photon Rabi oscillations in a
semiconductor quantum dot—metal nanoparticle heterodi-
mer are investigated in [23]. The demonstration of multipole
effect and the intensity of the plasmon–exciton dipole cou-
pling in the SQD-MNP nanosystem are shown in [24–27]. The optical properties of complex nanosystems, that when
a semiconductor quantum dot (SQD) is in the vicinity of a
metallic nanoparticle (MNP), are the area of considerable
current interest. So, the optics of the SQD become strongly
sensitive to the structural parameters of the nanosystem,
the intensity of the plasmon–exciton dipole coupling and
the dielectric constant of the environment when combining
semiconductor quantum dot (SQD) and plasmonic nano-
structures, and hence, it is carried out in the interdiscipli-
nary applications of nanoscience. The phenomena that have
been studied in these research areas are the phase control of
absorption and dispersion as well complete optical trans-
parency [1–4], Fano effects in energy absorption [5–8],
plasmonic electromagnetically induced transparency (EIT)
[9–12], the enhancement of nonlinear Kerr and suscepti-
bilities in several quantum systems [13–16]. The terahertz
generation enhancement from intraband transition in self-
assembled SQD molecules near a (MNP) is discussed in In this paper, we consider a single SQD in close vicinity
to three spherical MNPs. The present scheme is based
on a coupled SQD-MNPs nanosystem in the presence of
the pump, control and probe fields. The SQD is taken as
a four-level V-type system in which the distinct excitonic
transitions occur. Theoretically, we will study the effect
of the plasmon–exciton dipole coupling in the SQD-
MNP nanosystem and investigate the dependence of
the plasmon–exciton dipole coupling of the SQD-MNP
nanosystem on the position of MNPs with a SQD as well as
on the material parameters of the hybrid nanosystem. * Somia Abd‑Elnabi
somiaabdelnabi@Azhar.edu.eg 1
Department of Mathematics, Faculty of Science
and Women’s Section, Al-Azhar University, Cairo,
Nasr City, 11754, Egypt Introduction The 1
Department of Mathematics, Faculty of Science
and Women’s Section, Al-Azhar University, Cairo,
Nasr City, 11754, Egypt (0121 1634 Plasmonics (2022) 17:1633–1644 R1 , R2 and R3 . The schematic diagram of the nanosystem
is considered as a V-type four-level SQD structure. It is
composed of four states 1⟩ , 2⟩ , 3⟩ and 4⟩ with energies
ℏ𝜔1 , ℏ𝜔2 , ℏ𝜔3 and ℏ𝜔4 , respectively, as illustrated in
Fig. 1. The nanohybrid structure is subjected to the pump,
probe and control fields with amplitudes E2, E3 and E4 and
frequencies 휈2 , 휈3 and 휈4 , respectively. The weak probe
field derives the excitonic transition 1⟩↔3⟩ with reso-
nance frequency 휔13 , where 휔nm = 휔n −휔m and
( n, m = 1, 2, 3, 4) . The pump and control fields derive the
excitonic transitions 1⟩↔2⟩ and 3⟩↔4⟩ with reso-
nance frequencies 휔12 and 휔34 , respectively. The SQD is
situated at center-to-center distance r1, r2 and r3 from the paper is organized as follows: in Section 2, we describe the
SQD-MNP nanosystem, derive the density matrix equations
describing the dynamics of the system and obtain the form
of the plasmon–exciton dipole coupling for the SQD-MNP
nanosystem. In Section 3, we discuss our numerical results. Finally, we present our conclusions in Section 4. Theoretical Model and Description In this paper, we theoretically investigate coherent
light–matter interaction in a nanohybrid between a small
size of SQD and three spherical MNPs of different radii 1
Fig. 1 A schematic diagram of the SQD and three MNPs (hybrid system). SQD have four-level V-type configuration coupling with three fields Fig. 1 A schematic diagram of the SQD and three MNPs (hybrid system). SQD have four-level V-type configurati Fig. 1 A schematic diagram of the SQD and three MNPs (hybrid system). SQD have four-level V-type configuration coupling with three fields Plasmonics (2022) 17:1633–1644 1635 first, second and third spherical MNP, respectively. The
distance rj has an angle 휃j ( j = 1, 2, 3 ) with respect to the
Z− axis for the first, second and third spherical MNPs as
illustrated in Fig. 1, respectively. We consider a first spher-
ical MNP is positioned at center-to-center distance r12 and
r13 from the second and third spherical MNP, respectively,
while the second spherical MNP is at center-to-center dis-
tance r23 from the third spherical MNP. The excitonic tran-
sitions for the SQD 1⟩↔2⟩ , 1⟩↔3⟩ and 3⟩↔4⟩ are
characterized by the transition dipole moments 휇12 , 휇13
and 휇34 , respectively, where the optical excitations in the
SQD are excitons and the oscillating external fields give
rise to oscillations of conducting electrons in the MNP’s,
conventionally called localized surface plasmon (LSP). So,
excitons and plasmons are excited in the nanohybrid and
interact with each other via the dipole–dipole interaction,
which give rise to a renormalization of the field experi-
enced by both the SQD and MNPs. The dielectric constant
of the SQD is represented by 휖s , and it is surrounded by a
material with dielectric constant 휖B , while the three MNPs
are treating as classical dielectric particles with dielectric
function 휖mj(휔) and mj stands for MNP j where (j = 1, 2, 3) . The dielectric function 휖mj(휔) is obtained for the spherical
MNP as [28]: ( 퐄3 ) is along the Y-axis. 퐄ij
QD are the fields on SQD from
the three MNPs and given by: ( 퐄3 ) is along the Y-axis. Theoretical Model and Description 퐄ij
QD are the fields on SQD from
the three MNPs and given by: (4)
퐄ij
SQD =
1
4휋휖0휖Br3
j
[3(퐩ij⋅̂퐫j)̂퐫j −퐩ij
] (4) where the unite vector ̂퐫j along the vector 퐫j is given by: (5)
̂퐫j = cos 휃ĵZ + sin 휃ĵY
(j = 1, 2, 3). (5) The vector dipoles 퐩ij originate from the charge induced on
the surface of the MNPs and direct in the Z-axis, which is
given by: (6)
퐩ij = 훼j퐄ij, 훼j =
4휋휖0휖BR3
j 훾j
휀effmj
, 훾j =
휖mj(휔) −휖B
휖mj(휔) + 2휖B (6) where 퐄ij (for i = 2, 3, 4 & j = 1, 2, 3 ) are the fields acting on
the three MNPs and given by: (7)
퐄ij =
1
휀effmj
[
퐄i + 퐄SQD
ij
+ 퐄ik
ij + 퐄il
ij
] (7) mj =
휖mj(휔)+2휖B
3휖B where 휀effmj =
휖mj(휔)+2휖B
3휖B
is the screening of the dielectric
material of the three spherical MNPs. 퐄SQD
ij
(for i = 2, 3, 4 &
j = 1, 2, 3 ) are the fields from the SQD on the three MNPs
and given by: where 휀effmj =
휖mj(휔)+2휖B
3휖B
is the screening of the dielectric
material of the three spherical MNPs. 퐄SQD
ij
(for i = 2, 3, 4 &
j = 1, 2, 3 ) are the fields from the SQD on the three MNPs
and given by: (1)
휖mj(휔) = 1 −
휔2
pj
휔2 + i훾bj휔
(j = 1, 2, 3) (1)
휖mj(휔) = 1 −
휔2
pj
휔2 + i훾bj휔
(j = 1, 2, 3) (1) (8)
퐄SQD
ij
=
1
4휋휖0휖Br3
j
[
3(퐩i
SQD.̂퐫j)̂퐫j −퐩i
SQD
]
, i = q, 4 (8) where 휔pj is the plasma frequency for spherical MNP and 훾bj
is the damping constant. The Hamiltonian of the nanohybrid
system can be expressed as: HSQD = ℏ
4
∑
j=1
𝜔j𝜎jj −
[
훍12.퐄2
SQD𝜎12 + 훍13.퐄3
SQD훔13 + 훍34.퐄4
SQD𝜎34 + H.C. ] (2) (9)
pq
SQD = 휇1q(휌1q + 휌q1),
q = 2, 3
(10)
p4
SQD = 휇34(휌34 + 휌34),
i = 4. where 휎ij =∣i⟩⟨ j ∣ is the dipole transition operator between∣i⟩
and ⟨ j ∣ of the SQD. 퐄i
SQD (i = 2, 3, 4) are the fields felt by
the SQD polarized along the ∣1⟩⟺ ∣2⟩ , ∣1⟩⟺ ∣3⟩ and
∣3⟩⟺ ∣4⟩ transitions, respectively. Theoretical Model and Description (13) where: where: (14)
Ωeff
2 = [Ω2(Ψ2 + Γ2) + Λ2휌12
]
,
(15)
Ωeff
3 = [Ω3(Ψ3 + Γ3) + Λ3휌13
]
,
(16)
Ωeff
4 = [Ω4(Ψ4 + Γ4) + Λ4휌34
]
. (14) For the case (ZYZ), we have the same above equations
[14–27], but the subscript (q = 2, 3) in Ψq, Γq and Λq can
be exchanged to the subscript (q = 2, 4) and the subscript 4
in Ψ4, Γ4 and Λ4 can be exchanged to the subscript 3 (i.e.,
the value of Ωeff
3 and Ωeff
4 exchange). For another special
case (ZZY) under the condition 훼1 = 훼2 = 훼3 = 훼 and 휃1 = 0,
휃2 = 휋∕2, 휃3 = 휋, we can get the property Ωeff
4 = Ω4 , and
this means that the main factor for obtain this result is the
direction of the fields. For the case (ZYZ), we have the same above equations
[14–27], but the subscript (q = 2, 3) in Ψq, Γq and Λq can
be exchanged to the subscript (q = 2, 4) and the subscript 4
in Ψ4, Γ4 and Λ4 can be exchanged to the subscript 3 (i.e.,
the value of Ωeff
3 and Ωeff
4 exchange). For another special
case (ZZY) under the condition 훼1 = 훼2 = 훼3 = 훼 and 휃1 = 0,
휃2 = 휋∕2, 휃3 = 휋, we can get the property Ωeff
4 = Ω4 , and
this means that the main factor for obtain this result is the
direction of the fields. (15) For the case (ZZY), we have: (17)
Ω2 = 𝜇12E2
ℏ𝜀effs
, Ω3 = 𝜇13E3
ℏ𝜀effs
, Ω4 = 𝜇34E4
ℏ𝜀effs
,
(18)
Ψq = (1 +
3
∑
j=1
훼jAj),
(q = 2, 3)
(19)
Ψ4 = (1 +
3
∑
j=1
훼jCj),
this means that the main factor for obtain this result
direction of the fields. Theoretical Model and Description So, we have : pq
SQD = 휇1q(휌1q + 휌q1),
q = 2, 3 (9) (10)
p4
SQD = 휇34(휌34 + 휌34),
i = 4. (10) (3)
퐄i
SQD =
1
휀effs
[
퐄i +
3
∑
j=1
퐄ij
SQD
]
,
(i = 2, 3, 4) Also, the fields 퐄ik
ij and 퐄il
ij result in the interaction between
two polarized MNPs for ( j, k, l = 1, 2, 3 and
j ≠k ≠l, i = 2, 3, 4) and are given by: (3) where, 휀effs = [ 휖s+2휖B
3휖B ] is the screening of the dielectric mate-
rial of SQD. (11)
퐄ik
ij =
1
4휋휖0휖Br3
jk
[3(퐩ik.̂퐫jk)̂퐫jk −퐩ik
], ,
(12)
퐄il
ij =
1
4휋휖0휖Br3
jl
[3(퐩il.̂퐫jl)̂퐫jl −퐩il
] (11)
퐄ik
ij =
1
4휋휖0휖Br3
jk
[3(퐩ik.̂퐫jk)̂퐫jk −퐩ik
], , (11) Supposing that we have two cases for the direction of
the fields ( 퐄i ), the first case (ZZY) that means the direction
of the fields ( 퐄2, 퐄3 ) is along the Z-axis and the field ( 퐄4 )
is along the Y −axisand the second case (ZYZ): the direc-
tion of the fields ( 퐄2, 퐄4 ) is along the Z −axis and the field (12)
퐄il
ij =
1
4휋휖0휖Br3
jl
[3(퐩il.̂퐫jl)̂퐫jl −퐩il
] (12) 1 3 3 Plasmonics (2022) 17:1633–1644 1636 (25)
S1 = {3[(r2 cos 휃2 −r1 cos 휃1)∕r21]2 −1}∕4휋휖o휖Br3
21
S2 = {3[(r3 cos 휃3 −r2 cos 휃2)∕r32]2 −1}∕4휋휖o휖Br3
32
S3 = {3[(r3 cos 휃3 −r1 cos 휃1)∕r31]2 −1}∕4휋휖o휖Br3
31 (25)
S1 = {3[(r2 cos 휃2 −r1 cos 휃1)∕r21]2 −1}∕4휋휖o휖Br3
21
S2 = {3[(r3 cos 휃3 −r2 cos 휃2)∕r32]2 −1}∕4휋휖o휖Br3
32
S3 = {3[(r3 cos 휃3 −r1 cos 휃1)∕r31]2 −1}∕4휋휖o휖Br3
31 where ̂퐫jk = ̂퐫k −̂퐫j and ̂퐫jl = ̂퐫l −̂퐫j . By introducing 퐄i
SQD for
(i = 2, 3, 4) into Eq. (2), then the total Hamiltonian of the
SQD is expressed as: (25) where:
(13)
HSQD = ℏ
4
∑
j=1
𝜔j𝜎jj −ℏΩeff
2 𝜎12 −ℏΩeff
3 𝜎13 −ℏΩeff
4 𝜎34 + H.C. (26)
T1 = 3[r2 cos 휃2 −r1 cos 휃1][r2 sin 휃2 −r1 sin 휃1]∕4휋휖o휖Br5
21
T2 = 3[r3 cos 휃3 −r2 cos 휃2][r3 sin 휃3 −r2 sin 휃2]∕4휋휖o휖Br5
32
T3 = 3[r3 cos 휃3 −r1 cos 휃1][r3 sin 휃3 −r1 sin 휃1]∕4휋휖o휖Br5
31. Theoretical Model and Description Δ3 = 휈3 −휔31 is the frequency detuning for the i
(39)
훽5 = (훾2 + 훾3) −i(Δ2 −Δ3)
(40)
훽6 = (훾2 + 훾4) −i(Δ2 −Δ3 −Δ4)
(41)
훽7 = (훾3 + 훾4) + iΔ4 with the identity
4∑
n=1
휌nn = 1 and
(33)
⋅휌32 = −훽5휌32 −i Ωeff∗
2 휌31 + i Ωeff
3 휌12 + i Ωeff∗
4 휌42
(34)
⋅휌42 = −훽6휌42 −i Ωeff∗
2 휌41 + i Ωeff
4 휌32
(35)
⋅휌43 = −훽7휌43 −i Ωeff∗
3 휌41 −i Ωeff
4 ( 휌44 −휌33)
(36)
훽2 = 훾2 + iΔ2
(37)
훽3 = 훾3 + iΔ3
(38)
훽4 = 훾4 + i(Δ3 + Δ4)
where 훾2, 훾3 and 훾4 represent the radiative decay rates of the
excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission,
respectively. Δ3 = 휈3 −휔31 is the frequency detuning for the
weak probe field, and Δ2 = 휈2 −휔21 and Δ4 = 휈4 −휔43 are
the frequency detunings for the pump and control fields. In the following section, we present the results of numeri-
cal calculations of the plasmonic effects and dipole–dipole
interaction of the hybrid MNPs-SQD nanosystem, where the
SQD has a V-type four-level structure
(39)
훽5 = (훾2 + 훾3) −i(Δ2 −Δ3)
(40)
훽6 = (훾2 + 훾4) −i(Δ2 −Δ3 −Δ4)
(41)
훽7 = (훾3 + 훾4) + iΔ4 (39) (40) (41)
훽7 = (훾3 + 훾4) + iΔ4 (41) where 훾2, 훾3 and 훾4 represent the radiative decay rates of the
excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission, where 훾2, 훾3 and 훾4 represent the radiative decay rates of the
it ti
t t
2⟩3⟩
d4⟩d
t
t
i i 4
where 훾2, 훾3 and 훾4 represent the radiative decay rates of the
it ti
t t
2⟩3⟩
d4⟩d
t
t
i i where 훾2, 훾3 and 훾4 represent the radiative decay rates of the
excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission,
respectively. Δ3 = 휈3 −휔31 is the frequency detuning for the
weak probe field, and Δ2 = 휈2 −휔21 and Δ4 = 휈4 −휔43 are
the frequency detunings for the pump and control fields. with the identity with the identity respectively. Theoretical Model and Description Under the electric-dipole approximation and the rot
wave approximation, we define the equation of moti
density matrix elements (the master equation) of the
coupled to the three MNPs, as follows:
⋅휌22 = i Ωeff
2 휌12 −i Ω
eff∗
2 휌21 −2훾2휌22
⋅휌33 = i Ωeff
3 휌13 −i Ωeff∗
3 휌31 −i Ωeff
4 휌34 + i Ωeff∗
4 휌43 −2훾3 휌33 + (17)
Ω2 = 𝜇12E2
ℏ𝜀effs
, Ω3 = 𝜇13E3
ℏ𝜀effs
, Ω4 = 𝜇34E4
ℏ𝜀effs
,
(18)
Ψq = (1 +
3
∑
j=1
훼jAj),
(q = 2, 3) (17)
Ω2 = 𝜇12E2
ℏ𝜀effs
, Ω3 = 𝜇13E3
ℏ𝜀effs
, Ω4 = 𝜇34E4
ℏ𝜀effs
,
(18)
Ψq = (1 +
3
∑
j=1
훼jAj),
(q = 2, 3)
(19)
Ψ
(1
3
∑
C ) (17) Under the electric-dipole approximation and the rotating-
wave approximation, we define the equation of motion of
density matrix elements (the master equation) of the SQD
coupled to the three MNPs, as follows: (18) j=1
(19)
Ψ4 = (1 +
3
∑
j=1
훼jCj),
(27)
⋅휌22 = i Ωeff
2 휌12 −i Ω
eff∗
2 휌21 −2훾2휌22
(28)
⋅휌33 = i Ωeff
3 휌13 −i Ωeff∗
3 휌31 −i Ωeff
4 휌34 + i Ωeff∗
4 휌43 −2훾3 휌33 + 2훾4 휌44 (27)
⋅휌22 = i Ωeff
2 휌12 −i Ω
eff∗
2 휌21 −2훾2휌22 (27) (19) (19)
(28)
⋅휌33 = i Ωeff
3 휌13 −i Ωeff∗
3 휌31 −i Ωeff
4 휌34 + i Ωeff∗
4 휌43 −2훾3 휌33 + 2훾4 휌44 (20) (20)
Γq = S1훼1훼2(A1 + A2) + S2훼2훼3(A2 + A3) + S3훼3훼1(A3 + A1), (q = 2, 3)
(21)
Γ4 = T1훼1훼2(C1 + C2) + T2훼2훼3(C2 + C3) + T3훼3훼1(C3 + C1)
(29)
⋅휌44 = i Ωeff
4 휌34 −i Ωeff∗
4 휌43 −2훾4 휌44 (21)
Γ4 = T1훼1훼2(C1 + C2) + T2훼2훼3(C2 + C3) + T3훼3훼1(C3 + C1)
(29)
⋅휌44 = i Ωeff
4 휌34 −i Ωeff∗
4 휌43 −2훾4 휌44 (29) (22)
Λq =
( 𝜇2
1q
ℏ𝜀effs
)[ 3
∑
j=1
𝛼jA2
j + 2(S1𝛼1𝛼2A1A2 + S2𝛼2𝛼3A2A3 + S3𝛼3𝛼1A3A1)
]
, (q = 2, 3) (22)
Λq =
( 𝜇2
1q
ℏ𝜀effs
)[ 3
∑
j=1
𝛼jA2
j + 2(S1𝛼1𝛼2A1A2 + S2𝛼2𝛼3A2A3 + S3𝛼3𝛼1A3A1)
]
, (q = 2, 3) (22) (23)
Λ4 =
(
𝜇2
34
ℏ𝜀effs
)[ 3
∑
j=1
𝛼jBjCj + T1𝛼1𝛼2(C1B2 + C2B1) + T2𝛼2𝛼3(C2B3 + C3B2) + T3𝛼3𝛼1(C3B1 + C1B3)
] (23)
Λ4 =
(
𝜇2
34
ℏ𝜀effs
)[ 3
∑
j=1
𝛼jBjCj + T1𝛼1𝛼2(C1B2 + C2B1) + T2𝛼2𝛼3(C2B3 + C3B2) + T3𝛼3𝛼1(C3B1 + C1B3)
] (23)
Λ4 =
(
𝜇2
34
ℏ𝜀effs
)[ 3
∑
j=1
𝛼jBjCj + T1𝛼1𝛼2(C1B2 + C2B1) + T2𝛼2𝛼3(C2B3 + C3B2) + T3𝛼3𝛼1(C3B1 + C1B3)
] (23) (30)
⋅휌21 = −훽2휌21 −i Ωeff
2 ( 휌11 −휌22) −i Ωeff
3 휌23 where where where
(24)
Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3
j
Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3
j
Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3
j
(30)
⋅휌21 = −훽2휌21 −i Ωeff
2 ( 휌11 −휌22) −i Ωeff
3 휌23
(31)
⋅휌31 = −훽3휌31 −i Ωeff
2 휌32 + i Ωeff
3 ( 휌11 −휌33) + i Ωeff∗
4 휌41
(32)
⋅휌41 = −훽4휌41 −i Ωeff
2 휌42 −i Ωeff
3 휌43 + i Ωeff
4 휌31 where
(24)
Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3
j
Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3
j
Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3
j
(30)
⋅휌21 = −훽2휌21 −i Ωeff
2 ( 휌11 −휌22) −i Ωeff
3 휌23
(31)
⋅휌31 = −훽3휌31 −i Ωeff
2 휌32 + i Ωeff
3 ( 휌11 −휌33) + i Ωeff∗
4 휌41
(32)
⋅휌41 = −훽4휌41 −i Ωeff
2 휌42 −i Ωeff
3 휌43 + i Ωeff
4 휌31 (30) Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3
j
Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3
j
Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3
j Aj = (3 cos2 휃j −1)∕4휋휖o휖Br3
j
Bj = (3 sin 2휃j −1)∕4휋휖o휖Br3
j
Cj = (3 sin 휃j cos 휃j)∕4휋휖o휖Br3
j (24)
(31)
⋅휌31 = −훽3휌31 −i Ωeff
2 휌32 + i Ωeff
3 ( 휌11 −휌33) + i Ωeff∗
4 휌41 (32)
⋅휌41 = −훽4휌41 −i Ωeff
2 휌42 −i Ωeff
3 휌43 + i Ωeff
4 휌31 3 Plasmonics (2022) 17:1633–1644 1637 (33)
⋅휌32 = −훽5휌32 −i Ωeff∗
2 휌31 + i Ωeff
3 휌12 + i Ωeff∗
4 휌42
(34)
⋅휌42 = −훽6휌42 −i Ωeff∗
2 휌41 + i Ωeff
4 휌32
(35)
⋅휌43 = −훽7휌43 −i Ωeff∗
3 휌41 −i Ωeff
4 ( 휌44 −휌33)
(39)
훽5 = (훾2 + 훾3) −i(Δ2 −Δ3)
(40)
훽6 = (훾2 + 훾4) −i(Δ2 −Δ3 −Δ4)
(41)
훽7 = (훾3 + 훾4) + iΔ4 with the identity
4∑
n=1
휌nn = 1 and
(33)
⋅휌32 = −훽5휌32 −i Ωeff∗
2 휌31 + i Ωeff
3 휌12 + i Ωeff∗
4 휌42
(34)
⋅휌42 = −훽6휌42 −i Ωeff∗
2 휌41 + i Ωeff
4 휌32
(35)
⋅휌43 = −훽7휌43 −i Ωeff∗
3 휌41 −i Ωeff
4 ( 휌44 −휌33)
where 훾2, 훾3 and 훾4 represent the radiative decay rates of the
excitation states 2⟩ , 3⟩ and 4⟩ due to spontaneous emission,
respectively. Theoretical Model and Description Δ3 = 휈3 −휔31 is the frequency detuning for the
weak probe field, and Δ2 = 휈2 −휔21 and Δ4 = 휈4 −휔43 are
the frequency detunings for the pump and control fields. (36)
훽2 = 훾2 + iΔ2
(37)
훽3 = 훾3 + iΔ3
(38)
훽4 = 훾4 + i(Δ3 + Δ4) 훽2 = 훾2 + iΔ2
훽3 = 훾3 + iΔ3
훽4 = 훾4 + i(Δ3 + Δ In the following section, we present the results of numeri-
cal calculations of the plasmonic effects and dipole–dipole
interaction of the hybrid MNPs-SQD nanosystem, where the
SQD has a V-type four-level structure Fig. 2 The spectrum of the plasmon–exciton dipole interaction (Im휂3)
of the hybrid MNPs-SQD nanosystem versus probe field detuning
( Δ3 ). 휖s = 2, 휖B = 12, 휃2 = 2휋∕3, 휃3 = 3휋∕2, Ω2 = Ω4 = 6 ns−1and
Δ2 = Δ4 = Δ = 2 eV. Figure 2 (a1, a2) for (ZZY) and Fig. 2 (b1, b2)
for (ZYZ). Figure 2 (a1, b1) has: ℏ𝜔= 20 eV and Fig. 2 (a2, b2) has:
ℏ𝜔= 2.7 eV. 휃1 = 휋∕9 (dashed curve), 휃1 = 휋∕6 (solid curve) and
휃1 = 5휋∕6 (dashed-dotted curve) Fig. 2 The spectrum of the plasmon–exciton dipole interaction (Im휂3)
of the hybrid MNPs-SQD nanosystem versus probe field detuning
( Δ3 ). 휖s = 2, 휖B = 12, 휃2 = 2휋∕3, 휃3 = 3휋∕2, Ω2 = Ω4 = 6 ns−1and
Δ2 = Δ4 = Δ = 2 eV. Figure 2 (a1, a2) for (ZZY) and Fig. 2 (b1, b2) for (ZYZ). Figure 2 (a1, b1) has: ℏ𝜔= 20 eV and Fig. 2 (a2, b2) has:
ℏ𝜔= 2.7 eV. 휃1 = 휋∕9 (dashed curve), 휃1 = 휋∕6 (solid curve) and
휃1 = 5휋∕6 (dashed-dotted curve) 1 3 1638 Plasmonics (2022) 17:1633–1644 Numerical Results and Discussion Figure 3 (a2) displays the
effect of damping constant when it is large ( 훾bj = 1.6 eV). For case (ZZY), at 휃1 = 휋∕9 , it is observed a hole in the
left side which is converted into small peak at 휃1 = 휋∕6 . But at 휃1 = 휃1 = 5휋∕6 , we find different two peaks with
positive values in the left side and another different two
peaks with negative values in the right side. We have in this
figure zero absorption for all values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 )
at ( Δ3 = −3.2 ) approximately. In Fig. 3 (b2), , we observe
the middle peak has top value and another two peaks have in Fig. 2) versus probe field detuning ( Δ3 ). The data are as
in Fig. 2, in addition ℏ𝜔= 20 eV. We have Fig. 3 (a1, a2)
for case (ZZY) and Fig. 3 (b1, b2) for case (ZYZ), while
Fig. 3 (a1, b1) has the data: 훾bj = 0.026 eV and Fig. 3 (a2, b2)
has the data: 훾bj = 1.6 eV. At small damping constant ( 훾bj
= 0.026 eV), for case (ZZY) as in Fig. 3 (a1) we have an
the optical EIT for small 휃1 = 휋∕9 (dashed curve). When
increasing 휃1 = 휋∕6 (solid curve), the absorption has
three peaks and the optical EIT disappears, but at 휃1 =
5휋∕6 (dashed-dotted curve). It is observed four peaks
with negative values. But for case (ZYZ) for all values of
휃1 , we show three peaks only in the negative values with
different heights as in Fig. 3 (b1) . Figure 3 (a2) displays the
effect of damping constant when it is large ( 훾bj = 1.6 eV). For case (ZZY), at 휃1 = 휋∕9 , it is observed a hole in the
left side which is converted into small peak at 휃1 = 휋∕6 . But at 휃1 = 휃1 = 5휋∕6 , we find different two peaks with
positive values in the left side and another different two
peaks with negative values in the right side. We have in this
figure zero absorption for all values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 )
at ( Δ3 = −3.2 ) approximately. In Fig. Numerical Results and Discussion 훾3 = 1 ns−1, 훾4 = 0.01 ns−1, Ω3 = 0.01 ns−1, Ω2 = Ω4 = 6
ns−1 and ( r1, r2, r3) = 10 , 26, 42 nm, respectively. We have
Rj = R = 7 nm, 휖s = 2, 휖B = 12, 휃2 = 2휋∕3, 휃3 = 3휋∕2, and
Δ2 = Δ4 = Δ = 2 eV. Other parameters are indicated in the
figure captions and described in what follows. The numerical calculations for the set of density matrix
equations (27-35) at the steady state are done to obtain
the coherence 휌13 and 휌34 . We study the influence of the
strength of the plasmon–exciton dipole interaction as a
function of probe field Im Λ3휌13 = Im 휂3 and control field
ImΛ4휌34 = Im휂4 for different parameters of the hybrid
MNPs-SQD nanosystem. We consider three spherical
gold MNPs with radius aj . The parameters of the MNPs-
SQD are taken as ℏ𝜔pj = 9.02 eV, 훾bj = 0.026 eV, and
the dipole 휇12 = 휇13 = 휇34 = 0.65 e nm, 훾2 = 0.02 ns−1, Figure 2 shows the spectrum of the plasmon–exciton
dipole interaction (Im휂3) of the hybrid MNPs-SQD nano-
system for different values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 ) versus
probe field detuning ( Δ3 ). Figure 2 (a1, a2) is taken for case
(ZZY), and Fig. 2 (b1, b2) is taken for case (ZYZ), while
Fig. 2 (a1, b1) has the data: ℏ𝜔= 20 eV and Fig. 2 (a2, b2)
has the data: ℏ𝜔= 2.7 eV. Figure 2 (a1) shows different Fig. 3 The spectrum of the plasmon–exciton dipole interaction (Im휂4)
of the hybrid MNPs-SQD nanosystem versus probe field detuning
( Δ3 ). The data as in Fig. 2, in addition ℏ𝜔= 20 eV. Figure 3 ( a1, a2 )
for (ZZY) and Fig. 3 (b1, b2) for (ZYZ). Figure 3 (a1, b1) has: 훾bj
= 0.026 eV and Fig. 3 (a2, b2) has: 훾bj = 1.6 eV for (ZZY) and Fig. 3 (b1, b2) for (ZYZ). Figure 3 (a1, b1) has: 훾bj
= 0.026 eV and Fig. 3 (a2, b2) has: 훾bj = 1.6 eV Fig. 3 The spectrum of the plasmon–exciton dipole interaction (Im휂4)
of the hybrid MNPs-SQD nanosystem versus probe field detuning
( Δ3 ). The data as in Fig. 2, in addition ℏ𝜔= 20 eV. Numerical Results and Discussion Figure 3 ( a1, a2 ) 1 3 1639 Plasmonics (2022) 17:1633–1644 impacts and distinctive for the absorption spectra when
ℏ𝜔= 20 eV and has negative values for all 휃1 , at small 휃1 =
휋∕9 (dashed curve); the absorption has an optical EIT win-
dow at zero detuning ( Δ3 ) besides one peak on each side. With increasing 휃1 = 휋∕6 (solid curve), we have three peaks
and notice the optical EIT window disappearing, but at 휃1 =
5휋∕6 (dashed-dotted curve), the peaks are increasing to
four peaks. In Fig. 2 (a2) , the peaks are displacing under the
effect of angle 휃1 where they have four different peaks with
positive values (because of the decrease in ℏ𝜔= 2.7 eV). For
case (ZYZ), we notice three peaks only; the peaks for large
휃1 = 5휋∕6 have negative values in Fig. 2 (b1) . All the peaks
for all values of 휃1 have negative values for ( ℏ𝜔= 2.7 eV)
as in Fig. 2 (b2) , and also the peaks are displacing under the
effect of angle 휃1 like Fig. 2 (a2) . We conclude in this figure
the absorption spectrum is asymmetric about the vertical
axis at ( Δ3 = 0 ), we notice also the resonance frequency
( ℏ𝜔 ) as well the angles, and direction of the fields plays an
important role in the plasmon–exciton dipole coupling. in Fig. 2) versus probe field detuning ( Δ3 ). The data are as
in Fig. 2, in addition ℏ𝜔= 20 eV. We have Fig. 3 (a1, a2)
for case (ZZY) and Fig. 3 (b1, b2) for case (ZYZ), while
Fig. 3 (a1, b1) has the data: 훾bj = 0.026 eV and Fig. 3 (a2, b2)
has the data: 훾bj = 1.6 eV. At small damping constant ( 훾bj
= 0.026 eV), for case (ZZY) as in Fig. 3 (a1) we have an
the optical EIT for small 휃1 = 휋∕9 (dashed curve). When
increasing 휃1 = 휋∕6 (solid curve), the absorption has
three peaks and the optical EIT disappears, but at 휃1 =
5휋∕6 (dashed-dotted curve). It is observed four peaks
with negative values. But for case (ZYZ) for all values of
휃1 , we show three peaks only in the negative values with
different heights as in Fig. 3 (b1) . Numerical Results and Discussion 3 (b2), , we observe
the middle peak has top value and another two peaks have Figure 3 shows the spectrum of the plasmon–exciton
dipole interaction (Im휂4) of the hybrid MNPs-SQD
nanosystem for different values of 휃1(휋∕9 , 휋∕6 , 5휋∕6 ) (as Fig. 4 The spectrum of the plasmon–exciton dipole interaction (Im휂4)
of the hybrid MNPs-SQD nanosystem versus probe field detuning
( Δ3 ) at 휃1 = 0. Figure 4 (a1, a2, a3) for (ZZY) and Fig. 4 (b1, b2, b3) for
(ZYZ). Figure 4 (a1, b1)has : ℏ𝜔= 16 eV, Fig. 4 (a2, b2) has: ℏ𝜔= 6
eV and Fig. 4 (a3, b3) has: ℏ𝜔= 2.7 eV. The another data as in Fig. 2 Fig. 4 The spectrum of the plasmon–exciton dipole interaction (Im휂4)
of the hybrid MNPs-SQD nanosystem versus probe field detuning
( Δ3 ) at 휃1 = 0. Figure 4 (a1, a2, a3) for (ZZY) and Fig. 4 (b1, b2, b3) for (ZYZ). Figure 4 (a1, b1)has : ℏ𝜔= 16 eV, Fig. 4 (a2, b2) has: ℏ𝜔= 6
eV and Fig. 4 (a3, b3) has: ℏ𝜔= 2.7 eV. The another data as in Fig. 2 1 3 Plasmonics (2022) 17:1633–1644 1640 Fig. 4 (b1, b2, b3) is taken for case (ZYZ). Figure 4 (a1, b1)
has the data: ℏ𝜔= 16 eV, Fig. 4 (a2, b2) has: ℏ𝜔= 6 eV and
Fig. 4 (a3, b3) has: ℏ𝜔= 2.7 eV. We have different impacts
and distinctive for the resonance frequency ( ℏ𝜔 ) on the
spectrum (Im휂4) in this figure, when ℏ𝜔= 16 eV. Figure 4 (a1, b1) displays four peaks in the positive values
for case (ZZY) and three peaks in the negative values for
case (ZYZ), respectively, with different heights. But at
ℏ𝜔= 6 eV for case (ZZY), the spectrum exhibits a positive
high peak with a Fano-like lineshape in the positive ( Δ3 )
and a negative small peak with a Fano-like lineshape in the
negative ( Δ3 ) as in Fig. 4 (a2) . But in Fig. 4 (b2) , the small values for each value of 휃1with different values for
peaks. Then, in Fig. 3, the influence of the damping constant
on the plasmon–exciton dipole coupling is more obviously
for the case (ZZY), and also, the shape of the spectra is
asymmetric at ( Δ3 = 0 ). Numerical Results and Discussion 4 (b3) , the
spectrum exhibits a trapping at ( Δ3 = 0 ), and we notice also
a negative peak at a certain value ( Δ3 = −30 ) and also a
positive peak at ( Δ3 = 30 ) approximately. We conclude in
this figure the obvious role of the resonance frequency ( ℏ𝜔 )
or the dielectric function 휖mj(휔) for MNPs on the spectrum
of the plasmon–exciton dipole interaction (Im휂4) . So, it is
clear the influence of the metal nanoparticles is to enhance
different phenomena in the regime of exciton–plasmon
resonance. spectrum exhibits a negative high peak in the negative ( Δ3 )
and small peak with a Fano-like lineshape in the positive
( Δ3 ). At ℏ𝜔= 2.7 eV for case (ZZY), Fig. 4 (a3) displays a
positive peak and a negative peak in the negative ( Δ3 ) and
also in the positive ( Δ3 ). For case (ZYZ) in Fig. 4 (b3) , the
spectrum exhibits a trapping at ( Δ3 = 0 ), and we notice also
a negative peak at a certain value ( Δ3 = −30 ) and also a
positive peak at ( Δ3 = 30 ) approximately. We conclude in
this figure the obvious role of the resonance frequency ( ℏ𝜔 )
or the dielectric function 휖mj(휔) for MNPs on the spectrum
of the plasmon–exciton dipole interaction (Im휂4) . So, it is
clear the influence of the metal nanoparticles is to enhance
different phenomena in the regime of exciton–plasmon
resonance. dielectric constant 휖B is small and equal 휖s ( 휖B = 휖s = 2 )
at ℏ𝜔= 20 eV. The dashed curve is for Ω2 = Ω4 = 6ns−1
and the solid curve is for Ω2 = 4ns−1, Ω4 = 8ns−1 . The
another data are as in Fig. 4. Figure 5 (a1, a2, a3) is
taken for (Im휂3) , where the spectra have negative val-
ues, and Fig. 5 (b1, b2, b3) is taken for ( (Im휂4) , where
the spectra have positive values. Figure 5 (a1, b1) has
( Δ = 0 ), Fig. 5 (a2, b2) has ( Δ = 2 ) and Fig. 5 (a3, b3) has
( Δ = 5 ). The optical EIT, when ( Ω2 = Ω4 = 6ns−1 ) (in
Fig. 5 (a1, b1) ), is deep and disappears when increasing the
( Δ ) (as in Fig. Numerical Results and Discussion As well, the optical PEIT in the
spectrum appears at small damping constant and the hole in
the spectrum appears at large damping constant . Figure 4 shows the spectrum of the plasmon–exciton
dipole interaction (Im휂4) of the hybrid MNPs-SQD
nanosystem for different values of ℏ𝜔 (16, 6, 2.7) versus
probe field detuning ( Δ3 ) at 휃1 = 0. The another data are as
in Fig. 2. Figure 4 (a1, a2, a3) is taken for case (ZZY), and 1 3
Fig. 5 The spectrum of the plasmon–exciton dipole interaction
(Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY)
at ( 휖B = 휖s = 2 ), ℏ𝜔= 20 eV. Figure 5 (a1, a2, a3) is for (Im휂3) and
Fig. 5 (b1, b2, b3) for ( (Im휂4) . Figure 5 (a1, b1) has ( Δ = 0 ), Fig. 5
(a2, b2) has ( Δ = 2 ) and Fig. 5 (a3, b3) has ( Δ = 5 ). The dashed curve
for Ω2 = Ω4 = 6ns−1 and the solid curve for Ω2 = 4ns−1, Ω4 = 8ns−1 . The another data as in Fig. 4 Fig. 5 The spectrum of the plasmon–exciton dipole interaction
(Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY)
at ( 휖B = 휖s = 2 ), ℏ𝜔= 20 eV. Figure 5 (a1, a2, a3) is for (Im휂3) and
Fig. 5 (b1, b2, b3) for ( (Im휂4) . Figure 5 (a1, b1) has ( Δ = 0 ), Fig. 5 (a2, b2) has ( Δ = 2 ) and Fig. 5 (a3, b3) has ( Δ = 5 ). The dashed curve
for Ω2 = Ω4 = 6ns−1 and the solid curve for Ω2 = 4ns−1, Ω4 = 8ns−1 . The another data as in Fig. 4 1 3 1641 Plasmonics (2022) 17:1633–1644 spectrum exhibits a negative high peak in the negative ( Δ3 )
and small peak with a Fano-like lineshape in the positive
( Δ3 ). At ℏ𝜔= 2.7 eV for case (ZZY), Fig. 4 (a3) displays a
positive peak and a negative peak in the negative ( Δ3 ) and
also in the positive ( Δ3 ). For case (ZYZ) in Fig. Numerical Results and Discussion 5 (a3, b3) ) . As well, four peaks appeared for
( Ω2 = 4ns, Ω4 = 8ns−1 ) in Fig. 5 (a1, b1) and decreased to
only two peaks when increasing the ( Δ ) (in Fig. 5 (a3, b3) ). Then, the optical EIT is related by the change of ( Δ ), and
the value of the ( Δ ) plays an important role in the charac-
terization of the spectrum of the plasmon–exciton dipole
interaction (Im휂3) and (Im휂4). Figure 5 demonstrates the spectrum of the plas-
mon–exciton dipole interaction (Im휂3) and (Im휂4) of the
hybrid MNPs-SQD nanosystem for case (ZZY) when the 1 3
Fig. 6 The spectrum of the plasmon–exciton dipole interaction (Im휂3)
and (Im휂4) of the hybrid MNPs-SQD nanosystem at 휖s = 12 , Δ = 0
and 휃1 = 휋∕9 . Figure 6 (a1, a2) for (Im휂3) and Fig. 6 (b1, b2) for
( (Im휂4) . Figure 6 (a1, b1) for (ZZY) and Fig. 6 (a2, b2) for (ZYZ). The
dashed-dotted curve for ( 휖B = 2 ), the solid curve for ( 휖B = 6 ) and the
(dashed curve) for ( 휖B = 12 ) .The another data as in Fig. 2 Fig. 6 The spectrum of the plasmon–exciton dipole interaction (Im휂3)
and (Im휂4) of the hybrid MNPs-SQD nanosystem at 휖s = 12 , Δ = 0
and 휃1 = 휋∕9 . Figure 6 (a1, a2) for (Im휂3) and Fig. 6 (b1, b2) for ( (Im휂4) . Figure 6 (a1, b1) for (ZZY) and Fig. 6 (a2, b2) for (ZYZ). The
dashed-dotted curve for ( 휖B = 2 ), the solid curve for ( 휖B = 6 ) and the
(dashed curve) for ( 휖B = 12 ) .The another data as in Fig. 2 1 3 1642 Plasmonics (2022) 17:1633–1644 Figure 6 exhibits the spectrum of the plasmon–exciton
dipole interaction (Im휂3) and (Im휂4) of the hybrid MNPs-
SQD nanosystem when the dielectric constant 휖s is large
( 휖s = 12 ), unlike the above figures, at ( Δ = 0 and 휃1 = 휋∕9 ). Figure 6 (a1, a2) is taken for (Im휂3) and Fig. 6 (b1, b2) is
taken for ( (Im휂4) . Figure 6 (a1, b1) is for case (ZZY) and
Fig. 6 (a2, b2) is for case (ZYZ). Consent for Publication Not applicable. Consent for Publication Not applicable. Conflicts of Interest We investigated the dependence of the plasmon-
exciton dipole coupling of the SQD-MNP nanosystem on the distance
between the three metallic nanoparticles (MNPs). Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Conclusion We have derived a compound expression of the effective
Rabi frequencies based on the effect of the plasmon–exci-
ton dipole coupling in the SQD-MNPs nanosystem which
is composed of three sphere metallic nanoparticles (MNPs)
and semiconductor quantum dot (SQD) which have three
external fields. The strong exciton–plasmon interaction and
multipole effects are considered the main focus of this work. The direction and detunings of the three external fields play
an important role in the characterization of the SQD-MNPs
nanosystem. We investigated the dependence of the plas-
mon–exciton dipole coupling of the SQD-MNP nanosystem
on the distance between the three MNPs and also the dis-
tance between SQD and MNPs. The material parameters of
the hybrid nanosystem such as resonance frequency, damp-
ing constant and dielectric constant, are demonstrated for
many distinct characteristics and phenomena for the spec-
tra of the plasmon–exciton dipole interaction (ImΛ3휌13)
and (ImΛ4휌34) of the hybrid MNPs-SQD nanosystem. The Numerical Results and Discussion optical experiments on the hybrid SQD-MNPs nanosystem
may be analyzed by using the results obtained in this work. Author Contributions All authors contributed to the study conception,
design and preparation. All authors commented on previous versions
of the manuscript. All authors read and approved the final manuscript. Funding Information Open access funding provided by The Science,
Technology & Innovation Funding Authority (STDF) in cooperation
with The Egyptian Knowledge Bank (EKB). The authors declare that
no funds, grants or other support was received during the preparation
of this manuscript. Data Availability The datasets generated during and/or analyzed dur-
ing the current study are available from the corresponding author on
reasonable request. Numerical Results and Discussion The dashed-dotted curve
is for ( 휖B = 2 ), the solid curve for ( 휖B = 6 ) and the dashed
curve for ( 휖B = 12 ). The another data are as in Fig. 2. We see that when the dielectric constant 휖s is large, the
increase in the dielectric constant 휖B can be contributed to the enhancement of the optical EIT for case (ZZY) in the
spectrums of the plasmon–exciton dipole interaction ( Im휂3
and Im휂4 ); when 휖s = 휖B = 12 , the optical EIT becomes
more deep. The optical EIT for case (ZYZ) in the spec-
trums ( Im휂3 and Im휂4 ) is not available, and the three peaks
more extend range at increasing the dielectric constant 휖B in
Fig. 6 (a2, b2) . So, happening of the optical EIT is related by
the value of the dielectric constants 휖B , 휖s and the direction
of the fields. Figure 7 demonstrates the influence of the size
of the three spherical MNPs on the spectrum of the increase in the dielectric constant 휖B can be contributed to
of the three spherical MNPs on the spectrum of the
Fig. 7 The spectrum of the plasmon–exciton dipole interaction
(Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY),
at ( Rj = 4, 6 nm) and ( 휃1 = 0 ). Figure 7 (a1, a2) for (Im휂3) and
Fig. 7 (b1, b2) for ( (Im휂4) . Figure 7 (a1, b1) is plotted versus probe
field detuning ( Δ3 ) and Fig. 7 (a2, b2) is plotted versus probe field
detuning ( Δ4 ). The dashed curve for ( Rj = 4 ) and the solid curve for
( Rj = 6 ). The another data as in Fig. 2 Fig. 7 The spectrum of the plasmon–exciton dipole interaction
(Im휂3) and (Im휂4) of the hybrid MNPs-SQD nanosystem for (ZZY),
at ( Rj = 4, 6 nm) and ( 휃1 = 0 ). Figure 7 (a1, a2) for (Im휂3) and
Fig. 7 (b1, b2) for ( (Im휂4) . Figure 7 (a1, b1) is plotted versus probe field detuning ( Δ3 ) and Fig. 7 (a2, b2) is plotted versus probe field
detuning ( Δ4 ). The dashed curve for ( Rj = 4 ) and the solid curve for
( Rj = 6 ). The another data as in Fig. Numerical Results and Discussion 2 1 3 3 Plasmonics (2022) 17:1633–1644 1643 plasmon–exciton dipole interaction (Im휂3) and (Im휂4) of
the hybrid MNPs-SQD nanosystem for the case (ZZY),
and we take the radii with two different values ( R = 4, 6
nm) and ( 휃1 = 0 ), taking into consideration that the center-
to-center distances are constant. Figure 7 (a1, a2 ) is taken
for (Im휂3) and Fig. 7 (b1, b2) is taken for ( (Im휂4) . Fig-
ure 7 (a1, b1) is plotted versus probe field detuning ( Δ3 ) and
Fig. 7 (a2, b2) is plotted versus probe field detuning ( Δ4 ). The dashed curve is for ( R = 4 ) and the solid curve is for
( R = 6 ). The another data are as in Fig. 2. In Fig. 7 (a2, b2 ),
which is plotted versus probe field detuning ( Δ4 ), it does
not show peaks on the two sides of the spectrum as in
Fig. 7 (a1, b1) . We notice at ( R = 4 ), the spectrum has
small optical EIT; when increasing the radii ( R = 6 ), the
optical EIT becomes obvious as in Fig. 7 (a1, b1, a2, b2) ,
where we have from Eq. (22, 23) that the plasmon–exciton
dipole interaction ( Λ3, Λ4 ) depends on 훼j which contains
Rj (i.e., the size of spherical MNPs); when we take the
another values in these equations’ constant, the dipole
interaction for MNP of ( R = 6 ) is more powerful than the
dipole interaction for MNP of ( R = 4 ) because the MNP of
( R = 6 ) is the nearest to SQD unlike the MNP of ( R = 4 )
when the center-to-center distances are the same for the
two cases ( R = 6, 4 ) [also, see [8, 17, 19, 24, 27]. We
notice the ratio of the radius of MNP ( Rj ) to the center-to-
center distance ( rj ), which plays an important role in the
plasmon–exciton dipole interaction [24]. Then, the spec-
trum of the plasmon–exciton dipole interaction (Im휂3) and
(Im휂4) of the hybrid MNPs-SQD nanosystem is affected by
the size of the three spherical MNPs. optical experiments on the hybrid SQD-MNPs nanosystem
may be analyzed by using the results obtained in this work. Ethical Approval Not applicable. Ethical Approval Not applicable. Informed Consent Informed consent was obtained from all individual
participants included. References 1. Paspalakis E, Evangelou S, Yannopapas V, Terzis AF (2013)
Phase-dependent optical effects in a four-level quantum system
near a plasmonic nanostructure. Phys Rev A 88:053832 2. Kosionis S. G, Terzis A. F, Sadeghi S. M, Paspalakis E (2013) Opti-
cal response of a quantum dot-metal nanoparticle hybrid interacting
with a weak probe field. J. Phys. Condens. Matter 25:045304 3. Yang Yanlian, Guo Kangxian, Yang Tao, Li Keyin, Zhai Wangjian
(2019) Enhancement of linear and nonlinear optical absorp-
tion coefficients in spherical dome semiconductor nanoshells
by surface plasmon resonances. Physica B: Condensed Matter
556:158–162 4. Artuso RD, Bryant GW (2010) Strongly coupled quantum dot-
metal nanoparticle systems: Exciton-induced transparency, dis-
continuous response, and suppression as driven quantum oscillator
effects. Phys. Rev. B 82:195419 4. Artuso RD, Bryant GW (2010) Strongly coupled quantum dot-
metal nanoparticle systems: Exciton-induced transparency, dis-
continuous response, and suppression as driven quantum oscillator
effects. Phys. Rev. B 82:195419 1 3 1644 Plasmonics (2022) 17:1633–1644 5. Zhang W, Govorov AO (2011) Quantum theory of the nonlinear
Fano effect in hybrid metal-semiconductor nanostructures: The
case of strong nonlinearity Phys Rev B 84:081405fi 17. Carreno F, Anton MA, Melle S, Calderon OG, Cabrera-Granado
E, Cox J, Singh MR, Egatz-Gomez A (2014) Plasmon-enhanced
terahertz emission in self-assembled quantum dots by femtosec-
ond pulses. J Appl Phys 115:64304 6. Cui J, Ji B, Song X, Lin J (2018) Efficient modulation of multipo-
lar fano resonances in asymmetric ring-disk/split-ring-disk nano-
structure plasmonics 18. Sadeghi SM, Hatef A (2013) Simon Fortin-Deschenes, Michel
Meunier, Coherent confinement of plasmonic field in quantum
dot-metallic nanoparticle molecules. Nanotechnology 24:205201i p
7. Dongxing Zhao, Wu Jiarui, Gu Ying, Qihuang Gong (2014)
Tailoring double Fano profiles with plasmon-assisted quantum
interference in hybrid exciton-plasmon system. Appl Phys Lett
105:111112 19. Ryan D (2011) Artuso, Garnett W. Bryant, Using local fields to
tailor hybrid quantum-dot/metal nanoparticle systems, Physical
Review B 83:235406 20. Sadeghi SM (2010) Gain without inversion in hybrid quantum
dot-metallic nanoparticle systems. Nanotechnology 21:455401i 8. Zhang W, Govorov AO, Bryant GW (2006) Semiconductor-Metal
Nanoparticle Molecules: Hybrid Excitons and the Nonlinear Fano
Effect. Physical Review Letters 97:146804 21. Sadeghi SM (2014) Dynamics of plasmonic field polarization
induced by quantum coherence in quantum dot-metallic nanoshell
structures. Optics Lett 39 y
9. Hatef A, Sadeghi SM, Singh MR (2012) Plasmonic electromag-
netically induced transparency in metallic nanoparticle-quantum
dot hybrid systems. Nanotechnology 23:065701 22. References Sadeghi SM (2017) Chuanbin Mao, Quantum sensing using coher-
ent control of near-field polarization of quantum dot-metallic
nanoparticle molecules. Journal of Applied Physics 121:014309 10. Zamani N, Hatef A, Nadgaran H, Keshavarz A (2017) Control
of electromagnetically induced transparency via a hybrid semi-
conductor quantum dot-vanadium dioxide nanoparticle system. J
Nanophotonics 11(3):036011 23. Nugroho Bintoro S, Iskandar Alexander A, Malyshev Victor A,
Knoester Jasper (2019) Plasmon-assisted two-photon Rabi oscil-
lations in a semiconductor quantum dot-metal nanoparticle het-
erodimer. Phys Rev B 99:075302 11. Behroozian B (2020) Mohammad Reza Rezaie, Hassan Ranjbar
Askari, The dispersion behaviors of the tripod-type four-level
cylindrical quantum dot under phenomenon of electromagneti-
cally induced transparency. Phys. Scr. 95:065506 24. Yan JY, Zhang W, Duan S, Zhao XG, Govorov Alexander O
(2008) Optical properties of coupled metal-semiconductor and
metal-molecule nanocrystal complexes: Role of multipole effects. Phys Rev B 77:165301 12. Sadeghi SM, Deng L, Li X, Huang W-P (2009) Plasmonic (ther-
mal) electromagnetically induced transparency in metallic nano-
particle-quantum dot hybrid systems. Nanotechnology 20:365401i 25. Anton MA, Carreno F, Melle S, Calderon O.G, Cabrera-Granado
E (2013) Optical pumping of a single hole spin in a p-doped
quantum dot coupled to a metallic nanoparticle. Phys Rev B
87:195303 13. Evangelou S, Yannopapas V, Paspalakis E (2014) Modification of
Kerr nonlinearity in a four-level quantum system near a plasmonic
nanostructure. Journal of Modern Optics 61(18):1458–1464 26. Cox JD, Singh MR, Gumbs G, Anton MA, Carreno Fernando
(2012) Dipole-dipole interaction between a quantum dot and a
graphene nanodisk. Physical Review B 86:125452 p
14. Terzis AF, Kosionis SG, Boviatsis J, Paspalakis E (2016) Nonlin-
ear optical susceptibilities of semiconductor quantum dot - metal
nanoparticle hybrids. J Mod Opt 63(5):451–461 15. Lu Zhien, Ka-Di Zhu (2008) Enhancing Kerr nonlinearity of a
strongly coupled exciton-plasmon in hybrid nanocrystal mol-
ecules. J. Phys. B: At. Mol. Opt. Phys. 41:185503 27. Jiang X, Guo K, Liu G, Yang T, Yang Y (2017) Enhancement
of surface plasmon resonances on the nonlinear optical proper-
ties in a GaAs quantum dot. Superlattices and Microstructures
105:56–64 y
p
y
16. Kosionis SG, Terzis AF (2010) Paspalakis, Linear and nonlinear
optical properties of a two-subband system in a symmetric semi-
conductor quantum well. J Appl Phys 108:034316 28. Stefan A (2007) Maier, Plasmonics: Fundamentals and Applica-
tions Springer Science 1 3 3
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Causes and Consequences of Flavivirus RNA Methylation
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Frontiers in microbiology
| 2,017
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cc-by
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Causes and Consequences of
Flavivirus RNA Methylation Shelton S. Bradrick*
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States Shelton S. Bradrick* Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, United States Mosquito-borne flaviviruses are important human pathogens that represent global
threats to human health. The genomes of these positive-strand RNA viruses have been
shown to be substrates of both viral and cellular methyltransferases. N7-methylation of
the 5′ cap structure is essential for infection whereas 2′-O-methylation of the penultimate
nucleotide is required for evasion of host innate immunity. N6-methylation of internal
adenosine nucleotides has also been shown to impact flavivirus infection. Here, I
summarize recent progress made in understanding roles for methylation in the flavivirus
life-cycle and discuss relevant emerging hypotheses. Keywords: methylation, flavivirus, Zika virus, dengue virus, hepatitis C virus INTRODUCTION The genomes of mosquito-borne flaviruses are complex, multi-functional RNA molecules that
must be translated, replicated and packaged in the face of innate host defenses to accomplish
the ultimate viral goal: production of infectious particles to initiate new rounds of infection. Viral genomes must interface with viral proteins and host machinery to accomplish these critical
tasks. Such interactions are specified by RNA features within viral genomes, including sequences
and secondary/tertiary structures, and trans-acting factors that recognize these cis-acting features
(Campos et al., 2017). In addition to RNA sequence and structure, covalent modifications of
individual nucleotides represent another layer of cis-acting features that have been shown to impact
RNA function (Saletore et al., 2012). An RNA modification fundamental to flavivirus infection
is methylation, as evidenced by the existence of virus-encoded RNA methyltransferases (MTase)
(Dong et al., 2014). Moreover, a few recent studies implicate flavivirus genomes to be functionally
methylated by host enzymes. In this review I summarize the current state of knowledge of flavivirus
RNA methylation as well-effects of RNA methylation on flavivirus infection. Edited by:
Encarna Martinez-Salas,
Centro de Biología Molecular Severo
Ochoa (CSIC), Spain
Reviewed by:
Isabelle Imbert,
Aix-Marseille University, France
Graham John Belsham,
Technical University of Denmark,
Denmark
*Correspondence:
Shelton S. Bradrick
ssbradri@utmb.edu
Specialty section:
This article was submitted to
Virology,
a section of the journal
Frontiers in Microbiology
Received: 06 October 2017
Accepted: 16 November 2017
Published: 05 December 2017
Citation:
Bradrick SS (2017) Causes
and Consequences of Flavivirus RNA
Methylation. Front. Microbiol. 8:2374. doi: 10.3389/fmicb.2017.02374 Edited by:
Encarna Martinez-Salas,
Centro de Biología Molecular Severo
Ochoa (CSIC), Spain Reviewed by:
Isabelle Imbert,
Aix-Marseille University, France
Graham John Belsham,
Technical University of Denmark,
Denmark *Correspondence:
Shelton S. Bradrick
ssbradri@utmb.edu y
y
Multiple flaviviruses transmitted by arthropods represent serious human health concerns. These
include yellow fever virus (YFV), West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis
virus (JEV) and the four serotypes of dengue viruses (DENV) which are the most prevalent,
causing nearly 100 million symptomatic infections world-wide (Bhatt et al., 2013). These viruses,
comprising part of the flavivirus genus, belong to the Flaviviridae which includes the significant
blood-borne human pathogen within the hepacivirus genus, hepatitis C virus (HCV). The genomes
of viruses within this family share a similar organization: each contains a single open reading
frame flanked by untranslated regions (UTRs) of various sequence, length and structure. The viral
UTRs contain functional RNA elements that control viral translation and RNA synthesis (Garcia-
Blanco et al., 2016). Unique to members of the flavivirus genus is the presence of a so called “cap”
structure at the 5′ end of the genome. As discussed in detail below, methylation of the cap structure
and the adjacent penultimate nucleotide of the viral genome critically promotes virus infection by
multiple mechanisms. In contrast, HCV, the most prominent member of the hepacivirus genus, is
characterized by an uncapped genome that contains an internal ribosome entry site within the 5′
UTR (Tsukiyama-Kohara et al., 1992). Specialty section:
This article was submitted to
Virology,
a section of the journal
Frontiers in Microbiology Received: 06 October 2017
Accepted: 16 November 2017
Published: 05 December 2017 Keywords: methylation, flavivirus, Zika virus, dengue virus, hepatitis C virus MINI REVIEW published: 05 December 2017
doi: 10.3389/fmicb.2017.02374 ROLES FOR METHYLATION AT THE 5′
END OF THE FLAVIVIRUS GENOME Cellular mRNAs are modified in the nucleus with a 7-
methylguanosine (m7GpppN) cap structure attached to the
first base of the transcript via a 5′-5′ triphosphate linker
(Figure 1; Shatkin, 1976). This occurs early after the initiation
of transcription via recruitment and sequential action of capping
enzymes, including an RNA triphosphatase, guanylyltransferase
and N7-guanine MTase, to the C-terminal domain of elongating
RNA pol II (Phatnani and Greenleaf, 2006). The RNA
triphosphatase acts to remove the γ-phosphate from the 5′
nucleotide of the nascent RNA making it available for cap
addition by guanylyltransferase. Methylation of the guanosine
cap at N7 completes the reaction to generate a so called “type
0” cap structure (Wei C. M. et al., 1975). Importantly, in higher
eukaryotic organisms the mRNA is further modified by a separate
ribose MTase at the penultimate nucleotide with a 2′-O-methyl
group (Figure 1; type 1 cap) and to a lesser extent at the following
nucleotide (type 2 cap) (Wei and Moss, 1975). The 5′ cap
structure impacts every aspect of mRNA metabolism, including
splicing, nuclear export, translation, and decay (Cowling, 2010). In contrast, 2′-O-methylation is a mark that signifies an mRNA
as a “self” versus foreign molecule. FIGURE 1 | Depiction of the flavivirus RNA capping and methylation pathway. Nascent flavivirus genomes initiate with a 5′ triphosphorylated adenosine that
is dephosphorylated by the RNA triphosphatase (RTPase) activity of NS3. Next, the putative NS5 guanylyltransferase (GTase) attaches guanosine
monophosphate (GMP) via a 5′-5′ linkage. NS5 then methylates the guanine
N7 position to form the type 0 cap using S-adenosyl methionine (SAM) as a
cofactor. Methyl group donation by SAM converts it to S-adenosyl
homocysteine (SAH). NS5-mediated 2′-O-methylation of the adenosine
nucleotide generates the type I cap structure. Finally, hypothetical m6A
methylation of flaviviral RNA at the penultimate adenosine by the METTL3/14
protein complex would result in the formation m6Am. Flaviviruses do not have access to the nuclear m7G-capping
machinery and instead have evolved enzymatic activities to carry
out all the necessary steps to generate capped genomes. The
NS5 protein, in addition to its essential RNA-dependent RNA-
polymerase function, harbors guanylyltransferase and MTase
enzymes (Egloffet al., 2002; Ray et al., 2006; Issur et al., 2009). NS3 is similarly multifunctional, capable of protease, helicase
and RNA triphosphatase activities (Wengler and Wengler, 1993;
Bartelma and Padmanabhan, 2002). Citation: Bradrick SS (2017) Causes
and Consequences of Flavivirus RNA
Methylation. Front. Microbiol. 8:2374. doi: 10.3389/fmicb.2017.02374 December 2017 | Volume 8 | Article 2374 1 Frontiers in Microbiology | www.frontiersin.org Bradrick Flavivirus Methylation At the level of the individual cell all Flaviviridae use a
fundamental infection strategy: (i) virus particles attach to
various cellular receptors and are internalized via endocytosis, (ii)
endosome acidification causes fusion between the viral envelope
and endosomal membrane allowing for escape of the viral
nucleocapsid into the cytoplasm, (iii) the viral RNA dissociates
from capsid and engages the translational machinery to
synthesize viral proteins at the cytosolic face of the endoplasmic
reticulum (ER), (iv) viral proteins engage the positive-strand
genome to synthesize a negative strand intermediate, (v) the
negative-strand asymmetrically templates the synthesis of many
genomes, (vi) some of which associate with viral structural
proteins and bud into the ER to form immature viral particles
that (vii) transit through the golgi apparatus where they are
modified by host enzymes, and finally, (viii) mature virions
are secreted into the extracellular space. Note that multiple
phases of the life-cycle, including translation, RNA synthesis and
virus assembly, occur concurrently on separate genomes once
infection is established although completion of each process is a
prerequisite for the following to occur. Frontiers in Microbiology | www.frontiersin.org ROLES FOR METHYLATION AT THE 5′
END OF THE FLAVIVIRUS GENOME The latter of these catalyze
the first step in capping: removal of the γ-phosphate from the
5′ adenosine of nascent viral RNAs. NS5 is then believed to cap the RNA and performs sequential methylation reactions to
generate (i) m7GpppA (cap-0) and then (ii) m7GpppAm (cap-1)
(Ray et al., 2006). The cis- and trans-determinants of putative
guanylyltransferase activity have not been well-characterized
but the MTase reactions are relatively well-understood. Unlike December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 2 Bradrick Flavivirus Methylation translation initiation may occur via a non-canonical mechanism. First, as noted above cap methylation conferred a relatively small
translational advantage to WNV replicon RNAs (Ray et al., 2006). Second, depletion of eIF4E by RNA interference was reported
to not affect DENV replication or protein synthesis: whereas
DENV translation was reduced by ∼10%, cellular translation
was reduced by 60% due to eIF4E knockdown (Edgil et al.,
2006). These observations suggest that flaviviruses may use a
non-canonical pathway of translation initiation that depends
minimally on the presence of a 5′- m7Gppp. cellular MTase which is not believed to discriminate among
different RNA substrates, the flavivirus MTase exhibits substrate
specificity and will not efficiently methylate the cap of non-viral
RNAs (Dong et al., 2007). Cap methylation by NS5 requires the
second and third genome nucleotides to be GU and also the
presence of a 5′ stem loop which is structurally conserved across
all flaviviruses (Brinton and Dispoto, 1988). 2′-O-methylation
requires the first two nucleotides (AG) and is enhanced by
sequence within the first 20 residues of the genome. Notably,
m7GpppA-RNA is strongly preferred over GpppA-RNA as a
substrate for 2′-O-methylation, explaining the sequential order of
5′ end methylation reactions (Dong et al., 2008). Finally, it is hypothetically possible that cap methylation
protects viral genomes from recognition by factor(s) that sense
unmethylated cap structures as invading nucleic acid. No such
factor has yet been identified but there are many well-defined
“pattern recognition receptors” whose tasks are to detect invading
non-self nucleic acids and directly or indirectly, through innate
immune pathways, antagonize infection (Wu and Chen, 2014). A pertinent example of these factors is the IFIT family of proteins
discussed below. y
g
What are the functional consequences of cap and 2′-O-
methylation? Mutational analyses of the NS5 MTase have
identified residues that specifically ablate 2′-O-methylation, cap-
methylation, or both. ROLES FOR METHYLATION AT THE 5′
END OF THE FLAVIVIRUS GENOME Interestingly, loss of 2′-O-methylation can
be tolerated whereas cap methylation is essential for infection
(Zhou et al., 2007; Dong et al., 2010). In considering why cap
methylation is critical, it is worthwhile to consider data from
Ray et al. (2006) who measured the effects of GpppA, m7GpppA
and m7GpppAm caps on translation and RNA synthesis using
the WNV replicon system which encodes the Renilla luciferase
(RLuc) reporter. Compared to uncapped (pppA) WNV replicon
RNA, the addition of GpppA strongly (∼25-fold) enhanced the
accumulation of (RLuc) at 2 h post-transfection. Surprisingly,
m7GpppA enhanced replicon RNA translation by only twofold
compared to unmethylated GpppA cap, and m7GpppAm did not
further increase RLuc expression. Unexpectedly, no differences
in replicon RNA levels were detected for the differently capped
replicons. By 72 h post-transfection, each of the capped replicon
RNAs produced similar RLuc, indicating that methylation is
not required for initial negative-strand synthesis as this is
a prerequisite for synthesis of downstream positive-strand
synthesis and consequent production of RLuc. What are the functional consequences of 2′-O-methylation
at the penultimate nucleotide? As noted above, loss of this
methylation event does not cause virus lethality in contrast to
cap methylation. Key insights into this question were made by
Diamond and colleagues who observed that a 2′-O-methylation-
deficient NS5 mutant (E218A) WNV lacking m7GpppAm was
attenuated in immunocompetent mice and primary cells, whereas
animals and cells lacking the type I interferon (IFN) receptor
(IFNAR-/-) were fully susceptible to infection (Daffis et al., 2010). These authors went on to show that IFN-inducible proteins of
the IFIT family (murine IFIT1 and IFIT2) disproportionately
restricted WNV lacking 2′-O-methylation compared to WT
virus. Some IFIT proteins have been described to inhibit
translation via binding and interfering with the function of eIF3
(Hui et al., 2003), a complex of initiation factors that recruit
the 40S ribosomal subunit to mRNA via interaction with eIF4G
during initiation of translation (Hershey et al., 2012). More
recently, several groups have identified human IFIT1 as a protein
that binds directly to cap-0 and blocks translation (Kumar et al.,
2014; Abbas et al., 2017), presumably by hindering access of eIF4E
to the cap structure. This translational suppression coincides
with an accelerated innate immune response that compromises
infection (Schmid et al., 2015; Chang et al., 2016). ROLES FOR METHYLATION AT THE 5′
END OF THE FLAVIVIRUS GENOME Taken
together, these reports strongly suggest that 2′-O-methylation of
the cap is an epigenetic RNA modification that allows cells to
differentiate self versus non-self RNAs via IFIT proteins. Clearly
flaviviruses, and indeed many other types of viruses, have evolved
mechanisms to evade IFIT-mediated restriction by encoding their
own 2′-O-MTases. There are at least three non-mutually exclusive explanations
for a cap methylation requirement by flaviviruses. First, the
cap structure itself is known to protect RNA from 5′ to 3′
exonucleases such as Xrn1 (Hsu and Stevens, 1993) and likely
plays a significant role in preventing viral RNA decay. However,
it is not clear whether cap methylation plays a significant role
in stabilizing RNA. Indeed, the human decapping protein Dcp2
cannot act on an capped RNA substrate lacking N7-methylation
(Wang et al., 2002), suggesting that methylation actually enables
decapping which is a prerequisite for 5′-3′ decay (Wilusz et al.,
2001). Nevertheless, it is possible that cap methylation may
render flaviviral genomes resistant to cellular RNases by an
unknown mechanism. A role in stimulating viral translation initiation is a
plausible explanation for a cap methylation requirement by
flaviviruses. The canonical mRNA cap-binding protein, eIF4E, is
believed to be essential for translation of most cellular mRNAs
through indirect recruitment of the 40S ribosomal subunit and
associated initiation factors (Hershey et al., 2012). EIF4E strongly
discriminates between m7Gppp and Gppp, and early studies by
Shatkin and colleagues demonstrated that methylation enhanced
cap-dependent translation (Both et al., 1975; Muthukrishnan
et al., 1975). There are, however, a few clues that flavivirus Frontiers in Microbiology | www.frontiersin.org ROLES FOR INTERNAL ADENOSINE
METHYLATION IN INFECTION BY
FLAVIVIRUSES AND HCV It has been recognized for several decades that a prominent
modification to cellular mRNA across many diverse organisms
is the methylation of adenosine at the N6 position (Desrosiers December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 3 Bradrick Flavivirus Methylation et al., 1974; Perry and Kelley, 1974; Zhao et al., 2016). This occurs
at internal mRNA positions (m6A) and also at the penultimate
nucleotide of transcripts that initiate with A (Figure 1). The
latter is referred to as m6Am as it is also methylated at the 2′-
hydroxyl (Wei C. et al., 1975). In the past few years research on
m6A has greatly expanded and multiple studies have addressed
roles of m6A in virus infection. Several methyltransferase and
demethylase enzymes have been identified as well as proteins
that can recognize methyl groups in RNA (Zhao et al., 2016;
Meyer and Jaffrey, 2017). These factors are referred to as “writers,”
“erasers,” and “readers” of m6A. A key recent innovation is the
use of m6A-specific antibodies in RNA-immunoprecipitation to
allow transcriptome-wide mapping of m6A locations in RNA
molecules (Dominissini et al., 2012; Meyer et al., 2012; Linder
et al., 2015). This has enabled identification and functional
analysis of m6A sites by mutation of the low complexity
consensus motif DRACH (D = G/A/U; R = G/A; H = C/A/U). antagonism of ZIKV infection by these reader proteins and
YTHDF2 in particular. The authors speculated that YTHDF2
may bind to and destabilize ZIKV RNA. Finally, Lichinchi
et al. (2016b) reported that ZIKV infection alters the host m6A
methylome, implying that gene expression changes caused by
infection may be partly due to altered m6A patterns on cellular
mRNA. antagonism of ZIKV infection by these reader proteins and
YTHDF2 in particular. The authors speculated that YTHDF2
may bind to and destabilize ZIKV RNA. Finally, Lichinchi
et al. (2016b) reported that ZIKV infection alters the host m6A
methylome, implying that gene expression changes caused by
infection may be partly due to altered m6A patterns on cellular
mRNA. Many open questions remain in the nascent field addressing
roles for m6A in flavivirus infection. What is the mechanism
by which m6A inhibits ZIKV infection? Are similar effects
and modes of action operating during infection with other
flaviviruses? Does m6A impact cellular innate immune responses? How does infection impact functionality of the machinery that
regulates the m6A methylome? Does m6A control flavivirus
infection in vivo? ROLES FOR INTERNAL ADENOSINE
METHYLATION IN INFECTION BY
FLAVIVIRUSES AND HCV Notably,
depletion of these enzymes had no effect on HCV translation
or RNA synthesis, suggesting a role for m6A in opposing a late
stage of infection such as assembly or egress of infectious virus. Consistent with this idea, several known cytosolic reader proteins
(YTHDF1-3) suppressed viral titers, co-immunoprecipitated
HCV RNA and localized to lipid droplets which are known
sites of HCV assembly (Miyanari et al., 2007). Silent mutation
of four m6A sites within the envelope coding region enhanced
infection, providing further evidence for a restrictive role of m6A
in HCV infection. Gokhale et al. (2016) went on to map m6A
in the genomes of multiple mosquito-transmitted flaviviruses,
including DENV, YFV, WNV, and two divergent strains of ZIKV. Of note, this analysis revealed abundant m6A within the NS5
coding regions of these viruses. ROLES FOR INTERNAL ADENOSINE
METHYLATION IN INFECTION BY
FLAVIVIRUSES AND HCV In the context of HCV, how do YTHDF proteins
suppress late stages of the virus life-cycle? Lichinchi
et
al. (2016b)
observed
elevated
titer
and
viral RNA in supernatants of cells with reduced levels of
METTL3/METTL14 which implies that m6A modification
opposes virus infection. It should be noted that the overall effects
on ZIKV production may be considered mild (< ∼2-fold),
indicating that m6A acts as a moderate restriction factor for
infection in vitro. Nevertheless, the relatively small effects could
reflect a viral strategy that is able to counter, in part, otherwise
potent restriction by m6A and trans-acting reader proteins. One hypothesis is that the subgenomic flaviviral RNA (sfRNA),
a highly stable RNA fragment produced from decay of virus
genomes (Pijlman et al., 2008), could act to buffer the negative
impact of m6A by sequestering YTHDF reader proteins. It would
be of interest, for example, to test whether strains of DENV
that produce different amounts of sfRNA would be differentially
susceptible to inhibition by m6A (Manokaran et al., 2015). Of
course, this is only one hypothesis and there remains much work
to be done to gain a thorough understanding of how flavivirus
infections are affected by m6A. consensus motif DRACH (D = G/A/U; R = G/A; H = C/A/U). To date m6A mapping and some functional analyses
have been performed on multiple viruses including influenza
A virus (Courtney et al., 2017), human immunodeficiency
virus
(Kennedy
et
al.,
2016;
Lichinchi
et
al.,
2016a;
Tirumuru et al., 2016), HCV (Gokhale et al., 2016), YFV
(Gokhale et al., 2016), DENV (Gokhale et al., 2016), WNV
(Gokhale et al., 2016), and ZIKV (Gokhale et al., 2016; Lichinchi
et al., 2016b). Relevant to this discussion are the studies
conducted on Flaviviridae by Gokhale et al. (2016) and Lichinchi
et al. (2016b) who characterized functional roles of m6A in
HCV and ZIKV infection, respectively. To determine the
effects of m6A on infection, Gokhale et al. (2016) depleted
the key methylase (METTL3 plus its co-factor METTL14) and
demethylases (FTO and ALKBH5) by RNA interference and
assayed effects on HCV infection. Intriguingly, knockdown
of
METTL3/14
enhanced
infection
while
FTO
depletion
correspondingly reduced infection. These results are consistent
with an antiviral role for m6A in the HCV life-cycle. REFERENCES Yeast cells lacking 5′– > 3′ exoribonuclease
1 contain mRNA species that are poly(A) deficient and partially lack the
5′ cap structure. Mol. Cell. Biol. 13, 4826–4835. doi: 10.1128/MCB.13.8. 4826 Campos, R. K., Garcia-Blanco, M. A., and Bradrick, S. S. (2017). Roles of pro-
viral host factors in mosquito-borne flavivirus infections. Curr. Top. Microbiol. Immunol. doi: 10.1007/82_2017_26 [Epub ahead of print]. Hui, D. J., Bhasker, C. R., Merrick, W. C., and Sen, G. C. (2003). Viral stress-
inducible protein p56 inhibits translation by blocking the interaction of eIF3
with the ternary complex eIF2·GTP·Met-tRNAi. J. Biol. Chem. 278, 39477–
39482. doi: 10.1074/jbc.M305038200 Chang, D. C., Hoang, L. T., Mohamed Naim, A. N., Dong, H., Schreiber, M. J.,
Hibberd, M. L., et al. (2016). Evasion of early innate immune response by 2’-
O-methylation of dengue genomic RNA. Virology 499, 259–266. doi: 10.1016/j. virol.2016.09.022 Issur, M., Geiss, B. J., Bougie, I., Picard-Jean, F., Despins, S., Mayette, J., et al. (2009). The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a
two-step reaction to form the RNA cap structure. RNA 15, 2340–2350. doi:
10.1261/rna.1609709 Courtney, D. G., Kennedy, E. M., Dumm, R. E., Bogerd, H. P., Tsai, K., Heaton,
N. S., et al. (2017). Epitranscriptomic enhancement of influenza A virus gene
expression and replication. Cell Host Microbe 22, 377.e–386.e. doi: 10.1016/j. chom.2017.08.004 Kennedy, E. M., Bogerd, H. P., Kornepati, A. V. R., Kang, D., Ghoshal, D., Marshall,
J. B., et al. (2016). Posttranscriptional m6 A editing of HIV-1 mRNAs enhances
viral gene expression. Cell Host Microbe 19, 675–685. doi: 10.1016/j.chom.2016. 04.002 Cowling, V. H. (2010). Regulation of mRNA cap methylation. Biochem. J. 425,
295–302. doi: 10.1042/BJ20091352 Daffis, S., Szretter, K. J., Schriewer, J., Li, J., Youn, S., Errett, J., et al. (2010). 2′-
O methylation of the viral mRNA cap evades host restriction by IFIT family
members. Nature 468, 452–456. doi: 10.1038/nature09489 Kumar, P., Sweeney, T. R., Skabkin, M. A., Skabkina, O. V., Hellen, C. U. T.,
and Pestova, T. V. (2014). Inhibition of translation by IFIT family members
is determined by their ability to interact selectively with the 5′-terminal
regions of cap0-, cap1- and 5′ppp- mRNAs. Nucleic Acids Res. 42, 3228–3245. doi: 10.1093/nar/gkt1321 Desrosiers, R., Friderici, K., and Rottman, F. (1974). Identification of methylated
nucleosides in messenger RNA from Novikoffhepatoma cells. Proc. Natl. Acad. Sci. U. S. A. 71, 3971–3975. REFERENCES Edgil, D., Polacek, C., and Harris, E. (2006). Dengue virus utilizes a novel strategy
for translation initiation when cap-dependent translation is inhibited. J. Virol. 80, 2976–2986. doi: 10.1128/JVI.80.6.2976-2986.2006 Abbas, Y. M., Laudenbach, B. T., Martínez-Montero, S., Cencic, R., Habjan, M.,
Pichlmair, A., et al. (2017). Structure of human IFIT1 with capped RNA reveals
adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-
O methylations. Proc. Natl. Acad. Sci. U.S.A. 114, E2106–E2115. doi: 10.1073/
pnas.1612444114 Egloff, M.-P., Benarroch, D., Selisko, B., Romette, J.-L., and Canard, B. (2002). An RNA cap (nucleoside-2′-O-)-methyltransferase in the flavivirus RNA
polymerase NS5: crystal structure and functional characterization. EMBO J. 21,
2757–2768. doi: 10.1093/emboj/21.11.2757 O methylations. Proc. Natl. Acad. Sci. U.S.A. 114, E2106–E2115. doi: 10.1073/
pnas.1612444114 Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S., and Nicchitta, C. (2016). Bartelma, G., and Padmanabhan, R. (2002). Expression, purification, and
characterization of the RNA 5′-triphosphatase activity of dengue virus type 2
nonstructural protein 3. Virology 299, 122–132. doi: 10.1006/viro.2002.1504 Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S., and Nicchitta, C. (2016). Flavivirus RNA transactions from viral entry to genome replication Antiviral Garcia-Blanco, M. A., Vasudevan, S. G., Bradrick, S. S., and Nicchitta, C. (2016). Flavivirus RNA transactions from viral entry to genome replication. Antiviral
Res. 134, 244–249. doi: 10.1016/j.antiviral.2016.09.010 onstructural protein 3. Virology 299, 122–132. doi: 10.1006/viro.20 Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes,
C. L., et al. (2013). The global distribution and burden of dengue. Nature 496,
504–507. doi: 10.1038/nature12060 Gokhale, N. S., McIntyre, A. B. R., McFadden, M. J., Roder, A. E., Kennedy, E. M.,
Gandara, J. A., et al. (2016). N6 -methyladenosine in Flaviviridae viral RNA
genomes regulates infection. Cell Host Microbe 20, 654–665. doi: 10.1016/j. chom.2016.09.015 Both, G. W., Banerjee, A. K., and Shatkin, A. J. (1975). Methylation-dependent
translation of viral messenger RNAs in vitro. Proc. Natl. Acad. Sci. U.S.A. 72,
1189–1193. doi: 10.1073/pnas.72.3.1189 Hershey, J. W. B., Sonenberg, N., and Mathews, M. B. (2012). Principles of
translational control: an overview. Cold Spring Harb. Perspect. Biol. 4:a011528. doi: 10.1101/cshperspect.a011528 Brinton, M. A., and Dispoto, J. H. (1988). Sequence and secondary structure
analysis of the 5′-terminal region of flavivirus genome RNA. Virology 162,
290–299. doi: 10.1016/0042-6822(88)90468-0 Hsu, C. L., and Stevens, A. (1993). FUNDING infection with virulent viruses (Li et al., 2013; Züst et al.,
2013). Thus, understanding the basic molecular mechanisms of
flavivirus biology will hopefully lead to measures that reduce the
burden caused by these viruses. The author acknowledges Mariano Garcia-Blanco and the
University of Texas Medical Branch for support. ACKNOWLEDGMENT The author thanks Gaddiel Galarza-Munoza and Rafael Campos
for thoughtful comments. The author confirms being the sole contributor of this work and
approved it for publication. CONCLUSION Flavivirus RNA methylation critically impacts infection. Cap
methylation (m7GpppA) is essential for infection, at least in
part due to its role in stimulating virus translation. Methylation
at the 2′-hydroxyl of the penultimate adenosine (m7GpppAm)
is inessential for viability but allows the virus to escape the
inhibitory actions of IFIT proteins and likely other factor(s)
(Szretter et al., 2012). In contrast, internal m6A modifications
are somewhat deleterious to ZIKV and HCV infections in vitro
although there is much to be learned regarding roles for m6A
in infection. Studies addressing viral RNA methylation are
informative with respect to basic virus biology but may also allow
development of approaches to control infections by pathogenic
flaviruses for which there are no currently available therapeutics. Drugs specifically targeting flavivirus MTase enzymes could
be potent antivirals for the treatment of patients with acute
infections. Moreover, mutant flaviviruses lacking 2′-O-MTase
activity have shown promise as candidate vaccine strains because
they are attenuated yet induce robust immunity to heterologous In their companion article to the Gokhale et al. (2016)
study, Lichinchi et al. (2016b) and colleagues mapped locations
m6A on ZIKV RNA and investigated the roles of readers,
writers and erasers in infection. Depletion of METTL3 or
METTL14 enhanced ZIKV infection in 293T cells whereas
ALKBH5 and, to a lesser extent, FTO knockdown reduced
infection. Moreover, YTDHF1/2 expression negatively correlated
with ZIKV RNA levels released from infected cells, suggesting December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 4 Bradrick Flavivirus Methylation REFERENCES 33, 319–342. doi: 10.1146/annurev-cellbio-100616-
060758 Wang, Z., Jiao, X., Carr-Schmid, A., and Kiledjian, M. (2002). The hDcp2 protein
is a mammalian mRNA decapping enzyme. Proc. Natl. Acad. Sci. U.S.A. 99,
12663–12668. doi: 10.1073/pnas.192445599 Meyer, K. D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C. E., and Jaffrey, S. R. (2012). Comprehensive analysis of mRNA methylation reveals enrichment in
3′ UTRs and near stop codons. Cell 149, 1635–1646. doi: 10.1016/j.cell.2012. 05.003 Wei, C., Gershowitz, A., and Moss, B. (1975). N6, O2′-dimethyladenosine a novel
methylated ribonucleoside next to the 5′ terminal of animal cell and virus
mRNAs. Nature 257, 251–253. doi: 10.1038/257251a0 mRNAs. Nature 257, 251–253. doi: 10.1038/257251a0 Miyanari, Y., Atsuzawa, K., Usuda, N., Watashi, K., Hishiki, T., Zayas, M., et al. (2007). The lipid droplet is an important organelle for hepatitis C virus
production. Nat. Cell Biol. 9, 1089–1097. doi: 10.1038/ncb1631 Wei, C. M., Gershowitz, A., and Moss, B. (1975). Methylated nucleotides block
5′ terminus of HeLa cell messenger RNA. Cell 4, 379–386. doi: 10.1016/0092-
8674(75)90158-0 Muthukrishnan, S., Both, G. W., Furuichi, Y., and Shatkin, A. J. (1975). 5′-Terminal
7-methylguanosine in eukaryotic mRNA is required for translation. Nature 255,
33–37. doi: 10.1038/255033a0 Wei, C. M., and Moss, B. (1975). Methylated nucleotides block 5′-terminus of
vaccinia virus messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 72, 318–322. doi: 10.1073/pnas.72.1.318 Perry, R. P., and Kelley, D. E. (1974). Existence of methylated messenger RNA in
mouse L cells. Cell 1, 37–42. doi: 10.1016/0092-8674(74)90153-6 Wengler, G., and Wengler, G. (1993). The NS 3 nonstructural protein of flaviviruses
contains an RNA triphosphatase activity. Virology 197, 265–273. doi: 10.1006/
viro.1993.1587 Phatnani, H. P., and Greenleaf, A. L. (2006). Phosphorylation and functions of
the RNA polymerase II CTD. Genes Dev. 20, 2922–2936. doi: 10.1101/gad.147
7006 Wilusz, C. J., Wormington, M., and Peltz, S. W. (2001). The cap-to-tail guide
to mRNA turnover. Nat. Rev. Mol. Cell Biol. 2, 237–246. doi: 10.1038/3506
7025 Pijlman, G. P., Funk, A., Kondratieva, N., Leung, J., Torres, S., van der Aa, L.,
et al. (2008). A highly structured, nuclease-resistant, noncoding RNA produced
by flaviviruses is required for pathogenicity. Cell Host Microbe 4, 579–591. doi: 10.1016/j.chom.2008.10.007 Wu, J., and Chen, Z. J. (2014). Innate immune sensing and signaling of cytosolic
nucleic acids. Annu. Rev. Immunol. 32, 461–488. doi: 10.1146/annurev-
immunol-032713-120156 Zhao, B. S., Roundtree, I. A., and He, C. (2016). Post-transcriptional gene
regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42. REFERENCES doi: 10.1073/pnas.71.10.3971 Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., Salmon-Divon, M.,
Ungar, L., Osenberg, S., et al. (2012). Topology of the human and mouse m6A
RNA methylomes revealed by m6A-seq. Nature 485, 201–206. doi: 10.1038/
nature11112 Li, S.-H., Dong, H., Li, X.-F., Xie, X., Zhao, H., Deng, Y.-Q., et al. (2013). Rational
design of a flavivirus vaccine by abolishing viral RNA 2′-O methylation. J. Virol. 87, 5812–5819. doi: 10.1128/JVI.02806-12 Lichinchi, G., Gao, S., Saletore, Y., Gonzalez, G. M., Bansal, V., Wang, Y., et al. (2016a). Dynamics of the human and viral m6A RNA methylomes during
HIV-1 infection of T cells. Nat. Microbiol. 1, 16011. doi: 10.1038/nmicrobiol. 2016.11 Dong, H., Chang, D. C., Xie, X., Toh, Y. X., Chung, K. Y., Zou, G., et al. (2010). Biochemical and genetic characterization of dengue virus methyltransferase. Virology 405, 568–578. doi: 10.1016/j.virol.2010.06.039 Lichinchi, G., Zhao, B. S., Wu, Y., Lu, Z., Qin, Y., He, C., et al. (2016b). Dynamics
of human and viral RNA methylation during Zika virus infection. Cell Host
Microbe 20, 666–673. doi: 10.1016/j.chom.2016.10.002 Dong, H., Fink, K., Zust, R., Lim, S. P., Qin, C.-F., and Shi, P.-Y. (2014). Flavivirus RNA methylation. J. Gen. Virol. 95, 763–778. doi: 10.1099/vir.0.062
208-0 Dong, H., Ray, D., Ren, S., Zhang, B., Puig-Basagoiti, F., Takagi, Y., et al. (2007). Distinct RNA elements confer specificity to flavivirus RNA cap methylation
events. J. Virol. 81, 4412–4421. doi: 10.1128/JVI.02455-06 Linder, B., Grozhik, A. V., Olarerin-George, A. O., Meydan, C., Mason, C. E., and
Jaffrey, S. R. (2015). Single-nucleotide-resolution mapping of m6A and m6Am
throughout the transcriptome. Nat. Methods 12, 767–772. doi: 10.1038/nmeth. 3453 Dong, H., Ren, S., Li, H., and Shi, P.-Y. (2008). Separate molecules of west nile virus
methyltransferase can independently catalyze the N7 and 2′-O methylations of
viral RNA cap. Virology 377, 1–6. doi: 10.1016/j.virol.2008.04.026 Manokaran, G., Finol, E., Wang, C., Gunaratne, J., Bahl, J., Ong, E. Z., et al. (2015). Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 5 Bradrick Flavivirus Methylation for epidemiological fitness. Science 350, 217–221. doi: 10.1126/science.aab
3369 for epidemiological fitness. Science 350, 217–221. doi: 10.1126/science.aab
3369 Tsukiyama-Kohara, K., Iizuka, N., Kohara, M., and Nomoto, A. (1992). Internal ribosome entry site within hepatitis C virus RNA. J. Virol. 66,
1476–1483. Meyer, K. D., and Jaffrey, S. R. (2017). Rethinking m6 a readers, writers, and erasers. Annu. Rev. Cell Dev. Biol. REFERENCES doi: 10.1038/nrm.2016.132 Ray, D., Shah, A., Tilgner, M., Guo, Y., Zhao, Y., Dong, H., et al. (2006). West
nile virus 5′-cap structure is formed by sequential guanine N-7 and ribose
2′-O methylations by nonstructural protein 5. J. Virol. 80, 8362–8370. doi:
10.1128/JVI.00814-06 Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., et al. (2007). Structure
and function of flavivirus NS5 methyltransferase. J. Virol. 81, 3891–3903. doi: 10.1128/JVI.02704-06 Saletore, Y., Meyer, K., Korlach, J., Vilfan, I. D., Jaffrey, S., and Mason,
C. E. (2012). The birth of the Epitranscriptome: deciphering the function
of RNA modifications. Genome Biol. 13, 175. doi: 10.1186/gb-2012-13-
10-175 Züst, R., Dong, H., Li, X.-F., Chang, D. C., Zhang, B., Balakrishnan, T., et al. (2013). Rational design of a live attenuated dengue vaccine: 2′-O-methyltransferase
mutants are highly attenuated and immunogenic in mice and macaques. PLOS
Pathog. 9:e1003521. doi: 10.1371/journal.ppat.1003521 Schmid, B., Rinas, M., Ruggieri, A., Acosta, E. G., Bartenschlager, M.,
Reuter, A., et al. (2015). Live cell analysis and mathematical modeling identify
determinants of attenuation of dengue virus 2′-O-methylation mutant. PLOS
Pathog. 11:e1005345. doi: 10.1371/journal.ppat.1005345 Conflict of Interest Statement: The author declares that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Conflict of Interest Statement: The author declares that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Shatkin, A. J. (1976). Capping of eucaryotic mRNAs. Cell 9, 645–653. doi: 10.1016/
0092-8674(76)90128-8 Szretter, K. J., Daniels, B. P., Cho, H., Gainey, M. D., Yokoyama, W. M., Gale, M.,
et al. (2012). 2′-O methylation of the viral mRNA cap by west nile virus evades
ifit1-dependent and -independent mechanisms of host restriction in vivo. PLOS
Pathog. 8:e1002698. doi: 10.1371/journal.ppat.1002698 Copyright © 2017 Bradrick. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms. Tirumuru, N., Zhao, B. S., Lu, W., Lu, Z., He, C., and Wu, L. (2016). N6 -
methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag
protein expression. Frontiers in Microbiology | www.frontiersin.org REFERENCES Elife 5:e15528. doi: 10.7554/eLife.15528 December 2017 | Volume 8 | Article 2374 Frontiers in Microbiology | www.frontiersin.org 6
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